RLIMS-P Benchmarking Data Set for Extracting Phosphorylation Features from Abstracts (59 tagged abstracts) FT - PIR feature line TI - Title of the abstract AB - Abstract Each abstract (PMID) could be tagged for more than one feature line. Tagged evidences are highlighted in red. January 24, 2005        https://proteininformationresource.org/iprolink/

1. PMID:1377674
PIR:DVHUCF
FT - binding site | phosphate (Ser) (covalent) (by protein kinase C) | 686,790 (all)
TI - Phosphorylation of the cystic fibrosis transmembrane conductance
regulator.
AB - Regulation of epithelial chloride flux, which is defective in patients
with cystic fibrosis, may be mediated by phosphorylation of the cystic
fibrosis transmembrane conductance regulator (CFTR) by cyclic
AMP-dependent protein kinase (PKA) or protein kinase C (PKC). Part of the
R-domain of CFTR (termed CF-2) was expressed in and purified from
Escherichia coli. CF-2 was phosphorylated on seryl residues by PKA, PKC,
cyclic GMP-dependent protein kinase (PKG), and
calcium/calmodulin-dependent protein kinase I (CaM kinase I). Direct amino
acid sequencing and peptide mapping of CF-2 revealed that serines 660,
700, 737, and 813 as well as serine 768, serine 795, or both were
phosphorylated by PKA and PKG, and serines 686 and 790 were phosphorylated
by PKC. CFTR was phosphorylated in vitro by PKA, PKC, or PKG on the same
sites that were phosphorylated in CF-2. Kinetic analysis of
phosphorylation of CF-2 and of synthetic peptides confirmed that these
sites were excellent substrates for PKA, PKC, or PKG. CFTR was
immunoprecipitated from T84 cells labeled with 32Pi. Its phosphorylation
was stimulated in response to agents that activated either PKA or PKC.
Peptide mapping confirmed that CFTR was phosphorylated at several sites
identified in vitro. Thus, regulation of CFTR is likely to occur through
direct phosphorylation of the R-domain by protein kinases stimulated by
different second messenger pathways.
SO - J Biol Chem 1992 Jun 25;267(18):12742-52.

PIR:DVHUCF
FT - binding site | phosphate (Ser) (covalent) (by cAMP- and cGMP-dependent kinases) | 660,700,737,813 (all)
TI - Phosphorylation of the cystic fibrosis transmembrane conductance
regulator.
AB - Regulation of epithelial chloride flux, which is defective in patients
with cystic fibrosis, may be mediated by phosphorylation of the cystic
fibrosis transmembrane conductance regulator (CFTR) by cyclic
AMP-dependent protein kinase (PKA) or protein kinase C (PKC). Part of the
R-domain of CFTR (termed CF-2) was expressed in and purified from
Escherichia coli. CF-2 was phosphorylated on seryl residues by PKA, PKC,
cyclic GMP-dependent protein kinase (PKG), and
calcium/calmodulin-dependent protein kinase I (CaM kinase I). Direct amino
acid sequencing and peptide mapping of CF-2 revealed that serines 660,
700, 737, and 813 as well as serine 768, serine 795, or both were
phosphorylated by PKA and PKG, and serines 686 and 790 were phosphorylated
by PKC. CFTR was phosphorylated in vitro by PKA, PKC, or PKG on the same
sites that were phosphorylated in CF-2. Kinetic analysis of
phosphorylation of CF-2 and of synthetic peptides confirmed that these
sites were excellent substrates for PKA, PKC, or PKG. CFTR was
immunoprecipitated from T84 cells labeled with 32Pi. Its phosphorylation
was stimulated in response to agents that activated either PKA or PKC.
Peptide mapping confirmed that CFTR was phosphorylated at several sites
identified in vitro. Thus, regulation of CFTR is likely to occur through
direct phosphorylation of the R-domain by protein kinases stimulated by
different second messenger pathways.
SO - J Biol Chem 1992 Jun 25;267(18):12742-52.

2. PMID:164350
PIR:A31334
FT - binding site | phosphate (Ser) (covalent) (by cAMP-dependent kinase) | 1018 (all)
TI - The hormonal control of activity of skeletal muscle phosphorylase kinase.
Amino-acid sequences at the two sites of action of
adenosine-3':5'-monophosphate-dependent protein kinase.
AB - Two tryptic phosphopeptides containing the sites on the alpha and beta
subunits of phosphorylase kinase which are phosphorylated by protein
kinase, dependent on adenosine 3':5'-monophosphate (cyclic AMP), have been
isolated and their amino acid sequences have been determined. 32P-labelled
phosphorylase kinase, containing 1.9 mol phosphate per mol enzyme, was
digested with an equimolar quantity of trypsin for 2.5 min at pH 7.0, 20
degrees C. This treatment released nearly all the 32P radioactivity
associated with the beta subunit as trichloroacetic-acid-soluble material.
Only a small proportion of the 32P radioactivity associated with the alpha
subunit was solubilised, the remainder being removed in the
trichloroacetic acid pellet. The beta-subunit tryptic phosphopeptide was
completely resolved from traces of the alpha-subunit phosphopeptide by gel
filtration on Sephadex G-25. Further purification by peptide mapping
separated the phosphopeptide into four components, each derived from the
same nine-amino-acid segment of the betachain, which was found to possess
the sequence: Gln-Ser-Gly-Ser(P)-Val-Ile-Tyr-Pro-Leu-Lys. The four
components were produced by the partial cyclisation of the N-terminal
glutaminyl residue, and by the presence of two alleles for the beta
subunit in the rabbit population, which led to a valine-isoleucine
ambiguity. The alpha-subunit phosphopeptide was liberated from the
trichloroacetic acid pellet by redigestion with trypsin. It was the
largest component in the digest which remained soluble in 5%
trichloroacetic acid, and obtained in a highly purified form by a single
filtration on Sephadex G-50. The peptide comprised 39 amino acids of which
nine were serine and three were threonine residues. Only one residue, the
serine at position three from the amino terminus, was phosphorylated. The
amino-terminal sequence of the peptide was shown to be:
Arg-Leu-Ser(P)-Ile-Ser-Thr-Glu-Ser-Glx-Pro-Asx-Gly. The sequences confirm
the stoichiometry of the reaction and the absolute specificity of
cyclic-AMP-dependent protein kinase for just two of the 200 serine
residues in the enzyme. These results and an inspection of the rate of
phosphorylation of a number of skeletal muscle proteins, including each
enzyme of the glycolytic pathway, lead to the conclusion that
cyclic-AMP-dependent protein kinase is an extremely specific enzyme. The
molecular basis of this specificity is discussed.
SO - Eur J Biochem 1975 Feb 3;51(1):79-92.

3. PMID:1696913
PIR:SGHU1V
FT - binding site | phosphate (Ser) (covalent) (by cAMP-dependent kinase) | 397 (all)
TI - The phosphorylation of the two-chain form of vitronectin by protein kinase
A is heparin dependent.
AB - In circulating blood, vitronectin occurs in two forms: a single-chain (75
kDa) and an endogenously clipped two-chain form (65 kDa and 10 kDa) held
together by a disulfide bridge. The 75 kDa form was previously shown to be
phosphorylated at Ser378 by protein kinase A, released by physiologically
stimulated platelets. By contrast, at pH 7.5 the two-chain form is not
phosphorylated at all. Heparin or heparan sulfate are shown here to
modulate the conformation of clipped vitronectin at physiological pH,
exposing Ser378 and allowing its stoichiometric phosphorylation by the
kinase. At this pH the two-chain form of vitronectin in plasma exhibits a
higher affinity for heparin, and behaves as a flexible molecule, which can
conformationally respond to heparin and heparan sulfate, effectors
involved in vitronectin function.
SO - FEBS Lett 1990 Aug 20;269(1):221-5.

4. PMID:1737801
PIR:A40936
FT - binding site | phosphate (Ser) (covalent) (by MAP and cdc2 kinases) | 25 (all)
TI - Analysis of phosphoprotein p19 by liquid chromatography/mass spectrometry.
Identification of two proline-directed serine phosphorylation sites and a
blocked amino terminus.
AB - p19 is a highly conserved 19-kDa cytosolic protein that undergoes
phosphorylation in mammalian cells upon activation of several distinct
signal transduction pathways. Its expression is widespread but
developmentally regulated. To determine the in vivo phosphorylation
site(s) of p19, the protein was purified from bovine brain and resolved
into the unphosphorylated form (p19) and a mixture of the two predominant
phospho-forms (pp19). Proteolytic fragments of p19 and pp19 were examined
by liquid chromatography/mass spectrometry (LC/MS). We detected ion masses
corresponding to fragments spanning the entire amino acid sequence as
deduced from the cDNA except for those predicted to contain an unmodified
amino terminus. Instead, the digests revealed ions corresponding to
peptides lacking the initiator methionine and containing an N-acetylated
alanine at the amino terminus. The analysis of pp19, but not that of p19,
revealed two sets of ions representing peptides whose m/z values differed
by 80 atomic mass units, the incremental mass of a phosphate residue.
These putative phosphate-bearing peptides were sensitive to alkaline
phosphatase treatment. Using combined trypsin and V8 protease digestions,
the phosphorylation sites were mapped to Ser-25 and Ser-38, in the
peptides Leu-Ile-Leu-Ser*-Pro-Arg and Phe-Pro-Leu-Ser*-Pro-Pro-Lys,
respectively. Interestingly, both phosphoserines are in a very similar
sequence context, suggesting that a single proline-directed serine protein
kinase, possibly p34cdc2, is responsible for phosphorylation of both sites
in vivo.
SO - J Biol Chem 1992 Feb 15;267(5):3506-13.

PIR:A40936
FT - binding site | phosphate (Ser) (covalent) (by cdc2 kinase) | 38 (all)
TI - Analysis of phosphoprotein p19 by liquid chromatography/mass spectrometry.
Identification of two proline-directed serine phosphorylation sites and a
blocked amino terminus.
AB - p19 is a highly conserved 19-kDa cytosolic protein that undergoes
phosphorylation in mammalian cells upon activation of several distinct
signal transduction pathways. Its expression is widespread but
developmentally regulated. To determine the in vivo phosphorylation
site(s) of p19, the protein was purified from bovine brain and resolved
into the unphosphorylated form (p19) and a mixture of the two predominant
phospho-forms (pp19). Proteolytic fragments of p19 and pp19 were examined
by liquid chromatography/mass spectrometry (LC/MS). We detected ion masses
corresponding to fragments spanning the entire amino acid sequence as
deduced from the cDNA except for those predicted to contain an unmodified
amino terminus. Instead, the digests revealed ions corresponding to
peptides lacking the initiator methionine and containing an N-acetylated
alanine at the amino terminus. The analysis of pp19, but not that of p19,
revealed two sets of ions representing peptides whose m/z values differed
by 80 atomic mass units, the incremental mass of a phosphate residue.
These putative phosphate-bearing peptides were sensitive to alkaline
phosphatase treatment. Using combined trypsin and V8 protease digestions,
the phosphorylation sites were mapped to Ser-25 and Ser-38, in the
peptides Leu-Ile-Leu-Ser*-Pro-Arg and Phe-Pro-Leu-Ser*-Pro-Pro-Lys,
respectively. Interestingly, both phosphoserines are in a very similar
sequence context, suggesting that a single proline-directed serine protein
kinase, possibly p34cdc2, is responsible for phosphorylation of both sites
in vivo.
SO - J Biol Chem 1992 Feb 15;267(5):3506-13.

5. PMID:1778990
PIR:KIRTC2
FT - binding site | phosphate (Ser) (covalent) (by casein kinase II) (partial) | 11 (all)
TI - Phosphorylation of type II (beta) protein kinase C by casein kinase II.
AB - Rat brain type II (beta) protein kinase C (PKC) was phosphorylated by rat
lung casein kinase II (CK-II). Neither type I (gamma) nor type III (alpha)
PKC was significantly phosphorylated by CK-II. CK-II incorporated 0.2-0.3
mol of phosphate into 1 mol of type II PKC. This phosphate was located at
the single seryl residue (Ser-11) in the V1-variable region of the
regulatory domain of the PKC molecule. A glutamic acid cluster was located
at the carboxyl-terminal side of Ser-11, showing the consensus sequence
for phosphorylation by CK-II. The velocity of this phosphorylation was
enhanced by the addition of Ca2+, diolein, and phosphatidylserine, which
are all required for the activation of PKC. Phosphorylation of casein or
synthetic oligopeptides by CK-II was not affected by Ca2+, diolein, or
phosphatidylserine. Available evidence suggests that CK-II phosphorylates
preferentially the activated form of type II PKC. It remains unknown,
however, whether this reaction has a physiological significance.
SO - J Biochem (Tokyo) 1991 Oct;110(4):655-60.

6. PMID:1835085
PIR:IQECDK
FT - binding site | phosphate (Thr) (covalent) (by autophosphorylation) (partial) | 199 (all)
TI - DnaK as a thermometer: threonine-199 is site of autophosphorylation and is
critical for ATPase activity.
AB - DnaK, the sole Escherichia coli member of the highly conserved 70-kDa heat
shock protein (HSP70) family of proteins, autophosphorylates when
incubated with ATP in vitro. We show that threonine-199 is the amino acid
that becomes phosphorylated and we demonstrate that threonine-199 is
critical for the ATPase activity of DnaK. We also report that both the
ATPase and autophosphorylating activities of DnaK increase very strongly
over the range of temperatures that is physiologically relevant for E.
coli growth. The temperature dependence of either or both of these
activities could be of significance with respect to the postulated role of
DnaK as a molecular chaperone in helping cells ameliorate the deleterious
consequences of elevated temperature. Furthermore, we postulate that DnaK
plays a key role in regulation of the heat shock response by serving as a
cellular thermometer that directly senses the environmental temperature.
SO - Proc Natl Acad Sci U S A 1991 Nov 1;88(21):9513-7.

7. PMID:1869568
PIR:QRECCY
FT - binding site | phosphate (Asp) (covalent) | 57 (all)
TI - Crystal structure of Escherichia coli CheY refined at 1.7-A resolution.
AB - The three-dimensional structure of wild-type CheY from Escherichia coli
has been refined by stereochemically restrained least squares minimization
to a crystallographic R-factor of 15.1% at 1.7-A resolution. The structure
contains 1165 atoms, including all atoms of the protein, 147 water
molecules, and three sulfate ions. The final model has root mean square
deviations of 0.018 and 0.049 A from idealized bond lengths and angle
distances, respectively. Seven amino acid side chains have been modeled in
dual conformations. CheY folds as a compact (beta/alpha)5 globular
protein, with the phosphorylation region contained in a cavity on one face
of the molecule. This active site area is bordered by the carboxyl termini
of the three central beta-strands, by alpha 1, and by the loop connecting
beta 5 to alpha 5. The Lys-109 side chain of this loop extends into the
active site by virtue of its cis peptide bond conformation preceding
Pro-110. The epsilon-amino group of Lys-109 is in close bonding contact
with the carboxyl group of Asp-57, the residue that is phosphorylated in
the activation process of CheY. The details of the hydrogen bonding
network in the phosphorylation region indicate that structural
rearrangements must accompany the phosphorylation of Asp-57.
SO - J Biol Chem 1991 Aug 15;266(23):15511-9.

8. PMID:1939059
PIR:A33430
FT - binding site | phosphate (Ser) (covalent) (by cdc2 kinase) | 597,682,717 (all)
TI - Phosphorylation of caldesmon by p34cdc2 kinase. Identification of
phosphorylation sites.
AB - It has recently been shown that caldesmon from non-muscle (Yamashiro, S.,
Yamakita, Y., Hosoya, H., and Matsumura, F. (1991) Nature 349, 169-172)
and smooth muscle cells (Mak, A. S., Watson, M. H., Litwin, C. M. E., and
Wang, J. H. (1991) J. Biol. Chem. 266, 6678-6681) can be phosphorylated in
vitro by p34cdc2 kinase resulting in the inhibition of caldesmon binding
to F-actin and Ca(2+)-calmodulin. In this study, we have identified five
phosphorylation sites in smooth muscle caldesmon at Ser582, Ser667,
Thr673, Thr696, and Ser702. All the sites bear some resemblance to the
S(T)-P-X-X motif recognized by p34cdc2. The preferred site of
phosphorylation at Thr673 accounts for about 40% of the total
phosphorylation. Four of the sites occur in two pairs of closely spaced
sites, Ser667/Thr673 and Thr696/Ser702; phosphorylation of one site in
each pair inhibits strongly the phosphorylation of the second site in the
same pair, presumably due to the close proximity of the two sites. Similar
negative cooperativity in phosphorylation of Ser667 and Thr673 was
observed using a 22-residue synthetic peptide containing the two sites.
Phosphorylation of Ser667/Thr673 and Thr696/Ser702 account for about 90%
of the total level of phosphorylation and these sites are located within
the 10-kDa CNBr fragment at the COOH-terminal end of caldesmon known to
bind actin and Ca(2+)-calmodulin.
SO - J Biol Chem 1991 Oct 25;266(30):19971-5.

PIR:A33430
FT - binding site | phosphate (Thr) (covalent) (by cdc2 kinase) | 688,711 (all)
TI - Phosphorylation of caldesmon by p34cdc2 kinase. Identification of
phosphorylation sites.
AB - It has recently been shown that caldesmon from non-muscle (Yamashiro, S.,
Yamakita, Y., Hosoya, H., and Matsumura, F. (1991) Nature 349, 169-172)
and smooth muscle cells (Mak, A. S., Watson, M. H., Litwin, C. M. E., and
Wang, J. H. (1991) J. Biol. Chem. 266, 6678-6681) can be phosphorylated in
vitro by p34cdc2 kinase resulting in the inhibition of caldesmon binding
to F-actin and Ca(2+)-calmodulin. In this study, we have identified five
phosphorylation sites in smooth muscle caldesmon at Ser582, Ser667,
Thr673, Thr696, and Ser702. All the sites bear some resemblance to the
S(T)-P-X-X motif recognized by p34cdc2. The preferred site of
phosphorylation at Thr673 accounts for about 40% of the total
phosphorylation. Four of the sites occur in two pairs of closely spaced
sites, Ser667/Thr673 and Thr696/Ser702; phosphorylation of one site in
each pair inhibits strongly the phosphorylation of the second site in the
same pair, presumably due to the close proximity of the two sites. Similar
negative cooperativity in phosphorylation of Ser667 and Thr673 was
observed using a 22-residue synthetic peptide containing the two sites.
Phosphorylation of Ser667/Thr673 and Thr696/Ser702 account for about 90%
of the total level of phosphorylation and these sites are located within
the 10-kDa CNBr fragment at the COOH-terminal end of caldesmon known to
bind actin and Ca(2+)-calmodulin.
SO - J Biol Chem 1991 Oct 25;266(30):19971-5.

9. PMID:2040274
PIR:BGSH
FT - binding site | phosphate (Ser) (covalent) (partial) | 8,35,36,39 (all)
TI - Nuclear transition protein 1 from ram elongating spermatids. Mass
spectrometric characterization, primary structure and phosphorylation
sites of two variants.
AB - The ram transition protein 1 (TP1) is present in spermatid cell nuclei in
the nonphosphorylated, monophosphorylated and diphosphorylated forms. Its
primary structure was determined by automated Edman degradation of
S-carboxamidomethylated protein and of peptides generated by cleavage with
thermolysin and endoproteinase Lys-C. The ram TP1 is a small basic protein
of 54 residues and structurally very close to other mammalian TP1. The
mass spectrometric data obtained from the protein and its fragments reveal
that ram TP1 is indeed a mixture (approximately 5:1) of two structural
variants (Mr 6346 and 6300). These variants differ only by the nature of
the residue at position 27 (Cys in the major variant and Gly in the minor
variant). The study of phosphorylation sites has shown that four different
serine residues could be phosphorylated in the monophosphorylated TP1, at
positions 8, 35, 36 or 39. From previous physical studies, it has been
postulated that the Tyr32 surrounded by two highly conserved basic
clusters was responsible for the destabilization of chromatin by
intercalation of its phenol ring between the bases of double-stranded DNA.
The presence of three phosphorylatable serine residues in the very
conserved sequence 29-42 is another argument for the involvement of this
region in the interaction with DNA.
SO - Eur J Biochem 1991 May 23;198(1):13-20.

10. PMID:2108025
PIR:A31334
FT - binding site | phosphate (Ser) (covalent) (by autophosphorylation) | 972,985,1007 (all)
TI - Localization of phosphoserine residues in the alpha subunit of rabbit
skeletal muscle phosphorylase kinase.
AB - The alpha subunit of skeletal muscle phosphorylase kinase, as isolated,
carries phosphate at the serine residues 1018, 1020 and 1023. Employing
the S-ethyl-cysteine method, these residues are found to be phosphorylated
partially, i.e. differently phosphorylated species exist in muscle. Serine
1018 is a site which can be phosphorylated by the cyclic-AMP-dependent
protein kinase. The serine residues 972, 985 and 1007 are phosphorylated
by phosphorylase kinase itself when its activity is stimulated by
micromolar concentrations of Ca2+. These phosphorylation sites are not
identical to those found to be phosphorylated already in the enzyme as
prepared from freshly excised muscle. A 'multiphosphorylation loop'
uniquely present in this but not in the homologous beta subunit contains
all the phosphoserine residues so far identified in the alpha subunit.
SO - Eur J Biochem 1990 Mar 10;188(2):367-76.

PIR:A31334
FT - binding site | phosphate (Ser) (covalent) (by cAMP-dependent kinase) | 1018 (all)
TI - Localization of phosphoserine residues in the alpha subunit of rabbit
skeletal muscle phosphorylase kinase.
AB - The alpha subunit of skeletal muscle phosphorylase kinase, as isolated,
carries phosphate at the serine residues 1018, 1020 and 1023. Employing
the S-ethyl-cysteine method, these residues are found to be phosphorylated
partially, i.e. differently phosphorylated species exist in muscle. Serine
1018 is a site which can be phosphorylated by the cyclic-AMP-dependent
protein kinase. The serine residues 972, 985 and 1007 are phosphorylated
by phosphorylase kinase itself when its activity is stimulated by
micromolar concentrations of Ca2+. These phosphorylation sites are not
identical to those found to be phosphorylated already in the enzyme as
prepared from freshly excised muscle. A 'multiphosphorylation loop'
uniquely present in this but not in the homologous beta subunit contains
all the phosphoserine residues so far identified in the alpha subunit.
SO - Eur J Biochem 1990 Mar 10;188(2):367-76.

11. PMID:2109669
PIR:A60521
FT - binding site | phosphate (Ser) (covalent) (by phosphorylase b kinase) | 3 (all)
TI - Purification and characterization of glycogen phosphorylase B from
skeletal muscle of the mullet Liza ramada: amino acid sequence of the
phosphorylation site.
AB - 1. Skeletal muscle glycogen phosphorylase b has been purified from Liza
ramada (mullet). 2. The Mr of the purified enzyme subunit was found to be
97,000. By gel filtration a relative Mr of 190,000 was found. 3.
Proteolytic digestion of 32P-phosphorylated mullet phosphorylase gave a
[32P]-labelled peptide which is observed to contain Ser, its sequence
being -Gln-Ile-Ser-Val-Pro-. 4. During 'in vitro' phosphorylation of
mullet phosphorylase, 32P was incorporated in different protein bands
resolved by isoelectric focusing. The degree of radioactivity associated
with each one changed with the incubation time.
SO - Comp Biochem Physiol B 1990;95(2):295-301.

12. PMID:2122883
PIR:TIHUGK
FT - binding site | phosphate (Ser) (covalent) | 30 (all)
TI - Endogenous phosphorylation of the lipoprotein-associated coagulation
inhibitor at serine-2.
AB - Lipoprotein-associated coagulation inhibitor (LACI) inhibits activated
Factor X (Xa) directly and, in an Xa-dependent fashion, inhibits Factor
VIIa-tissue factor (TF), presumably by forming a quaternary
Xa-LACI-VIIa-TF complex. LACI isolated from the conditioned media of HepG2
cells grown in the presence of [32P]orthophosphate was observed to be
covalently phosphorylated. Dephosphorylation of 32P-LACI with phosphatase
resulted in an almost complete removal of the radiolabel. Phosphoamino
acid analysis of the purified 32P-LACI established that the
phosphorylation occurred on (a) serine residue(s). At its N-terminus, LACI
contains a cluster of acidic residues C-terminal to the serine-2 residue.
Such a site is characteristic of the sites phosphorylated by casein kinase
II (CKII) in protein substrates. Edman degradation of endogenously
labelled 32P-LACI revealed that the serine-2 residue was a major site of
phosphorylation. Phosphorylation of purified LACI by bovine CKII was
observed to occur in vitro; amino acid sequence analysis demonstrated that
CKII phosphorylated LACI at the serine-2 residue. Recombinant LACI
expressed from mouse C127 fibroblasts transfected using a
bovine-papilloma-virus expression vector was found to be endogenously
phosphorylated. By using site-directed mutagenesis, an altered form of
LACI was produced in which the serine-2 residue had been changed to
alanine. This altered LACI, although expressed in similar quantity to the
wild-type LACI, was not detectably phosphorylated. Using the altered LACI
in functional studies demonstrated that a serine residue at position 2,
and thus the phosphorylation of this site, was not essential for LACI's
inhibition of Xa and VIIa-TF activities.
SO - Biochem J 1990 Sep 15;270(3):621-5.

13. PMID:2171679
PIR:MMHUE4
FT - binding site | phosphate (Ser) (covalent) (by cAMP-dependent kinase) | 540,695 (all)
TI - Identification of two cAMP-dependent phosphorylation sites on erythrocyte
protein 4.1.
AB - In human erythrocytes, dibutyryl cyclic AMP induces the phosphorylation of
protein 4.1 on sites within the adjacent 16 kDa and 10 kDa chymotryptic
domains (Horne, W.C., Leto, T.L. and Marchesi, V.T. (1985) J. Biol. Chem.
260, 9073-9076). The 10 kDa domain also contains the
spectrin/actin-binding site (Correas, I., Leto, T.L., Speicher, D.W. and
Marchesi, V.T. (1986) J. Biol. Chem. 261, 3310-3315) and it has been shown
that phosphorylation of protein 4.1 by cyclic AMP-dependent protein kinase
inhibits the binding of protein 4.1 to spectrin and actin (Ling, E.,
Danilov, Y.N. and Cohen, C.M. (1988) J. Biol. Chem. 263, 2209-2216). In
this study, we have identified two sites on protein 4.1 which account for
80% of the phosphate incorporated into protein 4.1 during metabolic
labelling of erythrocytes in the presence of dibutyryl cyclic AMP. More
than 95% of the 32P incorporated into protein 4.1 was in the form of
phosphoserine. Reverse-phase HPLC of the peptides generated by digestion
of the isolated protein with trypsin or endoproteinase lysine C produced
two major radioactive peaks. The phosphorylation sites, identified by gas
phase sequencing of the purified phosphopeptides and confirmed by
determining the residues converted to S-ethylcysteine by reacting the
phosphopeptides with ethanethiol under alkaline conditions, were Ser-331,
in the 16 kDa domain and Ser-467, in the 10 kDa domain.
SO - Biochim Biophys Acta 1990 Oct 15;1055(1):87-92.

14. PMID:2210381
PIR:JH0216
FT - binding site | phosphate (Ser) (covalent) (by protein kinase A) | 73 (all)
TI - Analysis of the human, bovine and rat 33-kDa proteins and cDNA in retina
and pineal gland.
AB - A monoclonal antibody (mAb) was produced against a bovine retinal 33-kDa
protein. Several clones of 33-kDa protein were isolated from each library
of cDNA from human, bovine and rat retinas and rat pineal gland by mAb
screening and by hybridization with cDNA probes. Each of the four cDNA
sequences was determined and amino acid (aa) sequences were deduced from
the nucleotide sequences. The latter were nearly identical in rat retina
and rat pineal gland (99.6%) and were similar in human, bovine and rat
retina (more than 87%). Each of these cDNAs had one long ORF and encoded
245 or 246 aa. The deduced aa sequences in rat retina and rat pineal gland
were virtually identical and the sequences in human, bovine and rat retina
were highly homologous (more than 88%). The predicted Mr for each of these
proteins was 28,246 in the human, 28,176 in bovine, 28,143 in rat retina,
and 28,129 in rat pineal gland. Each of the sequences has a putative site
for phosphorylation by A kinase; we have confirmed that the putative site
is Ser73. These results show that the 33-kDa proteins in the retina and
pineal gland have the same sequences and the same phosphorylation site and
suggest that the functional role of this protein is the same in the retina
and pineal gland.
SO - Gene 1990 Jul 16;91(2):209-15.

15. PMID:2211670
PIR:A28822
FT - binding site | phosphate (Ser) (covalent) (by protein kinase C) | 887 (all)
TI - Feedback regulation of phospholipase C-beta by protein kinase C.
AB - Treatment of a variety of cells and tissues with
12-O-tetradecanoylphorbol-13-acetate (TPA), an activator of protein kinase
C (PKC) results in the inhibition of receptor-coupled inositol
phospholipid-specific phospholipase C (PLC) activity. To determine whether
or not the targets of TPA-activated PKC include one or more isozymes of
PLC, studies were carried out with PC12, C6Bu1, and NIH 3T3 cells, which
contain at least three PLC isozymes, PLC-beta, PLC-gamma, and PLC-delta.
Treatment of the cells with TPA stimulated the phosphorylation of serine
residues in PLC-beta, but the phosphorylation state of PLC-gamma and
PLC-delta was not changed significantly. Phosphorylation of bovine brain
PLC-beta by PKC in vitro resulted in a stoichiometric incorporation of
phosphate at serine 887, without any concomitant effect on PLC-beta
activity. We propose, therefore, that rather than having a direct effect
on enzyme activity, the phosphorylation of PLC-beta by PKC may alter its
interaction with a putative guanine nucleotide-binding regulatory protein
and thereby prevent its activation.
SO - J Biol Chem 1990 Oct 15;265(29):17941-5.

16. PMID:2226863
PIR:TPRBIC
FT - binding site | phosphate (Ser) (covalent) (by cAMP-dependent kinase) | 22,23 (all)
TI - A common motif of two adjacent phosphoserines in bovine, rabbit and human
cardiac troponin I.
AB - From rabbit and human cardiac troponin I N-terminal mono and
bisphosphorylated peptides were isolated which were obtained from Lys-C
proteinase digests. Two adjacent phosphoserine residues could be localized
in each phosphopeptide following further tryptic digestion. The previously
published sequence of rabbit cardiac troponin I had to be corrected. Two
adjacent phosphoserine residues are a common motif in the very similar
sequences of bovine, rabbit and human cardiac troponin I. The N-terminal
sequences are: AcADRSGGSTAG DTVPAPPPVR RRS(P)S(P)ANYRAY ATEPHAK (bovine),
AcADESTDAAG EARPAPAPVR RRS(P)S(P)ANYRAY ATEPHAK (rabbit),
(Ac,A,D/N,G,S,S,D/N,A,A,R) EPRPAPAPIR RRS(P)S(P)-NYRAY ATEPHAK (human).
SO - FEBS Lett 1990 Oct 29;273(1-2):41-5.

PIR:TPHUIC
FT - binding site | phosphate (Ser) (covalent) (by cAMP-dependent kinase) | 23,24 (all)
TI - A common motif of two adjacent phosphoserines in bovine, rabbit and human
cardiac troponin I.
AB - From rabbit and human cardiac troponin I N-terminal mono and
bisphosphorylated peptides were isolated which were obtained from Lys-C
proteinase digests. Two adjacent phosphoserine residues could be localized
in each phosphopeptide following further tryptic digestion. The previously
published sequence of rabbit cardiac troponin I had to be corrected. Two
adjacent phosphoserine residues are a common motif in the very similar
sequences of bovine, rabbit and human cardiac troponin I. The N-terminal
sequences are: AcADRSGGSTAG DTVPAPPPVR RRS(P)S(P)ANYRAY ATEPHAK (bovine),
AcADESTDA-AG EARPAPAPVR RRS(P)S(P)ANYRAY ATEPHAK (rabbit),
(Ac,A,D/N,G,S,S,D/N,A,A,R) EPRPAPAPIR RRS(P)S(P)-NYRAY ATEPHAK (human).
SO - FEBS Lett 1990 Oct 29;273(1-2):41-5.

17. PMID:2261989
PIR:S15815
FT - binding site | phosphate (Thr) (covalent) (by elongation factor 2 kinase) | 8,10 (all)
TI - Three phosphorylation sites in elongation factor 2.
AB - Elongation factor 2 (EF-2) of rabbit reticulocytes was phosphorylated in
vitro by incubation with partially purified EF-2 kinase and
[gamma-32P]ATP. After exhaustive tryptic hydrolysis 4 phosphopeptides were
revealed by two-dimensional peptide mapping. The phosphopeptides were
isolated by high performance liquid chromatography and sequenced. A
comparison of the primary structure of the phosphopeptides with that of
EF-2 showed that all 4 phosphopeptides originated from one region of EF-2
located near the N-terminus that contains 3 threonine residues: Thr-53,
Thr-56, Thr-58. A direct estimation of localization of radioactive
phosphate in the phosphopeptides demonstrated that all the enumerated
threonine residues in EF-2 can be phosphorylated in vitro.
SO - FEBS Lett 1990 Nov 26;275(1-2):209-12.

18. PMID:2346743
PIR:A34594
FT - binding site | phosphate (Ser) (covalent) (by protein kinase C) | 1 (all)
TI - Phosphorylation of bovine platelet myosin by protein kinase C.
AB - Bovine platelet myosin is phosphorylated by protein kinase C at multiple
sites. Most of the phosphate is incorporated in the 20,000-dalton light
chain although some phosphate is incorporated in the heavy chain.
Phosphorylation of the 20,000-dalton light chain of platelet myosin is 10
times faster than the phosphorylation of smooth muscle myosin. Platelet
myosin light chain is first phosphorylated at a threonine residue followed
by a serine residue. Dominant phosphorylation sites of the 20,000-dalton
light chain are estimated as serine-1, serine-2, and threonine-9.
Prolonged phosphorylation by protein kinase C resulted in an additional
phosphorylation site which, on the basis of limited proteolysis, appears
to be either serine-19 or threonine-18. Phosphorylation by protein kinase
C causes an inhibition of actin-activated ATPase activity of platelet
myosin prephosphorylated by myosin light chain kinase. Inhibition of
ATPase activity is due to a decreased affinity of myosin for actin, and no
change in Vmax is observed. It is shown that platelet myosin also exhibits
the 6S to 10S conformation transition as judged by viscosity and gel
filtration methods. Mg2(+)-ATPase activity of platelet myosin is
paralleled with the 10S-6S transition. Phosphorylation by protein kinase C
affects neither the 10S-6S transition nor the myosin filament formation.
Therefore, the inhibition of actin-activated ATPase activity of platelet
myosin is not due to the change in the myosin conformation.
SO - Biochemistry 1990 Mar 20;29(11):2713-20.

PIR:A34594
FT - binding site | phosphate (Ser) (covalent) (by protein kinase C) | 2 (all)
TI - Phosphorylation of bovine platelet myosin by protein kinase C.
AB - Bovine platelet myosin is phosphorylated by protein kinase C at multiple
sites. Most of the phosphate is incorporated in the 20,000-dalton light
chain although some phosphate is incorporated in the heavy chain.
Phosphorylation of the 20,000-dalton light chain of platelet myosin is 10
times faster than the phosphorylation of smooth muscle myosin. Platelet
myosin light chain is first phosphorylated at a threonine residue followed
by a serine residue. Dominant phosphorylation sites of the 20,000-dalton
light chain are estimated as serine-1, serine-2, and threonine-9.
Prolonged phosphorylation by protein kinase C resulted in an additional
phosphorylation site which, on the basis of limited proteolysis, appears
to be either serine-19 or threonine-18. Phosphorylation by protein kinase
C causes an inhibition of actin-activated ATPase activity of platelet
myosin prephosphorylated by myosin light chain kinase. Inhibition of
ATPase activity is due to a decreased affinity of myosin for actin, and no
change in Vmax is observed. It is shown that platelet myosin also exhibits
the 6S to 10S conformation transition as judged by viscosity and gel
filtration methods. Mg2(+)-ATPase activity of platelet myosin is
paralleled with the 10S-6S transition. Phosphorylation by protein kinase C
affects neither the 10S-6S transition nor the myosin filament formation.
Therefore, the inhibition of actin-activated ATPase activity of platelet
myosin is not due to the change in the myosin conformation.
SO - Biochemistry 1990 Mar 20;29(11):2713-20.

PIR:A34594
FT - binding site | phosphate (Thr) (covalent) (by protein kinase C) | 9 (all)
TI - Phosphorylation of bovine platelet myosin by protein kinase C.
AB - Bovine platelet myosin is phosphorylated by protein kinase C at multiple
sites. Most of the phosphate is incorporated in the 20,000-dalton light
chain although some phosphate is incorporated in the heavy chain.
Phosphorylation of the 20,000-dalton light chain of platelet myosin is 10
times faster than the phosphorylation of smooth muscle myosin. Platelet
myosin light chain is first phosphorylated at a threonine residue followed
by a serine residue. Dominant phosphorylation sites of the 20,000-dalton
light chain are estimated as serine-1, serine-2, and threonine-9.
Prolonged phosphorylation by protein kinase C resulted in an additional
phosphorylation site which, on the basis of limited proteolysis, appears
to be either serine-19 or threonine-18. Phosphorylation by protein kinase
C causes an inhibition of actin-activated ATPase activity of platelet
myosin prephosphorylated by myosin light chain kinase. Inhibition of
ATPase activity is due to a decreased affinity of myosin for actin, and no
change in Vmax is observed. It is shown that platelet myosin also exhibits
the 6S to 10S conformation transition as judged by viscosity and gel
filtration methods. Mg2(+)-ATPase activity of platelet myosin is
paralleled with the 10S-6S transition. Phosphorylation by protein kinase C
affects neither the 10S-6S transition nor the myosin filament formation.
Therefore, the inhibition of actin-activated ATPase activity of platelet
myosin is not due to the change in the myosin conformation.
SO - Biochemistry 1990 Mar 20;29(11):2713-20.

19. PMID:2394752
PIR:A38379
FT - binding site | phosphate (Ser) (covalent) (by protein kinase A) | 73 (all)
TI - Protein kinase A phosphorylates retinal phosducin on serine 73 in situ.
AB - Photoreceptors of vertebrate retinas contain a 33,000-dalton
phosphoprotein, phosducin, which complexes with the beta, gamma subunits
of the photoreceptor G-protein (guanine nucleotide-binding protein),
transducin. In situ, the retinal content of phosphorylated phosducin is
modulated by light in conjunction with light-triggered changes in
intracellular cyclic nucleotide concentration. In vitro, phosducin is
phosphorylated by either exogenous or endogenous protein kinase A.
32P-Labeled rat retina phosducin was isolated by immunoprecipitation
either after phosphorylation by protein kinase A in the presence of
[gamma-32P]ATP or after incubation of retinas in darkness with 32Pi. In
either case, phosphoamino acid analysis showed that greater than 98% of
32P was linked to serine, with less than 2% to threonine. Two-dimensional
peptide mapping showed that [32P]phosphoserine was associated with the
same characteristic set of tryptic peptides. Furthermore, Cleveland
peptide analysis using four different proteases showed that either sample
exhibited identical patterns of phosphopeptides which were characteristic
of the protease used. Identical phosphopeptide maps were also obtained
from 32P-labeled bovine retina phosducin, indicating that the serine
phosphorylation site for protein kinase A is conserved between rat and
bovine. Edman degradation of phosphopeptides derived from 32P-labeled
bovine phosducin showed that radioactive phosphate was incorporated into
serine residue 73 which is located within a consensus phosphorylation
sequence for protein kinase A (-R-K-M-S73(P)-). These observations are
uniformly in agreement with protein kinase A being the endogenous kinase
that phosphorylates phosducin in vivo.
SO - J Biol Chem 1990 Sep 15;265(26):15860-6.

20. PMID:2492519
PIR:HHHU84
FT - binding site | phosphate (Ser) (covalent) | 226,255 (all)
TI - Two human 90-kDa heat shock proteins are phosphorylated in vivo at
conserved serines that are phosphorylated in vitro by casein kinase II.
AB - Amino-terminal protein sequence analysis revealed that exponentially
growing human HeLa cells at 37 degrees C express two closely related
90-kDa "heat shock" proteins (hsp 90) in nearly equal amounts. Both hsp
90s begin with proline; the initial methionine residue is removed. The
alpha protein contains a 9-amino acid segment, TQTQDQPME, from residues 4
to 12, that is replaced by a 4-amino acid segment, VHHG, in the beta form.
The purified hsp 90 mixture contains 2 mol of phosphate/mol of
polypeptide. Both hsp 90 proteins are phosphorylated at two homologous
sites. For the alpha protein, these sites correspond to serine 231 and
serine 263. A 5-amino acid segment, ESEDK, between the two phosphorylation
sites is absent from the beta protein. The sequence between
phosphorylation sites of both hsp 90s is predicted to have alpha helical
structure. Dephosphorylated hsp 90 is phosphorylated at both sites by
casein kinase II from HeLa cells, calf thymus, or rabbit reticulocytes; no
other hsp 90 residues were phosphorylated by casein kinase II in vitro.
SO - J Biol Chem 1989 Feb 15;264(5):2431-7.
full paper-page: 2433 Both serine 226 and 255, of beta hsp 90 are phosphorylated (Fig. 4, F and C), while serine 261 is not (Fig. 4D).

PIR:HHHU86
FT - binding site | phosphate (Ser) (covalent) | 231,263 (all)
TI - Two human 90-kDa heat shock proteins are phosphorylated in vivo at
conserved serines that are phosphorylated in vitro by casein kinase II.
AB - Amino-terminal protein sequence analysis revealed that exponentially
growing human HeLa cells at 37 degrees C express two closely related
90-kDa "heat shock" proteins (hsp 90) in nearly equal amounts. Both hsp
90s begin with proline; the initial methionine residue is removed. The
alpha protein contains a 9-amino acid segment, TQTQDQPME, from residues 4
to 12, that is replaced by a 4-amino acid segment, VHHG, in the beta form.
The purified hsp 90 mixture contains 2 mol of phosphate/mol of
polypeptide. Both hsp 90 proteins are phosphorylated at two homologous
sites. For the alpha protein, these sites correspond to serine 231 and
serine 263. A 5-amino acid segment, ESEDK, between the two phosphorylation
sites is absent from the beta protein. The sequence between
phosphorylation sites of both hsp 90s is predicted to have alpha helical
structure. Dephosphorylated hsp 90 is phosphorylated at both sites by
casein kinase II from HeLa cells, calf thymus, or rabbit reticulocytes; no
other hsp 90 residues were phosphorylated by casein kinase II in vitro.
SO - J Biol Chem 1989 Feb 15;264(5):2431-7.

21. PMID:2500966
PIR:A43803
FT - binding site | phosphate (Ser) (covalent) (by cAMP-dependent kinase and protein kinase C) | 7,25,39,51,66 (all)
TI - Domain- and sequence-specific phosphorylation of vimentin induces
disassembly of the filament structure.
AB - We reported that stoichiometric phosphorylation by either cAMP-dependent
protein kinase or protein kinase C induces disassembly of vimentin
filaments [Inagaki, M., Nishi, Y., Nishizawa, K., Matsuyama, M., & Sato,
C. (1987) Nature 328, 649-652; Inagaki, M., Gonda, Y., Matsuyama, M.,
Nishizawa, K., Nishi, Y., & Sato, C. (1988) J. Biol. Chem. 263,
5970-5978]. In the present work, we attempted to identify the sites of
vimentin phosphorylated by each protein kinase. Sequential analysis of the
purified phosphopeptides, together with the known primary sequence,
revealed that Ser-8, Ser-9, Ser-20, Ser-25, Ser-33, and Ser-41 were
specifically phosphorylated by protein kinase C, whereas Ser-46 was
phosphorylated preferentially by cAMP-dependent protein kinase. Both
kinases reacted with Ser-6, Ser-24, Ser-38, Ser-50, and Ser-65. Specific
phosphorylation sites for protein kinase C are mostly located close to the
amino-terminal side of arginine while those for cAMP-dependent protein
kinase are located close to the carboxyl-terminal side of arginine. The
phosphorylation sites exclusively occur in the amino-terminal
non-alpha-helical head domain, particularly at the beta-turn region. These
results provide clues to the molecular mechanisms of
phosphorylation-dependent disassembly of vimentin filaments.
SO - Biochemistry 1989 Apr 4;28(7):2974-9.

PIR:A43803
FT - binding site | phosphate (Ser) (covalent) (by protein kinase C) | 9,10,21,26,34,42 (all)
TI - Domain- and sequence-specific phosphorylation of vimentin induces
disassembly of the filament structure.
AB - We reported that stoichiometric phosphorylation by either cAMP-dependent
protein kinase or protein kinase C induces disassembly of vimentin
filaments [Inagaki, M., Nishi, Y., Nishizawa, K., Matsuyama, M., & Sato,
C. (1987) Nature 328, 649-652; Inagaki, M., Gonda, Y., Matsuyama, M.,
Nishizawa, K., Nishi, Y., & Sato, C. (1988) J. Biol. Chem. 263,
5970-5978]. In the present work, we attempted to identify the sites of
vimentin phosphorylated by each protein kinase. Sequential analysis of the
purified phosphopeptides, together with the known primary sequence,
revealed that Ser-8, Ser-9, Ser-20, Ser-25, Ser-33, and Ser-41 were
specifically phosphorylated by protein kinase C, whereas Ser-46 was
phosphorylated preferentially by cAMP-dependent protein kinase. Both
kinases reacted with Ser-6, Ser-24, Ser-38, Ser-50, and Ser-65. Specific
phosphorylation sites for protein kinase C are mostly located close to the
amino-terminal side of arginine while those for cAMP-dependent protein
kinase are located close to the carboxyl-terminal side of arginine. The
phosphorylation sites exclusively occur in the amino-terminal
non-alpha-helical head domain, particularly at the beta-turn region. These
results provide clues to the molecular mechanisms of
phosphorylation-dependent disassembly of vimentin filaments.
SO - Biochemistry 1989 Apr 4;28(7):2974-9.

PIR:A43803
FT - binding site | phosphate (Ser) (covalent) (by cAMP-dependent kinase) | 47 (all)
TI - Domain- and sequence-specific phosphorylation of vimentin induces
disassembly of the filament structure.
AB - We reported that stoichiometric phosphorylation by either cAMP-dependent
protein kinase or protein kinase C induces disassembly of vimentin
filaments [Inagaki, M., Nishi, Y., Nishizawa, K., Matsuyama, M., & Sato,
C. (1987) Nature 328, 649-652; Inagaki, M., Gonda, Y., Matsuyama, M.,
Nishizawa, K., Nishi, Y., & Sato, C. (1988) J. Biol. Chem. 263,
5970-5978]. In the present work, we attempted to identify the sites of
vimentin phosphorylated by each protein kinase. Sequential analysis of the
purified phosphopeptides, together with the known primary sequence,
revealed that Ser-8, Ser-9, Ser-20, Ser-25, Ser-33, and Ser-41 were
specifically phosphorylated by protein kinase C, whereas Ser-46 was
phosphorylated preferentially by cAMP-dependent protein kinase. Both
kinases reacted with Ser-6, Ser-24, Ser-38, Ser-50, and Ser-65. Specific
phosphorylation sites for protein kinase C are mostly located close to the
amino-terminal side of arginine while those for cAMP-dependent protein
kinase are located close to the carboxyl-terminal side of arginine. The
phosphorylation sites exclusively occur in the amino-terminal
non-alpha-helical head domain, particularly at the beta-turn region. These
results provide clues to the molecular mechanisms of
phosphorylation-dependent disassembly of vimentin filaments.
SO - Biochemistry 1989 Apr 4;28(7):2974-9.

22. PMID:2530230
PIR:MWAXIC
FT - binding site | phosphate (Ser) (covalent) | 311 (all)
TI - The localization and sequence of the phosphorylation sites of Acanthamoeba
myosins I. An improved method for locating the phosphorylated amino acid.
AB - The actin-activated Mg2+-ATPase activities of Acanthamoeba myosins IA, IB,
and IC are expressed only when a single site in their heavy chains is
phosphorylated by a myosin I heavy chain-specific kinase. We show that
phosphorylation occurs at Ser-315 in the myosin IB heavy chain, Ser-311 in
myosin IC, and a threonine residue at a corresponding position in myosin
IA whose amino acid sequence is as yet unknown. The most obvious feature
common to the three substrates is a basic amino acid(s) 2 or 3 residues
before the site of phosphorylation. The phosphorylation site is located
between the ATP- and actin-binding sites, which corresponds to the middle
of the 50-kDa domain of skeletal muscle myosin subfragment 1. The sequence
similarity between the region surrounding the phosphorylation site of
myosin I and subfragment 1 is much lower than the average sequence
similarity between myosin I and subfragment 1. This is consistent with the
hypothesis that the conformation of this region of myosin I differs from
that of the corresponding region in skeletal muscle myosin and that
phosphorylation converts the conformation of the actomyosin I complex into
a conformation comparable to that present in actosubfragment 1 without
phosphorylation. The protein sequences obtained in the course of this work
led to the conclusion that the myosin I genes previously identified as
myosin IB and IL (myosin-like) heavy chains actually are the myosin IC and
IB heavy chains, respectively. Finally, we report a modification of the
method for monitoring the appearance of 32Pi during sequencing of
32P-labeled peptides that results in almost complete recovery of the
radioactivity, thus allowing unequivocal assignment of the position of the
phosphorylated residue.
SO - J Biol Chem 1989 Nov 15;264(32):19340-8.

23. PMID:2552036
PIR:A34957
FT - binding site | phosphate (Ser) (covalent) (by cAMP-dependent kinase) | 56 (all)
TI - ARPP-21, a cyclic AMP-regulated phosphoprotein enriched in
dopamine-innervated brain regions. I. Amino acid sequence of ARPP-21B from
bovine caudate nucleus.
AB - ARPP-21 (cAMP-regulated phosphoprotein, Mr = 21,000 as determined by
SDS-PAGE) is a major cytosolic substrate for cAMP-stimulated protein
phosphorylation in dopamine-innervated regions of the rat CNS. It has
recently been purified to homogeneity from bovine caudate nucleus and
characterized (Hemmings and Greengard, 1989). ARPP-21 is isolated as 2
isoforms, ARPP-21A and ARPP-21B. The amino acid sequence of purified
bovine ARPP-21B has now been determined by gas-phase sequencing. The
S-14C-carboxymethylated protein was subjected to enzymatic cleavage with
trypsin, chymotrypsin, subtilisin, and endoproteinase Lys-C. The resulting
peptides were purified by high-performance liquid chromatography, and
selected peptides were subjected to amino acid analysis and/or amino acid
sequencing by automated Edman degradation. ARPP-21B consists of a single
NH2-terminal blocked polypeptide chain of 88 residues, with a calculated
molecular mass of 9561 Da, including an NH2-terminal acetyl group inferred
by deblocking with an acylaminopeptidase. This molecular mass is
significantly lower than earlier estimates based on SDS-PAGE or
hydrodynamic measurements. The seryl residue phosphorylated by
cAMP-dependent protein kinase (Hemmings et al., 1989) is located at
position 55. The molecule contains 1 cysteinyl residue, at position 71,
and contains no methionyl, tyrosyl, phenylalanyl, tryptophanyl, or
histidinyl residues. Determination of the primary structure of ARPP-21,
one of several phosphoproteins localized to dopaminoceptive neurons in the
basal ganglia, provides a framework for further investigations into the
molecular mechanisms involved in dopaminergic neurotransmission.
SO - J Neurosci 1989 Oct;9(10):3631-7.

24. PMID:2558715
PIR:A56968
FT - binding site | phosphate (Ser) (covalent) (by calmodulin-dependent kinase II) | 411 (all)
TI - Identification of the site on calcineurin phosphorylated by
Ca2+/CaM-dependent kinase II: modification of the CaM-binding domain.
AB - The catalytic subunit of the Ca2+/calmodulin- (CaM) dependent
phosphoprotein phosphatase calcineurin (CN) was phosphorylated by an
activated form of Ca2+/CaM-dependent protein kinase II (CaM-kinase II)
incorporating approximately 1 mol of phosphoryl group/mol of catalytic
subunit, in agreement with a value previously reported (Hashimoto et al.,
1988). Cyanogen bromide cleavage of radiolabeled CN followed by peptide
fractionation using reverse-phase high-performance liquid chromatography
yielded a single labeled peptide that contained a phosphoserine residue.
Microsequencing of the peptide allowed both the determination of the
cleavage cycle that released [32P]phosphoserine and the identity of amino
acids adjacent to it. Comparison of this sequence with the sequences of
methionyl peptides deduced from the cDNA structure of CN (Kincaid et al.,
1988) allowed the phosphorylated serine to be uniquely identified.
Interestingly, the phosphoserine exists in the sequence
Met-Ala-Arg-Val-Phe-Ser(P)-Val-Leu-Arg-Glu, part of which lies within the
putative CaM-binding site. The phosphorylated serine residue was resistant
to autocatalytic dephosphorylation, yet the slow rate of hydrolysis could
be powerfully stimulated by effectors of CN phosphatase activity. The
mechanism of dephosphorylation may be intramolecular since the initial
rate was the same at phosphoCN concentrations of 2.5-250 nM.
SO - Biochemistry 1989 Nov 28;28(24):9243-7.

25. PMID:2721673
PIR:UDCH
FT - binding site | phosphate (Ser) (covalent) (partial) | 103 (all)
TI - The cysteine proteinase inhibitor chicken cystatin is a phosphoprotein.
AB - Peptide maps obtained by reversed-phase HPLC of tryptic digests of
isoelectric form 1 (pI = 6.5) and 2 (pI = 5.6) of chicken egg white
cystatin revealed that the difference was located only in a single peptide
(residues Ser-74-Lys-91). Ser-80 of cystatin 2 was subsequently identified
as being modified by phosphorylation. Moreover, alkaline phosphatase
treatment of a mixture of native cystatin forms 1 and 2 was shown by
ion-exchange chromatography to cause the disappearance of isoelectric form
2 with a concomitant increase in form 1. Thus, the existence of two
isoelectric forms of chicken cystatin is due to the phosphorylated form 2
and non-phosphorylated form 1.
SO - FEBS Lett 1989 May 8;248(1-2):162-8.

26. PMID:278975
PIR:TMRBA
FT - binding site | phosphate (Ser) (covalent) (partial) | 283 (all)
TI - Specific phosphorylation at serine-283 of alpha tropomyosin from frog
skeletal and rabbit skeletal and cardiac muscle.
AB - Tropomyosin, extracted from the leg muscle of frogs that had been injected
with [32P]orthophosphate, was fractionated into two components, alpha and
beta, on a CM-cellulose column. Radioactivity was associated only with the
alpha component. A single phosphorylation site was located at serine-283
(pentultimate at the COOH-terminal end) of the frog alpha tropomyosin. The
same phosphorylated peptide was recovered in low yields from both rabbit
skeletal alpha and cardiac tropomyosin. The presence of covalently bound
phosphate in alpha tropomyosin and its absence in the beta component of
rabbit skeletal muscle was suggested by 31P NMR spectroscopy. The amino
acid sequences around the phosphorylation sites of frog and rabbit
tropomyosin are identical. Because this sequence is not similar to any
other known phosphorylation site in proteins, this indicates the existence
of either specific kinase or phosphatase that can distinguish between
alpha and beta tropomyosins. In a model proposed for the head-to-tail
overlap of alpha tropomyosin molecules, one O-phosphoserine-283 residue
could form a salt linkage with lysine-6 on one side of the overlap region
and another with lysine-12 on the other side. This would predict a
difference in the stability of polymers of phosphorylated and
nonphosphorylated alphaalpha and alphabeta dimers of tropomyosin.
SO - Proc Natl Acad Sci U S A 1978 Aug;75(8):3588-92.

27. PMID:2834385
PIR:KIZMPO
FT - binding site | phosphate (Thr) (covalent) (by bifunctional regulatory protein) | 527 (all)
TI - Sequence of the phosphothreonyl regulatory site peptide from inactive
maize leaf pyruvate, orthophosphate dikinase.
AB - The regulatory site peptide sequence of phosphorylated inactive pyruvate,
orthophosphate dikinase from maize leaf tissue was determined by automated
Edman degradation analysis of 32P-labeled peptides purified by
reversed-phase high performance liquid chromatography. The overlapping
phosphopeptides were products of a digestion of the
[beta-32P]ADP-inactivated dikinase with either trypsin or Pronase E. The
sequence is
Thr-Glu-Arg-Gly-Gly-Met-Thr(P)-Ser-His-Ala-Ala-Val-Val-Ala-Arg. The
phosphothreonine residue, which appeared as either an anomalous proline or
an unidentifiable phenylthiohydantoin derivative during sequencing, was
verified by two-dimensional phosphoamino acid analysis of the
phosphopeptides and by resequencing the tryptic peptide after
dephosphorylation with exogenous alkaline phosphatase. This sequence,
starting at position 4, is completely homologous to the previously
published sequence of the tryptic dodecapeptide harboring the
catalytically essential (phospho)histidyl residue in the active-site
domain of the dikinase from the nonphotosynthetic bacterium, Bacteroides
symbiosus (Goss, N.H., Evans, C.T., and Wood, H.G. (1980) Biochemistry 19,
5805-5809). These comparative results indicate that the regulatory
phosphothreonine causing complete inactivation of maize leaf dikinase is
separated from the critical active-site (phospho)histidine by just one
intervening residue in the primary sequence.
SO - J Biol Chem 1988 May 15;263(14):6683-7.

28. PMID:2846551
PIR:A31780
FT - binding site | phosphate (Ser) (covalent) (by cAMP-dependent kinase) | 467 (all)
TI - Phosphorylation of myocardial fructose-6-phosphate,2-kinase:
fructose-2,6-bisphosphatase by cAMP-dependent protein kinase and protein
kinase C. Activation by phosphorylation and amino acid sequences of the
phosphorylation sites.
AB - Phosphorylation of pure
fructose-6-phosphate,2-kinase:fructose-2,6-bisphosphatase from bovine
heart by cAMP-dependent protein kinase and protein kinase C was
investigated. The major enzyme form (subunit Mr of 58,000) was rapidly
phosphorylated by both cAMP-dependent protein kinase and protein kinase C,
incorporating 0.8 and 1.0 mol/mol of subunit, respectively. The rate of
phosphorylation of the heart enzyme by cAMP-dependent protein kinase was
10 times faster than that of the rat liver enzyme. The minor enzyme
(subunit Mr of 54,000), however, was phosphorylated only by protein kinase
C and was phosphorylated much more slowly with a phosphate incorporation
of less than 0.1 mol/mol of subunit. Phosphorylation by either
cAMP-dependent protein kinase or protein kinase C activated the enzyme,
but each phosphorylation affected different kinetic parameters.
Phosphorylation by cAMP-dependent protein kinase lowered the Km value for
fructose 6-phosphate from 87 to 42 microM without affecting the Vmax,
whereas the phosphorylation by protein kinase C increased the Vmax value
from 55 to 85 milliunits/mg without altering the Km value. The
phosphorylated peptides were isolated, and their amino acid sequences were
determined. The phosphorylation sites for both cAMP-dependent protein
kinase and protein kinase C were located in a single peptide whose
sequence was Arg-Arg-Asn-Ser-(P)-Phe-Thr-Pro-Leu-Ser-Ser-Ser-Asn-Thr(P)-Ile-
Arg-Arg-Pro.
The seryl residue nearest the N terminus was the residue specifically
phosphorylated by cAMP-dependent protein kinase, whereas the threonine
residue nearest the C terminus was phosphorylated by protein kinase C.
SO - J Biol Chem 1988 Nov 15;263(32):16796-801.

PIR:A31780
FT - binding site | phosphate (Thr) (covalent) (by protein kinase C) | 476 (all)
TI - Phosphorylation of myocardial fructose-6-phosphate,2-kinase:
fructose-2,6-bisphosphatase by cAMP-dependent protein kinase and protein
kinase C. Activation by phosphorylation and amino acid sequences of the
phosphorylation sites.
AB - Phosphorylation of pure
fructose-6-phosphate,2-kinase:fructose-2,6-bisphosphatase from bovine
heart by cAMP-dependent protein kinase and protein kinase C was
investigated. The major enzyme form (subunit Mr of 58,000) was rapidly
phosphorylated by both cAMP-dependent protein kinase and protein kinase C,
incorporating 0.8 and 1.0 mol/mol of subunit, respectively. The rate of
phosphorylation of the heart enzyme by cAMP-dependent protein kinase was
10 times faster than that of the rat liver enzyme. The minor enzyme
(subunit Mr of 54,000), however, was phosphorylated only by protein kinase
C and was phosphorylated much more slowly with a phosphate incorporation
of less than 0.1 mol/mol of subunit. Phosphorylation by either
cAMP-dependent protein kinase or protein kinase C activated the enzyme,
but each phosphorylation affected different kinetic parameters.
Phosphorylation by cAMP-dependent protein kinase lowered the Km value for
fructose 6-phosphate from 87 to 42 microM without affecting the Vmax,
whereas the phosphorylation by protein kinase C increased the Vmax value
from 55 to 85 milliunits/mg without altering the Km value. The
phosphorylated peptides were isolated, and their amino acid sequences were
determined. The phosphorylation sites for both cAMP-dependent protein
kinase and protein kinase C were located in a single peptide whose
sequence was Arg-Arg-Asn-Ser-(P)-Phe-Thr-Pro-Leu-Ser-Ser-Ser-Asn-Thr(P)-Ile
Arg-Arg-Pro.
The seryl residue nearest the N terminus was the residue specifically
phosphorylated by cAMP-dependent protein kinase, whereas the threonine
residue nearest the C terminus was phosphorylated by protein kinase C.
SO - J Biol Chem 1988 Nov 15;263(32):16796-801.

29. PMID:2874140
PIR:WHRTY
FT - binding site | phosphate (Ser) (covalent) (by unidentified kinase) | 8 (all)
TI - Identification of four phosphorylation sites in the N-terminal region of
tyrosine hydroxylase.
AB - As reported previously [Vulliet et al. (1985) FEBS Lett. 182 335-339],
tyrosine hydroxylase purified from rat pheochromocytoma is phosphorylated
at an identical site (site A) by cyclic AMP-dependent protein kinase, the
calmodulin-dependent multiprotein kinase and protein kinase C, while the
calmodulin-dependent multiprotein kinase also phosphorylates another
unique site (site C). Preparations of tyrosine hydroxylase purified from
this source are also contaminated with traces of a fourth protein kinase
which phosphorylates another unique site (site E). We have isolated
tryptic peptides containing each of these sites and determined their amino
acid sequences. By comparison of these data with the known cDNA sequence
for rat tyrosine hydroxylase, we have been able to identify these sites as
Ser-8 (site E), Ser-19 (site C), and Ser-40 (site A). In some preparations
of tyrosine hydroxlyase, cyclic AMP-dependent protein kinase also
phosphorylated a secondary site which was identified as ser-153. All of
these phosphorylation sites are in the amino-terminal region, where there
is no significant homology with the closely related enzyme, phenylalanine
hydroxylase. Our data also establish that the initiator methionine is
removed by post-translational processing to leave pro-2 as the
amino-terminus of the mature protein. The significance of these results
for the mechanism of action of extracellular signals on catecholamine
biosynthesis is discussed.
SO - J Biol Chem 1986 Aug 15;261(23):10489-92.

PIR:WHRTY
FT - binding site | phosphate (Ser) (covalent) (by calmodulin-dependent kinase) | 19 (all)
TI - Identification of four phosphorylation sites in the N-terminal region of
tyrosine hydroxylase.
AB - As reported previously [Vulliet et al. (1985) FEBS Lett. 182 335-339],
tyrosine hydroxylase purified from rat pheochromocytoma is phosphorylated
at an identical site (site A) by cyclic AMP-dependent protein kinase, the
calmodulin-dependent multiprotein kinase and protein kinase C, while the
calmodulin-dependent multiprotein kinase also phosphorylates another
unique site (site C). Preparations of tyrosine hydroxylase purified from
this source are also contaminated with traces of a fourth protein kinase
which phosphorylates another unique site (site E). We have isolated
tryptic peptides containing each of these sites and determined their amino
acid sequences. By comparison of these data with the known cDNA sequence
for rat tyrosine hydroxylase, we have been able to identify these sites as
Ser-8 (site E), Ser-19 (site C), and Ser-40 (site A). In some preparations
of tyrosine hydroxlyase, cyclic AMP-dependent protein kinase also
phosphorylated a secondary site which was identified as ser-153. All of
these phosphorylation sites are in the amino-terminal region, where there
is no significant homology with the closely related enzyme, phenylalanine
hydroxylase. Our data also establish that the initiator methionine is
removed by post-translational processing to leave pro-2 as the
amino-terminus of the mature protein. The significance of these results
for the mechanism of action of extracellular signals on catecholamine
biosynthesis is discussed.
SO - J Biol Chem 1986 Aug 15;261(23):10489-92.

PIR:WHRTY
FT - binding site | phosphate (Ser) (covalent) (by cAMP-dependent kinase) | 40,153 (all)
TI - Identification of four phosphorylation sites in the N-terminal region of
tyrosine hydroxylase.
AB - As reported previously [Vulliet et al. (1985) FEBS Lett. 182 335-339],
tyrosine hydroxylase purified from rat pheochromocytoma is phosphorylated
at an identical site (site A) by cyclic AMP-dependent protein kinase, the
calmodulin-dependent multiprotein kinase and protein kinase C, while the
calmodulin-dependent multiprotein kinase also phosphorylates another
unique site (site C). Preparations of tyrosine hydroxylase purified from
this source are also contaminated with traces of a fourth protein kinase
which phosphorylates another unique site (site E). We have isolated
tryptic peptides containing each of these sites and determined their amino
acid sequences. By comparison of these data with the known cDNA sequence
for rat tyrosine hydroxylase, we have been able to identify these sites as
Ser-8 (site E), Ser-19 (site C), and Ser-40 (site A). In some preparations
of tyrosine hydroxlyase, cyclic AMP-dependent protein kinase also
phosphorylated a secondary site which was identified as ser-153. All of
these phosphorylation sites are in the amino-terminal region, where there
is no significant homology with the closely related enzyme, phenylalanine
hydroxylase. Our data also establish that the initiator methionine is
removed by post-translational processing to leave pro-2 as the
amino-terminus of the mature protein. The significance of these results
for the mechanism of action of extracellular signals on catecholamine
biosynthesis is discussed.
SO - J Biol Chem 1986 Aug 15;261(23):10489-92.

30. PMID:2914943
PIR:EQBOA
FT - binding site | phosphate (Ser) (covalent) (partial) | 239,245,247 (all)
TI - The isolation and chemical characterization of phosphorylated
enkephalin-containing peptides from bovine adrenal medulla.
AB - There is increasing evidence that the opioid peptide precursor,
proenkephalin A, and its products undergo extensive post-translational
modification, in addition to the cleavage at dibasic amino acid sites. We
have used an antiserum directed toward the C terminus of Met-enkephalin
Arg6-Phe7 in a radioimmunoassay to monitor the purification to homogeneity
of four peptide B variants from bovine adrenal medulla, using gel
filtration, anion exchange chromatography, and reverse phase high
performance liquid chromatography. Amino acid sequence analysis, together
with immunochemical data, confirmed that each comprised the primary
sequence, proenkephalin A-(209-239). In addition, three of the four
variants were shown to be phosphorylated by alkaline phosphatase
digestion, microphosphate analysis, and ethanethiol derivatization coupled
with amino acid sequence analysis; these variants were shown to have 1, 2,
or 3 phosphate groups per peptide chain, which corresponded to their
increasing acidic nature. The phosphorylation sites were clustered
together at positions Ser7, Ser13, and Ser15 and were in close association
with acidic residues. The clustering of phosphorylated residues is unique
among regulatory peptide precursors. This region of proenkephalin A is
well conserved, which suggests that it constitutes an important novel
functional domain.
SO - J Biol Chem 1989 Feb 25;264(6):3061-5.

31. PMID:3121625
PIR:FMSP32
FT - binding site | phosphate (Thr) (covalent) | 2 (all)
TI - Tandem mass spectrometry reveals that three photosystem II proteins of
spinach chloroplasts contain N-acetyl-O-phosphothreonine at their NH2
termini.
AB - Photosystem II cores of spinach contain four phosphoproteins (8.3, 32, 34,
and 44 kDa). Tryptic digestion of core particles released four
phosphopeptides which were purified by affinity chromatography on
Fe3+-chelating Sepharose and reverse-phase high pressure liquid
chromatography. One peptide, derived from the 8.3-kDa protein, has been
found to be the NH2 terminus of the psbH gene product (Michel, H. P., and
Bennett, J. (1987) FEBS Lett. 212, 103-108). The other three peptides were
found to be blocked at the NH2 terminus. We now report the use of tandem
mass spectrometry to obtain the sequence of the three other peptides, to
locate the phosphorylated residue, and to identify the blocking group. The
three peptides correspond to the NH2 termini of D1, D2, and CPa-2; and
each begins with N-acetyl-O-phosphothreonine. Comparison with sequences
deduced from cloned genes indicates that D1 and D2 have lost their
initiating N-formylmethionyl residues. The result for D1 contradicts the
view that translation of D1 begins at the second AUG of the mRNA (Bloom,
M., Brot, N., Cohen, B. N., and Weissbach, H. (1986) Methods Enzymol. 118,
309-315) and supports the view that processing of pre-D1 to its mature
form involves loss of amino acids from the COOH terminus (Marder, J. B.,
Goloubinoff, P., and Edelman, M. (1984) J. Biol. Chem. 259, 3900-3908). In
contrast, CPa-2 is processed at the NH2 terminus by cleaving off the first
14 amino acids. These results also establish that the NH2 termini of D1,
D2, and CPa-2 are exposed to the stromal side of the thylakoids.
SO - J Biol Chem 1988 Jan 25;263(3):1123-30.

PIR:F2SPD2
FT - binding site | phosphate (Thr) (covalent) | 2 (all)
TI - Tandem mass spectrometry reveals that three photosystem II proteins of
spinach chloroplasts contain N-acetyl-O-phosphothreonine at their NH2
termini.
AB - Photosystem II cores of spinach contain four phosphoproteins (8.3, 32, 34,
and 44 kDa). Tryptic digestion of core particles released four
phosphopeptides which were purified by affinity chromatography on
Fe3+-chelating Sepharose and reverse-phase high pressure liquid
chromatography. One peptide, derived from the 8.3-kDa protein, has been
found to be the NH2 terminus of the psbH gene product (Michel, H. P., and
Bennett, J. (1987) FEBS Lett. 212, 103-108). The other three peptides were
found to be blocked at the NH2 terminus. We now report the use of tandem
mass spectrometry to obtain the sequence of the three other peptides, to
locate the phosphorylated residue, and to identify the blocking group. The
three peptides correspond to the NH2 termini of D1, D2, and CPa-2; and
each begins with N-acetyl-O-phosphothreonine. Comparison with sequences
deduced from cloned genes indicates that D1 and D2 have lost their
initiating N-formylmethionyl residues. The result for D1 contradicts the
view that translation of D1 begins at the second AUG of the mRNA (Bloom,
M., Brot, N., Cohen, B. N., and Weissbach, H. (1986) Methods Enzymol. 118,
309-315) and supports the view that processing of pre-D1 to its mature
form involves loss of amino acids from the COOH terminus (Marder, J. B.,
Goloubinoff, P., and Edelman, M. (1984) J. Biol. Chem. 259, 3900-3908). In
contrast, CPa-2 is processed at the NH2 terminus by cleaving off the first
14 amino acids. These results also establish that the NH2 termini of D1,
D2, and CPa-2 are exposed to the stromal side of the thylakoids.
SO - J Biol Chem 1988 Jan 25;263(3):1123-30.

32. PMID:3185734
PIR:QRECCS
FT - binding site | phosphate (His) (covalent) (by autophosphorylation) | 48 (all)
TI - Histidine phosphorylation and phosphoryl group transfer in bacterial
chemotaxis.
AB - A cascade of protein phosphorylation, initiated by autophosphorylation of
the CheA protein, may be important in the signal transduction pathway of
bacterial chemotaxis. A proteolytic fragment of CheA cannot
autophosphorylate, but can still transfer phosphate to proteins that
generate excitation and adaptation signals. The site of CheA
phosphorylation is His 48; mutants altered at this position are
non-chemotactic. Similar mechanisms of transient protein phosphorylation
and phosphoryl group transfer seem to be involved in processing sensory
data and in activating specific gene expression.
SO - Nature 1988 Nov 10;336(6195):139-43.

33. PMID:3198618
PIR:A34594
FT - binding site | phosphate (Ser) (covalent) (by myosin-light-chain kinase) | 19 (all)
TI - Sites phosphorylated in myosin light chain in contracting smooth muscle.
AB - Purified smooth muscle myosin light chain can be phosphorylated at
multiple sites by myosin light chain kinase and protein kinase C. We have
determined the sites phosphorylated on myosin light chain in intact bovine
tracheal smooth muscle. Stimulation with 10 microM carbachol resulted in
66 +/- 5% monophosphorylated and 11 +/- 2% diphosphorylated myosin light
chain after 1 min, and 47 +/- 4% monophosphorylated and 5 +/- 2%
diphosphorylated myosin light chain after 30 min. Myosin heavy chain
contained 0.06 +/- 0.01 mol of phosphate/mol of protein which did not
change with carbachol. At both 1 and 30 min the monophosphorylated myosin
light chain contained only phosphoserine whereas the diphosphorylated
myosin light chain contained both phosphoserine and phosphothreonine.
Two-dimensional peptide mapping of tryptic digests of monophosphorylated
and diphosphorylated myosin light chain obtained from carbachol-stimulated
tissue was similar to the peptide maps of purified light chain
monophosphorylated and diphosphorylated, respectively, by myosin light
chain kinase; these maps were distinct from the map obtained with tracheal
light chain phosphorylated by protein kinase C. Phosphorylation of
tracheal smooth muscle myosin light chain by myosin light chain kinase
yields the tryptic phosphopeptide AT(P)S(P)NVFAMFDQSQIQEFK with S(P) the
phosphoserine in the monophosphorylated myosin light chain and T(P)S(P) the
phosphotreonine and phosphoserine in the diphosphorylated myosin light
chain. Thus, stimulation of tracheal smooth muscle with a high
concentration of carbachol results in formation of both monophosphorylated
and diphosphorylated myosin light chain although the amount of
diphosphorylated light chain is substantially less than monophosphorylated
light chain. In the intact muscle, myosin light chain is phosphorylated at
sites corresponding to myosin light chain kinase phosphorylation.
SO - J Biol Chem 1988 Dec 15;263(35):19166-73.

PIR:A34594
FT - binding site | phosphate (Thr) (covalent) (by myosin-light-chain kinase) (partial) | 18 (all)
TI - Sites phosphorylated in myosin light chain in contracting smooth muscle.
AB - Purified smooth muscle myosin light chain can be phosphorylated at
multiple sites by myosin light chain kinase and protein kinase C. We have
determined the sites phosphorylated on myosin light chain in intact bovine
tracheal smooth muscle. Stimulation with 10 microM carbachol resulted in
66 +/- 5% monophosphorylated and 11 +/- 2% diphosphorylated myosin light
chain after 1 min, and 47 +/- 4% monophosphorylated and 5 +/- 2%
diphosphorylated myosin light chain after 30 min. Myosin heavy chain
contained 0.06 +/- 0.01 mol of phosphate/mol of protein which did not
change with carbachol. At both 1 and 30 min the monophosphorylated myosin
light chain contained only phosphoserine whereas the diphosphorylated
myosin light chain contained both phosphoserine and phosphothreonine.
Two-dimensional peptide mapping of tryptic digests of monophosphorylated
and diphosphorylated myosin light chain obtained from carbachol-stimulated
tissue was similar to the peptide maps of purified light chain
monophosphorylated and diphosphorylated, respectively, by myosin light
chain kinase; these maps were distinct from the map obtained with tracheal
light chain phosphorylated by protein kinase C. Phosphorylation of
tracheal smooth muscle myosin light chain by myosin light chain kinase
yields the tryptic phosphopeptide AT(P)S(P)NVFAMFDQSQIQEFK with S(P) the
phosphoserine in the monophosphorylated myosin light chain and T(P)S(P) the
phosphotreonine and phosphoserine in the diphosphorylated myosin light
chain. Thus, stimulation of tracheal smooth muscle with a high
concentration of carbachol results in formation of both monophosphorylated
and diphosphorylated myosin light chain although the amount of
diphosphorylated light chain is substantially less than monophosphorylated
light chain. In the intact muscle, myosin light chain is phosphorylated at
sites corresponding to myosin light chain kinase phosphorylation.
SO - J Biol Chem 1988 Dec 15;263(35):19166-73.

34. PMID:3224509
PIR:PL0040
FT - binding site | phosphate (Ser) (covalent) | 10 (all)
TI - The sequence around the phosphorylation site of the porcine heart type
phosphorylase isoenzyme.
AB - 1. A tetradecapeptide containing the phosphorylation site was obtained
from 32P-labelled pig heart phosphorylase a isoenzyme by
alpha-chymotryptic digestion. 2. The peptide was purified by Mono S
cation-exchange chromatography and reversed-phase HPLC. 3. The
phosphorylated residue was identified as Ser and the sequence was
determined: T D G E R R K Q I S V R G L. 4. The sequence was compared to
the known sequences of muscle and liver type isophosphorylases and the
structural consequences of the amino acid residue exchanges were
predicted.
SO - Comp Biochem Physiol B 1988;91(4):717-21.

35. PMID:3345839
PIR:S00347
FT - binding site | phosphate (Ser) (covalent) | 8 (all)
TI - Primary structure of the site on bovine hormone-sensitive lipase
phosphorylated by cyclic AMP-dependent protein kinase.
AB - The primary structure of a region on hormone-sensitive lipase was
determined to be: Lys-Thr-Glu-Pro-Met-Arg-Arg-Ser-
Val-Ser-Glu-Ala-Ala-Leu-Thr-Gln-Pro-Glu-Gly-Pro-Leu-Gly-Thr-Asp-Ser-Leu-Ly
s. Ser-8 was the only residue in the intact protein phosphorylated by
cyclic AMP-dependent protein kinase. However, Ser-10 also appeared to be
present in a phosphorylated form, suggesting that it is a target for a
distinct protein kinase in vivo.
SO - FEBS Lett 1988 Feb 29;229(1):68-72.

36. PMID:3680273
PIR:ACRYD1
FT - binding site | phosphate (Ser) (covalent) (by cAMP-dependent kinase) | 382 (all)
TI - Determination of the sites of cAMP-dependent phosphorylation on the
nicotinic acetylcholine receptor.
AB - The nicotinic acetylcholine receptor is a substrate for cAMP-dependent
protein kinase both in vitro and in vivo. Recently, it has been
demonstrated that phosphorylation of the nicotinic receptor by this kinase
increases its rate of rapid desensitization. We now report the
identification of the cAMP-dependent phosphorylation sites on the gamma
and delta subunits. Two-dimensional phosphopeptide mapping of the
phosphorylated gamma and delta subunits, after limit proteolysis with
thermolysin, indicated that each subunit is phosphorylated on a single
site. Phosphoamino acid analysis of the 32P-labeled subunits demonstrates
that phosphorylation had occurred exclusively on serine residues. Purified
phosphorylated subunits were cleaved with cyanogen bromide and the
resultant phosphopeptides were purified by reverse-phase high performance
liquid chromatography. Shorter phosphopeptides, obtained by secondary
digestion with trypsin, were purified and subjected to both automated
gas-phase sequencing and manual Edman degradation. The results demonstrate
that the gamma subunit was phosphorylated at Ser-353, contained within the
sequence Arg-Arg-Ser(P)-Ser-Phe-Ile and that the delta subunit was
phosphorylated at Ser-361, contained within the sequence
Arg-Ser-Ser(P)-Ser-Val-Gay-Tyr-Ser-Lys. Determination of the sites
phosphorylated within the structure of the gamma and delta subunits should
contribute to the molecular characterization of the regulation of
desensitization of the nicotinic acetylcholine receptor by protein
phosphorylation.
SO - J Biol Chem 1987 Dec 5;262(34):16748-53.

37. PMID:3928373
PIR:A33369
FT - binding site | phosphate (Thr) (covalent) | 6 (all)
TI - Multisite phosphorylation of glycogen synthase from rabbit skeletal
muscle. Identification of the sites phosphorylated by casein kinase-I.
AB - A casein kinase was highly purified from rabbit skeletal muscle whose
substrate specificity and enzymatic properties were virtually identical to
those of casein kinase-I from rabbit reticulocytes. Prolonged incubation
of glycogen synthase with high concentrations of skeletal muscle casein
kinase-I and Mg-ATP resulted in the incorporation of greater than 6 mol
phosphate/mol subunit and decreased the activity ratio (+/- glucose-6P)
from 0.8 to less than 0.02. The sites phosphorylated by casein kinase-I
were all located in the N and C-terminal cyanogen bromide peptides, termed
CB-1 and CB-2. At an incorporation of 6 mol phosphate/mol subunit,
approximately equal to 2 mol/mol was present in CB-1 and approximately
equal to 4 mol/mol in CB-2. Within CB-1, casein kinase-I phosphorylated
the serines that were 3, 7 and 10 residues from the N-terminus of glycogen
synthase, with minor phosphorylation at threonine-5. Within CB-2,
approximately equal to 90% of the phosphate incorporated was located
between residues 28 and 53, and at least five of the seven serine residues
in this region were phosphorylated. The remaining 10% of phosphate
incorporated into CB-2 was located between residues 98 and 123, mainly at
a serine residue(s). Two of the major sites labelled by casein kinase-I
(serine-3 and serine-10 of CB-1) are not phosphorylated by any other
protein kinase. This will enable the role of casein kinase-I as a glycogen
synthase kinase in vivo to be evaluated.
SO - Eur J Biochem 1985 Aug 15;151(1):39-48.

PIR:A33369
FT - binding site | phosphate (Ser) (covalent) | 4,8,11,645,649,653,657,698,711 (4,8,11
TI - Multisite phosphorylation of glycogen synthase from rabbit skeletal
muscle. Identification of the sites phosphorylated by casein kinase-I.
AB - A casein kinase was highly purified from rabbit skeletal muscle whose
substrate specificity and enzymatic properties were virtually identical to
those of casein kinase-I from rabbit reticulocytes. Prolonged incubation
of glycogen synthase with high concentrations of skeletal muscle casein
kinase-I and Mg-ATP resulted in the incorporation of greater than 6 mol
phosphate/mol subunit and decreased the activity ratio (+/- glucose-6P)
from 0.8 to less than 0.02. The sites phosphorylated by casein kinase-I
were all located in the N and C-terminal cyanogen bromide peptides, termed
CB-1 and CB-2. At an incorporation of 6 mol phosphate/mol subunit,
approximately equal to 2 mol/mol was present in CB-1 and approximately
equal to 4 mol/mol in CB-2. Within CB-1, casein kinase-I phosphorylated
the serines that were 3, 7 and 10 residues from the N-terminus of glycogen
synthase, with minor phosphorylation at threonine-5. Within CB-2,
approximately equal to 90% of the phosphate incorporated was located
between residues 28 and 53, and at least five of the seven serine residues
in this region were phosphorylated. The remaining 10% of phosphate
incorporated into CB-2 was located between residues 98 and 123, mainly at
a serine residue(s). Two of the major sites labelled by casein kinase-I
(serine-3 and serine-10 of CB-1) are not phosphorylated by any other
protein kinase. This will enable the role of casein kinase-I as a glycogen
synthase kinase in vivo to be evaluated.
SO - Eur J Biochem 1985 Aug 15;151(1):39-48.

38. PMID:3944083
PIR:A32541
FT - binding site | phosphate (Ser) (covalent) | 21 (all)
TI - The primary structure and functional characterization of the neutral
histidine-rich polypeptide from human parotid secretion.
AB - The neutral histidine-rich polypeptide (HRP) from human parotid secretion
was isolated by ion-exchange and gel-filtration chromatography. The
complete amino acid sequence determined by automated Edman degradation of
the protein, tryptic and Staphylococcus aureus V8 protease peptides, and
digestion with carboxypeptidase A is: NH2-Asp-Pse-His-Glu-Lys-Arg-His-His-Gly-Tyr-Arg-Arg-Lys-Phe-His-Glu-Lys-His-His-Ser-His-Arg-Glu-Phe-Pro-Phe-Tyr-Gly-Asp-Tyr-Gly-Ser-Asn-Tyr-Leu-Tyr-Asp-Asn-COOH, where Pse
represents phosphoserine. The polypeptide contains 38 residues and has Mr
4929. The charged amino acids predominate with 7 histidine, 4 arginine, 3
lysine, 3 aspartic acid, 3 glutamic acid residues, and 1 phosphoserine.
Assuming minimal charge contributions from histidine and one negative
charge from phosphoserine at pH 7, the net charge of HRP is balanced by an
equal contribution of basic and acidic residues. Furthermore, the
distribution of hydrophilic and hydrophobic residues along the polypeptide
chain indicates that there is no structural polarity. The polypeptide
lacks threonine, alanine, valine, cysteine, methionine, and isoleucine.
HRP did not display sequence similarity with any protein sequence in the
National Biomedical Research Foundation Data Bank. HRP is an active
inhibitor of hydroxyapatite crystal growth from solutions supersaturated
with respect to calcium phosphate salts and therefore must play a role in
the stabilization of mineral-solute interactions in oral fluid. In
addition, HRP is a potent inhibitor of Candida albicans germination and
therefore may be a significant component of the antimicrobial host defense
system in the oral cavity.
SO - J Biol Chem 1986 Jan 25;261(3):1177-82.

39. PMID:6290217
PIR:PZRB1
FT - binding site | phosphate (Ser) (covalent) (by cAMP-dependent kinase) (partial) | 67 (all)
TI - Complete primary structure of protein phosphatase inhibitor-1 from rabbit
skeletal muscle.
AB - The complete primary structure of protein phosphatase inhibitor-1 has been
determined. The protein consists of a single polypeptide chain of 165
residues, molecular weight 18640. The threonine residue that must be
phosphorylated for activation is at position 35 and the active cyanogen
bromide peptide, CB-1, comprises residues 2-66. The N-terminal methionine
is acetylated and 40% of the inhibitor-1 molecules lack the C-terminal
dipeptide Ala-Val. Serine-67 is substantially phosphorylated in vivo, but
this phosphoserine residue does not appear to influence the activity of
inhibitor-1.
SO - Eur J Biochem 1982 Aug;126(2):235-46.

PIR:PZRB1
FT - binding site | phosphate (Thr) (covalent) (by cAMP-dependent kinase) | 35 (all)
TI - Complete primary structure of protein phosphatase inhibitor-1 from rabbit
skeletal muscle.
AB - The complete primary structure of protein phosphatase inhibitor-1 has been
determined. The protein consists of a single polypeptide chain of 165
residues, molecular weight 18640. The threonine residue that must be
phosphorylated for activation is at position 35 and the active cyanogen
bromide peptide, CB-1, comprises residues 2-66. The N-terminal methionine
is acetylated and 40% of the inhibitor-1 molecules lack the C-terminal
dipeptide Ala-Val. Serine-67 is substantially phosphorylated in vivo, but
this phosphoserine residue does not appear to influence the activity of
inhibitor-1.
SO - Eur J Biochem 1982 Aug;126(2):235-46.

40. PMID:6304091
PIR:OKBOG
FT - binding site | phosphate (Thr) (covalent) (by autophosphorylation) | 59 (all)
TI - Amino acid sequence around a "hinge" region and its "autophosphorylation"
site in bovine Lung cGMP-dependent protein kinase.
AB - An exposed "hinge" region of cGMP-dependent protein kinase is known to be
susceptible to both limited proteolysis and autophosphorylation. A
91-residue fragment has been isolated from this region and its amino acid
sequence has been compared with the analogous regions of the
cAMP-dependent protein kinases. Although a resemblance among these
sequences is not striking, the phosphorylation sites are in corresponding
regions toward the NH2 termini, and there are indications of homology in
the vicinity of their autophosphorylation sites. As in the cAMP-dependent
protein kinase, the site of autophosphorylation and the site of
susceptibility to limited proteolysis are very near each other in the
primary structure. The actual site of autophosphorylation (the underlined
threonine residue in Pro-Arg-Thr-Thr-Arg) is quite different from those in
the regulatory subunit of Type II cAMP-dependent kinase or the site in
Type I regulatory subunit that can be phosphorylated by the cGMP-dependent
protein kinase.
SO - J Biol Chem 1983 May 10;258(9):5531-6.

41. PMID:6311252
PIR:OKBO2C
FT - binding site | phosphate (Thr) (covalent) | 198 (all)
TI - Amino acid sequence of the catalytic subunit of bovine type II adenosine
cyclic 3',5'-phosphate dependent protein kinase.
AB - The 350-residue amino acid sequence of the catalytic subunit of bovine
cardiac muscle adenosine cyclic 3',5'-phosphate dependent protein kinase
is described. The protein has a molecular weight of 40 862, which includes
an N-tetradecanoyl (myristyl) group blocking the NH2 terminus and
phosphate groups at threonine-197 and serine-338. Seven methionyl bonds in
the S-carboxymethylated protein were cleaved with cyanogen bromide to
yield eight primary peptides. These fragments, and subpeptides generated
by cleavage with trypsin, pepsin, chymotrypsin, thermolysin, and
Myxobacter AL-1 protease II, were purified and analyzed to yield the
majority of the sequence. The primary peptides were aligned by analyses of
overlapping peptides, particularly of methione-containing tryptic peptides
generated after in vitro [14C]methyl exchange labeling of methionyl
residues in the intact protein.
SO - Biochemistry 1983 Jul 19;22(15):3702-9.

PIR:OKBO2C
FT - binding site | phosphate (Ser) (covalent) | 339 (all)
TI - Amino acid sequence of the catalytic subunit of bovine type II adenosine
cyclic 3',5'-phosphate dependent protein kinase.
AB - The 350-residue amino acid sequence of the catalytic subunit of bovine
cardiac muscle adenosine cyclic 3',5'-phosphate dependent protein kinase
is described. The protein has a molecular weight of 40 862, which includes
an N-tetradecanoyl (myristyl) group blocking the NH2 terminus and
phosphate groups at threonine-197 and serine-338. Seven methionyl bonds in
the S-carboxymethylated protein were cleaved with cyanogen bromide to
yield eight primary peptides. These fragments, and subpeptides generated
by cleavage with trypsin, pepsin, chymotrypsin, thermolysin, and
Myxobacter AL-1 protease II, were purified and analyzed to yield the
majority of the sequence. The primary peptides were aligned by analyses of
overlapping peptides, particularly of methione-containing tryptic peptides
generated after in vitro [14C]methyl exchange labeling of methionyl
residues in the intact protein.
SO - Biochemistry 1983 Jul 19;22(15):3702-9.

42. PMID:6349995
PIR:HSIN21
FT - binding site | phosphate (Ser) (covalent) (partial) | 1 (all)
TI - Primary structure of histone H2A from nucleated erythrocyte of the marine
worm Sipunculus nudus. Presence of two forms of H2A in the sipunculid
chromatin.
AB - The complete amino acid sequence (123 residues) of histone H2A from
erythrocytes of the marine worm Sipunculus nudus, has been established
from data provided by automated sequence analysis of large fragments
generated by V8 staphylococcal protease digestion of histone H2A and by
limited hydrolysis of the protein with alpha-chymotrypsin and from
structural studies of tryptic peptides of the protein. By comparison with
calf homologous histone, the sipunculid histone H2A shows 6 deletions and
13 substitutions. Six of the substitutions are non-conservative. Most of
the evolutionary changes are mainly observed in the basic amino-terminal
and carboxy-terminal regions of the molecule, which are the primary
DNA-binding sites. Few conservative point changes are observed in the
central region (residues 18-118) which interacts strongly with histone H2B
to form the dimer H2A-H2B. 60% of the H2A molecules were found
phosphorylated on the amino-terminal residue, N-acetyl-serine. The high
content of phosphorylated histone H2A in the sipunculid erythrocyte
chromatin could probably be related to smaller repeat length (177 +/- 5
base pairs) of nucleosomal DNA and to nuclear inactivation and chromatin
condensation.
SO - Eur J Biochem 1983 Sep 1;135(1):113-21.

43. PMID:6698958
PIR:R3RTS6
FT - binding site | phosphate (Ser) (covalent) (by ribosomal protein S6 kinase) | 235,236 (all)
TI - Phosphorylation of hepatic ribosomal protein S6 on 80 and 40 S ribosomes.
Primary structure of S6 in the region of the major phosphorylation sites
for cAMP-dependent protein kinases.
AB - Rat liver ribosomes and 40 S ribosomal subunits were phosphorylated with
the purified catalytic subunit of cAMP-dependent protein kinase.
Phosphorylation of ribosomal protein S6 plateaued at around 2 mol of
phosphate/mol of protein with both substrates. Peptide map analyses showed
that the most prominent phosphorylation sites associated with 40 S
substrates were the adjacent serines in the Arg-Leu-Ser-Ser-Leu-Arg
segment of S6. The first serine residue appeared to be the preferred site
as has been established previously for 80 S ribosomes (Wettenhall, R.E.H.,
and Cohen, P. (1982) FEBS Lett. 140, 263-269). Additional phosphorylation
sites were apparent from the peptide maps. One of these was associated
with the triphosphopeptide (termed T1a) having the sequence
Arg-Leu-Ser-Ser-Leu-Arg-Ala-Ser-Thr-Ser-Lys. A larger fragment of S6
(termed Tlc) isolated from mild tryptic digests of whole ribosomes,
consisted of the T1a sequence extended by the sequence
Ser-Glu-Glu-Ser-Gln-(Lys) at the COOH terminus. A comparison of the size
and chromatographic and isoelectric focusing properties of the T1a/T1c
peptides and prominent tryptic peptides of S6 from insulin-stimulated
hepatocytes indicated a relationship between these peptides. Thus, it
appeared that some of the potential phosphorylation sites in the T1a/T1c
region of S6 are phosphorylated by an insulin-regulated kinase in
hepatocytes.
SO - J Biol Chem 1984 Feb 25;259(4):2084-91.

44. PMID:697844
PIR:SYRTPP
FT - binding site | phosphate (Ser) (covalent) | 66 (all)
TI - Phosphorylation, a factor controlling the synthesis of
L-erythrodihydrobiopterin (BH2).
AB - Isolation of brain enzyme in presence of 0.8 M NaF allowed recovery of the enzyme phosphorylated at residue 67 (serine) as determined by a new assay for phosphate.
SO - Biochem Biophys Res Commun 1978 Jul 28;83(2):593-8.

45. PMID:7107568
PIR:SBMQPI
FT - binding site | phosphate (Ser) (covalent) | 21,22 (all)
TI - Phosphoproteins in the parotid saliva from the subhuman primate Macaca
fascicularis. Isolation and characterization of a proline-rich
phosphoglycoprotein and the complete covalent structure of a proline-rich
phosphopeptide.
AB - Parotid saliva from the cynomolgus monkey (Macaca fascicularis) and from
pooled human collections displayed the same groups of proteins when
fractionated by anion exchange and gel filtration chromatography. We have
isolated and characterized a proline-rich phosphoglycoprotein (MPRP) and a
proline-rich phosphopeptide (M-statherin) from macaque parotid saliva.
MPRP has an apparent molecular weight of 16,900 and displays an unusual
chemical composition. It is enriched in proline, glycine, and acidic amino
acids, but lacks cysteine, methionine, and tyrosine. MPRP contains 25%
(w/w) carbohydrate with 7.0 mol of neutral hexoses, 5.3 mol of
galactosamine, 5.9 mol of sialic acid, and 3 mol of phosphorus/mol of
protein. M-statherin is a 42-residue phosphopeptide with a high proline,
glutamic acid, and tyrosine content, but which lacks threonine, valine,
cysteine, methionine, isoleucine, and histidine. The complete covalent
structure of M-statherin (Mr = 5,368) is:
NH2-Asp-Pse-Pse-Glu-Glu-Lys-Phe-Leu-Arg-Arg-Leu-Arg-Arg-Phe-Asp-Glu-Gly-Ar
g-Tyr-
-Gly-Pro-Tyr-Gln-Pro-Phe-Ala-Pro-Gln-Pro-Leu-Tyr-Pro-Gln-Pro-Tyr-Gln-Pro-T
yr-Gln-Pro-Gln-Tyr-COOH This is the first complete amino acid sequence of
a component in the salivary secretion of a subhuman primate. Phosphoserine
occurs at residues 2 and 3. All 13 acidic and basic amino acids are
located in the NH2-terminal half of the molecule. The carboxyl-terminal
half of the molecule is hydrophobic where the tripeptide Tyr-Gln-Pro is
repeated three times, the dipeptide Gln-Pro occurs twice, and the
tripeptides Tyr-Gly-Pro, Phe-Ala-Pro, and Leu-Tyr-Pro occur once.
Evaluation of secondary structure by the Chou-Fasman method predicts an
alpha helix in the NH2-terminal half (residue 4-16) and a beta pleated
sheet in the carboxyl-terminal half (residues 22-26; 38-42) of the
molecule. Both MPRP and M-statherin inhibit spontaneous and seeded
precipitation from solutions supersaturated with respect to calcium
phosphate salts. This suggests that these macaque compounds may function
by maintaining saliva supersaturated with respect to calcium phosphate
salts, a necessary requirement for stabilization of hydroxyapatite in the
surface layers of teeth.U
SO - J Biol Chem 1982 Aug 25;257(16):9271-82.

46. PMID:7615564
PIR:A35702
FT - binding site | phosphate (Ser) (covalent) | 3 (all)
TI - Reactivation of phosphorylated actin depolymerizing factor and
identification of the regulatory site.
AB - Actin depolymerizing factor (ADF) occurs naturally in two forms, one of
which contains a phosphorylated Ser and does not bind G-actin or
depolymerize F-actin. Removal of this phosphate in vitro by alkaline
phosphatase restores full F-actin depolymerizing activity. To identify the
phosphorylation site, [32P]pADF was purified and digested with
endoproteinase Lys-C. The digest contained only one 32P-labeled peptide.
Further digestion with endoproteinase Asp-N and mass spectrometric
analysis showed that this peptide came from the N terminus of ADF.
Alkaline phosphatase treatment of one Asp-N peptide (mass 753) converted
it to a peptide of mass 673, demonstrating that this peptide contains the
phosphate group. Tandem mass spectrometric sequence analysis of this
peptide identified the phosphorylated Ser as the encoded Ser3 (Ser2 in the
processed protein). HeLa cells, transfected with either chick wild-type
ADF cDNA or a cDNA mutated to code for Ala in place of Ser24 or Thr25,
express and phosphorylate the exogenous ADF. Cells also expressed high
levels of mutant ADF when Ser3 was deleted or converted to either Ala or
Glu. However, none of these mutants was phosphorylated, confirming that
Ser3 in the encoded ADF is the single in vivo regulatory site.
SO - J Biol Chem 1995 Jul 21;270(29):17582-7.

47. PMID:7822248
PIR:A45100
FT - binding site | phosphate (Thr) (covalent) (by raf-1 kinase) | 292,386 (all)
TI - Mitogen-activated protein (MAP) kinase phosphorylation of MAP kinase
kinase: determination of phosphorylation sites by mass spectrometry and
site-directed mutagenesis.
AB - Mitogen-activated protein kinase kinase (MKK) phosphorylates and activates
mitogen-activated protein kinase (MAPK) in response to stimulation of
various eukaryotic signaling pathways. Conversely, a recent report showed
that MAPK phosphorylates MKK in vitro [Matsuda, S., Gotoh, Y., and
Nishida, E. (1993) J. Biol. Chem. 268, 3277-3281]. To gain insight into
the function of this feedback phosphorylation, we identified the major
sites targeted for phosphorylation by MAPK and examined whether such a
modification plays a role in regulating the basal and stimulated MKK
activities. Two phosphopeptides generated by tryptic digestion of
MAPK-phosphorylated MKK were identified by electrospray ionization mass
spectrometry. Cyanogen bromide cleavage also yielded two phosphopeptides
whose sequence overlapped with the tryptic phosphopeptides. Both sets of
phosphopeptides contained candidate MAPK target sites at Thr292 and Thr386
that fit the consensus sequence ProXThr*Pro. Replacement of either Thr292
or Thr386 with alanine by site-directed mutagenesis reduced the phosphate
incorporation respectively to 32 or 75% that of wild type MKK. Replacement
of both threonine residues with alanine reduced phosphate incorporation to
2.5% that of wild type enzyme. Comparison of MAPK-phosphorylated vs.
unphosphorylated MKK showed no significant differences in basal or
Raf-1-stimulated MKK activity. We conclude that the phosphorylation of MKK
at Thr292 and Thr386 does not interfere with catalysis in vitro.
SO - J Biochem (Tokyo) 1994 Aug;116(2):304-14.

48. PMID:7909431
PIR:DVHU1
FT - binding site | phosphate (Ser) (covalent) (by protein kinase C) | 661,667,671 (all)
TI - Phosphorylation by protein kinase C and cyclic AMP-dependent protein
kinase of synthetic peptides derived from the linker region of human
P-glycoprotein.
AB - Specific sites in the linker region of human P-glycoprotein phosphorylated
by protein kinase C (PKC) were identified by means of a synthetic peptide
substrate, PG-2, corresponding to residues 656-689 from this region of the
molecule. As PG-2 has several sequences of the type recognized by the
cyclic AMP-dependent protein kinase (PKA), PG-2 was also tested as a
substrate for PKA. PG-2 was phosphorylated by purified PKC in a
Ca2+/phospholipid-dependent manner, with a Km of 1.3 microM, and to a
maximum stoichiometry of 2.9 +/- 0.1 mol of phosphate/mol of peptide.
Sequence analysis of tryptic fragments of PG-2 phosphorylated by PKC
identified Ser-661, Ser-667 and Ser-671 as the three sites of
phosphorylation. PG-2 was also found to be phosphorylated by purified PKA
in a cyclic AMP-dependent manner, with a Km of 21 microM, and to a maximum
stoichiometry of 2.6 +/- 0.2 mol of phosphate/mol of peptide. Ser-667,
Ser-671 and Ser-683 were phosphorylated by PKA. Truncated peptides of PG-2
were utilized to confirm that Ser-661 was PKC-specific and Ser-683 was
PKA-specific. Further studies showed that PG-2 acted as a competitive
substrate for the P-glycoprotein kinase present in membranes from
multidrug-resistant human KB cells. The membrane kinase phosphorylated
PG-2 mainly on Ser-661, Ser-667 and Ser-671. These results show that human
P-glycoprotein can be phosphorylated by at least two protein kinases,
stimulated by different second-messenger systems, which exhibit both
overlapping and unique specificities for phosphorylation of multiple sites
in the linker region of the molecule.
SO - Biochem J 1994 Apr 1;299 ( Pt 1):309-15.

PIR:DVHU1
FT - binding site | phosphate (Ser) (covalent) (by cAMP-dependent kinase) | 667,671,683 (all)
TI - Phosphorylation by protein kinase C and cyclic AMP-dependent protein
kinase of synthetic peptides derived from the linker region of human
P-glycoprotein.
AB - Specific sites in the linker region of human P-glycoprotein phosphorylated
by protein kinase C (PKC) were identified by means of a synthetic peptide
substrate, PG-2, corresponding to residues 656-689 from this region of the
molecule. As PG-2 has several sequences of the type recognized by the
cyclic AMP-dependent protein kinase (PKA), PG-2 was also tested as a
substrate for PKA. PG-2 was phosphorylated by purified PKC in a
Ca2+/phospholipid-dependent manner, with a Km of 1.3 microM, and to a
maximum stoichiometry of 2.9 +/- 0.1 mol of phosphate/mol of peptide.
Sequence analysis of tryptic fragments of PG-2 phosphorylated by PKC
identified Ser-661, Ser-667 and Ser-671 as the three sites of
phosphorylation. PG-2 was also found to be phosphorylated by purified PKA
in a cyclic AMP-dependent manner, with a Km of 21 microM, and to a maximum
stoichiometry of 2.6 +/- 0.2 mol of phosphate/mol of peptide. Ser-667,
Ser-671 and Ser-683 were phosphorylated by PKA. Truncated peptides of PG-2
were utilized to confirm that Ser-661 was PKC-specific and Ser-683 was
PKA-specific. Further studies showed that PG-2 acted as a competitive
substrate for the P-glycoprotein kinase present in membranes from
multidrug-resistant human KB cells. The membrane kinase phosphorylated
PG-2 mainly on Ser-661, Ser-667 and Ser-671. These results show that human
P-glycoprotein can be phosphorylated by at least two protein kinases,
stimulated by different second-messenger systems, which exhibit both
overlapping and unique specificities for phosphorylation of multiple sites
in the linker region of the molecule.
SO - Biochem J 1994 Apr 1;299 ( Pt 1):309-15.

49. PMID:8061611
PIR:GERTM1
FT - binding site | phosphate (Ser) (covalent) (by casein kinase II) | 22,25,28 (all)
TI - Conserved phosphorylation of serines in the Ser-X-Glu/Ser(P) sequences of
the vitamin K-dependent matrix Gla protein from shark, lamb, rat, cow, and
human.
AB - The present studies demonstrate that matrix Gla protein (MGP), a 10-kDa
vitamin K-dependent protein, is phosphorylated at 3 serine residues near
its N-terminus. Phosphoserine was identified at residues 3, 6, and 9 of
bovine, human, rat, and lamb MGP by N-terminal protein sequencing. All 3
modified serines are in tandemly repeated Ser-X-Glu sequences. Two of the
serines phosphorylated in shark MGP, residues 2 and 5, also have glutamate
residues in the n + 2 position in tandemly repeated Ser-X-Glu sequences,
whereas the third, shark residue 3, would acquire an acidic phosphoserine
in the n + 2 position upon phosphorylation of serine 5. The recognition
motif found for MGP phosphorylation, Ser-X-Glu/Ser(P), has been seen
previously in milk caseins, salivary proteins, and a number of regulatory
peptides. A review of the literature has revealed an intriguing dichotomy
in the extent of serine phosphorylation among secreted proteins that are
phosphorylated at Ser-X-Glu/Ser(P) sequences. Those phosphoproteins
secreted into milk or saliva are fully phosphorylated at each target
serine, whereas phosphoproteins secreted into the extracellular
environment of cells are partially phosphorylated at target serine
residues, as we show here for MGP and others have shown for regulatory
peptides and the insulin-like growth factor binding protein 1. We propose
that the extent of serine phosphorylation regulates the activity of
proteins secreted into the extracellular environment of cells, and that
partial phosphorylation can therefore be explained by the need to ensure
that the phosphoprotein be poised to gain or lose activity with regulated
changes in phosphorylation status.(ABSTRACT TRUNCATED AT 250 WORDS)
SO - Protein Sci 1994 May;3(5):822-30.

PIR:GEBOM
FT - binding site | phosphate (Ser) (covalent) (by casein kinase II) | 22,25,28 (all)
TI - Conserved phosphorylation of serines in the Ser-X-Glu/Ser(P) sequences of
the vitamin K-dependent matrix Gla protein from shark, lamb, rat, cow, and
human.
AB - The present studies demonstrate that matrix Gla protein (MGP), a 10-kDa
vitamin K-dependent protein, is phosphorylated at 3 serine residues near
its N-terminus. Phosphoserine was identified at residues 3, 6, and 9 of
bovine, human, rat, and lamb MGP by N-terminal protein sequencing. All 3
modified serines are in tandemly repeated Ser-X-Glu sequences. Two of the
serines phosphorylated in shark MGP, residues 2 and 5, also have glutamate
residues in the n + 2 position in tandemly repeated Ser-X-Glu sequences,
whereas the third, shark residue 3, would acquire an acidic phosphoserine
in the n + 2 position upon phosphorylation of serine 5. The recognition
motif found for MGP phosphorylation, Ser-X-Glu/Ser(P), has been seen
previously in milk caseins, salivary proteins, and a number of regulatory
peptides. A review of the literature has revealed an intriguing dichotomy
in the extent of serine phosphorylation among secreted proteins that are
phosphorylated at Ser-X-Glu/Ser(P) sequences. Those phosphoproteins
secreted into milk or saliva are fully phosphorylated at each target
serine, whereas phosphoproteins secreted into the extracellular
environment of cells are partially phosphorylated at target serine
residues, as we show here for MGP and others have shown for regulatory
peptides and the insulin-like growth factor binding protein 1. We propose
that the extent of serine phosphorylation regulates the activity of
proteins secreted into the extracellular environment of cells, and that
partial phosphorylation can therefore be explained by the need to ensure
that the phosphoprotein be poised to gain or lose activity with regulated
changes in phosphorylation status.(ABSTRACT TRUNCATED AT 250 WORDS)
SO - Protein Sci 1994 May;3(5):822-30.

PIR:GEHUM
FT - binding site | phosphate (Ser) (covalent) (by casein kinase II) | 22,25,28 (all)
TI - Conserved phosphorylation of serines in the Ser-X-Glu/Ser(P) sequences of
the vitamin K-dependent matrix Gla protein from shark, lamb, rat, cow, and
human.
AB - The present studies demonstrate that matrix Gla protein (MGP), a 10-kDa
vitamin K-dependent protein, is phosphorylated at 3 serine residues near
its N-terminus. Phosphoserine was identified at residues 3, 6, and 9 of
bovine, human, rat, and lamb MGP by N-terminal protein sequencing. All 3
modified serines are in tandemly repeated Ser-X-Glu sequences. Two of the
serines phosphorylated in shark MGP, residues 2 and 5, also have glutamate
residues in the n + 2 position in tandemly repeated Ser-X-Glu sequences,
whereas the third, shark residue 3, would acquire an acidic phosphoserine
in the n + 2 position upon phosphorylation of serine 5. The recognition
motif found for MGP phosphorylation, Ser-X-Glu/Ser(P), has been seen
previously in milk caseins, salivary proteins, and a number of regulatory
peptides. A review of the literature has revealed an intriguing dichotomy
in the extent of serine phosphorylation among secreted proteins that are
phosphorylated at Ser-X-Glu/Ser(P) sequences. Those phosphoproteins
secreted into milk or saliva are fully phosphorylated at each target
serine, whereas phosphoproteins secreted into the extracellular
environment of cells are partially phosphorylated at target serine
residues, as we show here for MGP and others have shown for regulatory
peptides and the insulin-like growth factor binding protein 1. We propose
that the extent of serine phosphorylation regulates the activity of
proteins secreted into the extracellular environment of cells, and that
partial phosphorylation can therefore be explained by the need to ensure
that the phosphoprotein be poised to gain or lose activity with regulated
changes in phosphorylation status.(ABSTRACT TRUNCATED AT 250 WORDS)
SO - Protein Sci 1994 May;3(5):822-30.

50. PMID:8125092
PIR:A40936
FT - binding site | phosphate (Ser) (covalent) (by MAP and cdc2 kinases) | 25 (all)
TI - Cell-cycle-regulated phosphorylation of oncoprotein 18 on Ser16, Ser25 and
Ser38.
AB - Oncoprotein 18 (Op18) has been independently identified due to its
increased phosphorylation in response to external signals and its
up-regulated expression in acute leukemia. We have identified two serine
residues of Op18 that are phosphorylated after triggering by the T cell
antigen receptor. One of these residues, Ser25, was shown to be a likely
substrate for the mitogen-activated protein (MAP) kinase, while the other
residue, Ser16, was shown to be phosphorylated in response to increased
intracellular calcium. Our previous site-mapping studies of Op18 also
revealed that basal phosphorylation of Op18 is mainly located on Ser38,
which was found to be the primary in vitro phosphorylation site of
p13suc1-precipitated cdc2 kinase activities. These findings raised the
possibility that Op18 may be a substrate for both receptor-regulated
calcium-induced protein kinases and the MAP kinase family, as well as
being a substrate for the cell-cycle-regulated cdc2 kinase family. In the
present report we have performed site-mapping studies of
cell-cycle-regulated fluctuations of Op18 phosphorylation. The results
reveal that S-phase progression of a synchronised leukemic T cell line is
associated with increased phosphorylation of both the Ser25 and Ser38
residues. Moreover, during mitosis, a burst of phosphorylation was
observed and at this stage of the cell cycle a major fraction of Op18 was
phosphorylated at multiple sites. Phosphorylation of Op18 during mitosis
was located primarily on Ser38 and to lesser extent on Ser25, Ser16 and at
an unidentified C-terminal residue. In vitro phosphorylation experiments,
employing two distinct members of the cdc2 kinase family, were consistent
with involvement of both p34-cdc2 and p33-cdk2 in cell-cycle-regulated
phosphorylation of Ser25 and Ser38 of Op18. Most importantly, the ratio of
Ser25/Ser38 phosphorylation observed in vitro, using either p34-cdc2 or
p33-cdk2, was found to be the same as the ratio observed in intact cells
during all phases of the cell cycle. These findings suggest that Op18 may
be a physiological substrate for several members of the cdc2 kinase family
during both the S-phase and the mitotic phase of the cell cycle.
SO - Eur J Biochem 1994 Mar 1;220(2):359-68.

PIR:A40936
FT - binding site | phosphate (Ser) (covalent) (by cdc2 kinase) | 38 (all)
TI - Cell-cycle-regulated phosphorylation of oncoprotein 18 on Ser16, Ser25 and
Ser38.
AB - Oncoprotein 18 (Op18) has been independently identified due to its
increased phosphorylation in response to external signals and its
up-regulated expression in acute leukemia. We have identified two serine
residues of Op18 that are phosphorylated after triggering by the T cell
antigen receptor. One of these residues, Ser25, was shown to be a likely
substrate for the mitogen-activated protein (MAP) kinase, while the other
residue, Ser16, was shown to be phosphorylated in response to increased
intracellular calcium. Our previous site-mapping studies of Op18 also
revealed that basal phosphorylation of Op18 is mainly located on Ser38,
which was found to be the primary in vitro phosphorylation site of
p13suc1-precipitated cdc2 kinase activities. These findings raised the
possibility that Op18 may be a substrate for both receptor-regulated
calcium-induced protein kinases and the MAP kinase family, as well as
being a substrate for the cell-cycle-regulated cdc2 kinase family. In the
present report we have performed site-mapping studies of
cell-cycle-regulated fluctuations of Op18 phosphorylation. The results
reveal that S-phase progression of a synchronised leukemic T cell line is
associated with increased phosphorylation of both the Ser25 and Ser38
residues. Moreover, during mitosis, a burst of phosphorylation was
observed and at this stage of the cell cycle a major fraction of Op18 was
phosphorylated at multiple sites. Phosphorylation of Op18 during mitosis
was located primarily on Ser38 and to lesser extent on Ser25, Ser16 and at
an unidentified C-terminal residue. In vitro phosphorylation experiments,
employing two distinct members of the cdc2 kinase family, were consistent
with involvement of both p34-cdc2 and p33-cdk2 in cell-cycle-regulated
phosphorylation of Ser25 and Ser38 of Op18. Most importantly, the ratio of
Ser25/Ser38 phosphorylation observed in vitro, using either p34-cdc2 or
p33-cdk2, was found to be the same as the ratio observed in intact cells
during all phases of the cell cycle. These findings suggest that Op18 may
be a physiological substrate for several members of the cdc2 kinase family
during both the S-phase and the mitotic phase of the cell cycle.
SO - Eur J Biochem 1994 Mar 1;220(2):359-68.

PIR:A40936
FT - binding site | phosphate (Ser) (covalent) (by protein kinase A and calmodulin-dependent kinase) | 16 (all)
TI - Cell-cycle-regulated phosphorylation of oncoprotein 18 on Ser16, Ser25 and
Ser38.
AB - Oncoprotein 18 (Op18) has been independently identified due to its
increased phosphorylation in response to external signals and its
up-regulated expression in acute leukemia. We have identified two serine
residues of Op18 that are phosphorylated after triggering by the T cell
antigen receptor. One of these residues, Ser25, was shown to be a likely
substrate for the mitogen-activated protein (MAP) kinase, while the other
residue, Ser16, was shown to be phosphorylated in response to increased
intracellular calcium. Our previous site-mapping studies of Op18 also
revealed that basal phosphorylation of Op18 is mainly located on Ser38,
which was found to be the primary in vitro phosphorylation site of
p13suc1-precipitated cdc2 kinase activities. These findings raised the
possibility that Op18 may be a substrate for both receptor-regulated
calcium-induced protein kinases and the MAP kinase family, as well as
being a substrate for the cell-cycle-regulated cdc2 kinase family. In the
present report we have performed site-mapping studies of
cell-cycle-regulated fluctuations of Op18 phosphorylation. The results
reveal that S-phase progression of a synchronised leukemic T cell line is
associated with increased phosphorylation of both the Ser25 and Ser38
residues. Moreover, during mitosis, a burst of phosphorylation was
observed and at this stage of the cell cycle a major fraction of Op18 was
phosphorylated at multiple sites. Phosphorylation of Op18 during mitosis
was located primarily on Ser38 and to lesser extent on Ser25, Ser16 and at
an unidentified C-terminal residue. In vitro phosphorylation experiments,
employing two distinct members of the cdc2 kinase family, were consistent
with involvement of both p34-cdc2 and p33-cdk2 in cell-cycle-regulated
phosphorylation of Ser25 and Ser38 of Op18. Most importantly, the ratio of
Ser25/Ser38 phosphorylation observed in vitro, using either p34-cdc2 or
p33-cdk2, was found to be the same as the ratio observed in intact cells
during all phases of the cell cycle. These findings suggest that Op18 may
be a physiological substrate for several members of the cdc2 kinase family
during both the S-phase and the mitotic phase of the cell cycle.
SO - Eur J Biochem 1994 Mar 1;220(2):359-68.

51. PMID:8132603
PIR:MMECZB
FT - binding site | phosphate (His) (covalent) (by autophosphorylation) | 243 (all)
TI - Identification of the site of phosphorylation on the osmosensor, EnvZ, of
Escherichia coli.
AB - EnvZ is a membrane-bound histidine kinase that functions as an osmotic
sensor capable of phosphorylating the regulator protein OmpR in
Escherichia coli. To characterize the site of phosphorylation
biochemically, we overexpressed a 36-kDa truncated EnvZ protein (Glu-106
to Gly-450) that formed inclusion bodies in the cell. After
solubilization, the inclusion body form of EnvZ was cleaved into two major
fragments with molecular weights of 25,000 and 10,000. The 25-kDa
fragment, EnvZc, was purified and found to exist as a dimer. N-terminal
sequence analysis established that cleavage had occurred at Arg-214,
indicating that EnvZc contained most of the cytoplasmic domain of EnvZ.
After labeling EnvZc with [gamma-32P]ATP, the protein was proteolytically
digested, and the resulting peptides were separated by reverse phase
chromatography using high performance liquid chromatography. One major
radioactive peptide containing greater than 90% of the recovered
peptide-associated radioactivity was isolated. Amino acid analysis of this
purified peptide indicated that the composition was consistent with a
peptide that contained His-243. The amino acid sequence of this peptide
was determined to be MAGVSHDLRTP (residues 238-248). These results
indicate that His-243 is the major site of phosphorylation on EnvZ and
represents the first biochemical characterization of the site of
phosphorylation of a membrane histidine kinase of the two-component
regulatory family of molecules in bacteria.
SO - J Biol Chem 1994 Mar 25;269(12):8728-33.

52. PMID:8218377
PIR:HSHUP2
FT - binding site | phosphate (Ser) (covalent) (partial) | 59 (all)
TI - Phosphorylation of human sperm protamines HP1 and HP2: identification of
phosphorylation sites.
AB - Human sperm is characterized by a high heterogeneity of its basic nuclear
protein complement of pro-protamines, protamines and histones. This
heterogeneity is increased by the persistence of phosphorylated protamines
in mature spermatozoa. Alkaline phosphatase treatment of whole protein
indicated that protamines HP1 and HP2 were phosphorylated to various
degrees. Presence of non-phosphorylated and phosphorylated protamines HP1
and HP2 was further demonstrated by electrospray mass spectrometry.
Phosphorylation sites of mono- and di-phosphorylated protamine HP1 were
identified by automatic Edman degradation of the protein after
phosphoserine derivatization to S-ethylcysteine. In both phosphorylated
forms, Ser-10 was found phosphorylated; in the di-phosphorylated form,
Ser-8 was identified as the second site of phosphorylation. In protamine
HP2, the unique site of phosphorylation (Ser-14) was located after limited
acid hydrolysis of enzymic peptides and thin-layer electrophoresis.
SO - Biochim Biophys Acta 1993 Nov 10;1203(1):109-14.

PIR:HSHUP1
FT - binding site | phosphate (Ser) (covalent) (partial) | 9,11 (all)
TI - Phosphorylation of human sperm protamines HP1 and HP2: identification of
phosphorylation sites.
AB - Human sperm is characterized by a high heterogeneity of its basic nuclear
protein complement of pro-protamines, protamines and histones. This
heterogeneity is increased by the persistence of phosphorylated protamines
in mature spermatozoa. Alkaline phosphatase treatment of whole protein
indicated that protamines HP1 and HP2 were phosphorylated to various
degrees. Presence of non-phosphorylated and phosphorylated protamines HP1
and HP2 was further demonstrated by electrospray mass spectrometry.
Phosphorylation sites of mono- and di-phosphorylated protamine HP1 were
identified by automatic Edman degradation of the protein after
phosphoserine derivatization to S-ethylcysteine. In both phosphorylated
forms, Ser-10 was found phosphorylated; in the di-phosphorylated form,
Ser-8 was identified as the second site of phosphorylation. In protamine
HP2, the unique site of phosphorylation (Ser-14) was located after limited
acid hydrolysis of enzymic peptides and thin-layer electrophoresis.
SO - Biochim Biophys Acta 1993 Nov 10;1203(1):109-14.

53. PMID:8226879
PIR:QRBOT1
FT - binding site | phosphate (Ser) (covalent) (by proline-directed kinase) | 202,209,242,248,411 (
TI - Brain proline-directed protein kinase phosphorylates tau on sites that are
abnormally phosphorylated in tau associated with Alzheimer's paired
helical filaments.
AB - Brain proline-directed protein kinase (BPDK), which contains a catalytic
subunit homologous to and displaying site-specific phosphorylation similar
to p34cdc2 kinase (Lew, J., Winkfein, R. J., Paudel, H. K., and Wang, J.
H. (1992) J. Biol. Chem. 267, 25922-25926), has been examined for possible
involvement in tau phosphorylation. Immunoblot analyses using peptide
antibodies specific for BPDK have revealed the presence of the kinase in
bovine brain microtubules purified extensively by repeated polymerization
and depolymerization cycles. When the microtubule proteins are separated
into the tubulin and microtubule-associated protein fractions, BPDK is
found exclusively in the latter fraction. BPDK phosphorylates both tau and
MAP2, the former protein being phosphorylated to a stoichiometry of 3.8
mol of phosphate/mol of tau. Analysis of the phosphopeptides isolated from
the tryptic digest of the phosphorylated bovine tau has revealed seven
phosphorylation sites. Based on the sequence alignment between bovine and
human tau proteins, these sites correspond to Ser-195, Ser-202, Thr-205,
Thr-231, Ser-235, Ser-396, and Ser-404 of human tau. Mass spectrometric
analysis of the tau protein isolated from Alzheimer's paired helical
filaments (PHFs) has determined three abnormal phosphorylation sites and
two phosphopeptides containing a total of five abnormal phosphates
(Hasegawa, M., Morishima-Kawashima, M., Takio, K., Suzuki, M., Titani, K.,
and Ihara, Y. (1992) J. Biol. Chem. 267, 17047-17054). Two of the sites in
tau phosphorylated by BPDK, Thr-231 and Ser-235, are among the abnormal
phosphorylation sites, and the other sites phosphorylated by BPDK are
within phosphopeptides from PHF-tau. These results suggest that BPDK may
be one of the kinases responsible for the abnormal
phosphorylation-associated PHF-tau.
SO - J Biol Chem 1993 Nov 5;268(31):23512-8.

PIR:QRBOT1
FT - binding site | phosphate (Thr) (covalent) (by proline-directed kinase) | 212 (all)
TI - Brain proline-directed protein kinase phosphorylates tau on sites that are
abnormally phosphorylated in tau associated with Alzheimer's paired
helical filaments.
AB - Brain proline-directed protein kinase (BPDK), which contains a catalytic
subunit homologous to and displaying site-specific phosphorylation similar
to p34cdc2 kinase (Lew, J., Winkfein, R. J., Paudel, H. K., and Wang, J.
H. (1992) J. Biol. Chem. 267, 25922-25926), has been examined for possible
involvement in tau phosphorylation. Immunoblot analyses using peptide
antibodies specific for BPDK have revealed the presence of the kinase in
bovine brain microtubules purified extensively by repeated polymerization
and depolymerization cycles. When the microtubule proteins are separated
into the tubulin and microtubule-associated protein fractions, BPDK is
found exclusively in the latter fraction. BPDK phosphorylates both tau and
MAP2, the former protein being phosphorylated to a stoichiometry of 3.8
mol of phosphate/mol of tau. Analysis of the phosphopeptides isolated from
the tryptic digest of the phosphorylated bovine tau has revealed seven
phosphorylation sites. Based on the sequence alignment between bovine and
human tau proteins, these sites correspond to Ser-195, Ser-202, Thr-205,
Thr-231, Ser-235, Ser-396, and Ser-404 of human tau. Mass spectrometric
analysis of the tau protein isolated from Alzheimer's paired helical
filaments (PHFs) has determined three abnormal phosphorylation sites and
two phosphopeptides containing a total of five abnormal phosphates
(Hasegawa, M., Morishima-Kawashima, M., Takio, K., Suzuki, M., Titani, K.,
and Ihara, Y. (1992) J. Biol. Chem. 267, 17047-17054). Two of the sites in
tau phosphorylated by BPDK, Thr-231 and Ser-235, are among the abnormal
phosphorylation sites, and the other sites phosphorylated by BPDK are
within phosphopeptides from PHF-tau. These results suggest that BPDK may
be one of the kinases responsible for the abnormal
phosphorylation-associated PHF-tau.
SO - J Biol Chem 1993 Nov 5;268(31):23512-8.
full paper-page: 23516 The amino acid sequence of phosphopeptide 3 determined is shown in Table I and contains 3 phosphorylated residues. This peptide extends from residues 202 to 216 in the bovine tau sequence (46), and the phosphorylation sites are Ser-202, Ser-209, and Thr-212.

54. PMID:8300603
PIR:HSBO3
FT - binding site | phosphate (Arg) (covalent) | 2,128,129,131 (all)
TI - Ca(2+)-calmodulin-dependent phosphorylation of arginine in histone 3 by a
nuclear kinase from mouse leukemia cells.
AB - A Ca(2+)-calmodulin dependent histone 3 kinase was partially purified from
a low salt (150 mM NaCl) nuclear extract of mouse leukemia cells by
calmodulin-Sepharose affinity chromatography. In vitro, the kinase
activity transferred gamma-phosphate from ATP to histone 3 to form an
acid-labile and alkaline-stable linkage. Under the assay conditions 1.8
mol of phosphate are incorporated per mol of histone 3. Upon modification
of arginine residues with phenylglyoxal prior to phosphorylation, a
considerable decrease in the amount of phosphate transferred to histone 3
was observed. Amino acid analysis revealed that H3 was phosphorylated on
arginine residues. To identify the phosphorylated peptide(s), histone 3
was cleaved with cyanogen bromide prior to phosphorylation. The
phosphorylated mixture was then separated by gel filtration
high-performance liquid chromatography under denaturing conditions.
Fragments I (N-terminal 10.3-kDa peptide) and III (C-terminal 1.7-kDa
peptide) were both phosphorylated. Amino acid sequencing further revealed
that the molar yields of 3 of the 4 arginines present in the
phosphorylated cyanogen bromide fragment III were reduced by a factor of
about 10 compared with the corresponding arginines from the
unphosphorylated fragment. In the case of fragment I, 25 cycles of Edman
degradation revealed that the recovery of only arginine 2 was reduced by a
factor of 20. The putative phosphorylation sites are arginines 2, 128,
129, and 131. The sequence information offered an indirect evidence that
these arginines were the sites of phosphorylation. The kinase described in
this report represents a first member of a potentially important new class
of kinases which are Ca(2+)-calmodulin dependent and which phosphorylate
arginine.
SO - J Biol Chem 1994 Jan 28;269(4):2722-7.

55. PMID:8376365
PIR:A40936
FT - binding site | phosphate (Ser) (covalent) (by protein kinase A and calmodulin-dependent kinase) | 16 (all)
TI - Multiple phosphorylation of stathmin. Identification of four sites
phosphorylated in intact cells and in vitro by cyclic AMP-dependent
protein kinase and p34cdc2.
AB - Stathmin is a ubiquitous, highly conserved phosphoprotein which most
likely acts as a relay integrating various intracellular pathways
regulating cell proliferation, differentiation, and functions. At least 14
molecular forms of stathmin have been identified so far, which migrate as
2 unphosphorylated and 12 increasingly phosphorylated spots (M(r) =
19,000-23,000; pI = 6.2-5.6) on two-dimensional electrophoretic gels, and
whose pattern may reflect the state of activation of cells. We found that
stathmin could be phosphorylated in vitro by at least three different
protein kinases: cAMP-dependent protein kinase, p34cdc2, and casein kinase
II, cAMP-dependent protein kinase catalyzed the phosphorylation of
stathmin on serines 16 (K-R-A-S) and 63 (R-R-K-S), whereas p34cdc2 induced
phosphorylation on serines 25 (I-L-S-P-R) and 38 (P-L-S-P-P-K-K-K).
Interestingly, phosphorylation by both kinases together yielded all of the
phosphoforms of stathmin identified so far. Two-dimensional phosphopeptide
analysis allowed us to demonstrate that the same four sites were
exclusively found to be phosphorylated in vivo, in brain tissue as well as
in control or nerve growth factor-stimulated PC12 cells. In this latter
case, the major site phosphorylated in response to nerve growth factor
being serine 25, it is likely that a kinase such as a mitogen-activated
protein kinase, known to be activated by growth factors, might directly
phosphorylate stathmin. The phosphopeptide map analysis allowed further
identification of the specific combinations among the four sites whose
phosphorylation is responsible for the characteristic two-dimensional
polyacrylamide gel electrophoresis migration of the resulting stathmin
forms both in vitro and in vivo and revealed the existence of likely
structural interactions between the sites phosphorylated. In conclusion,
our results show that phosphorylation of serines 16, 25, 38, and 63
accounts for all of the major functional stathmin forms observed in vivo.
The present identification of these sites will foster a better
understanding of some intracellular mechanisms involved in the diverse
physiological regulation of the proliferation, differentiation, and
functions of cells, including the role of stathmin in these processes as a
relay integrating diverse signaling pathways.
SO - J Biol Chem 1993 Sep 25;268(27):20076-84.

PIR:A40936
FT - binding site | phosphate (Ser) (covalent) (by protein kinase A) | 63 (all)
TI - Multiple phosphorylation of stathmin. Identification of four sites
phosphorylated in intact cells and in vitro by cyclic AMP-dependent
protein kinase and p34cdc2.
AB - Stathmin is a ubiquitous, highly conserved phosphoprotein which most
likely acts as a relay integrating various intracellular pathways
regulating cell proliferation, differentiation, and functions. At least 14
molecular forms of stathmin have been identified so far, which migrate as
2 unphosphorylated and 12 increasingly phosphorylated spots (M(r) =
19,000-23,000; pI = 6.2-5.6) on two-dimensional electrophoretic gels, and
whose pattern may reflect the state of activation of cells. We found that
stathmin could be phosphorylated in vitro by at least three different
protein kinases: cAMP-dependent protein kinase, p34cdc2, and casein kinase
II, cAMP-dependent protein kinase catalyzed the phosphorylation of
stathmin on serines 16 (K-R-A-S) and 63 (R-R-K-S), whereas p34cdc2 induced
phosphorylation on serines 25 (I-L-S-P-R) and 38 (P-L-S-P-P-K-K-K).
Interestingly, phosphorylation by both kinases together yielded all of the
phosphoforms of stathmin identified so far. Two-dimensional phosphopeptide
analysis allowed us to demonstrate that the same four sites were
exclusively found to be phosphorylated in vivo, in brain tissue as well as
in control or nerve growth factor-stimulated PC12 cells. In this latter
case, the major site phosphorylated in response to nerve growth factor
being serine 25, it is likely that a kinase such as a mitogen-activated
protein kinase, known to be activated by growth factors, might directly
phosphorylate stathmin. The phosphopeptide map analysis allowed further
identification of the specific combinations among the four sites whose
phosphorylation is responsible for the characteristic two-dimensional
polyacrylamide gel electrophoresis migration of the resulting stathmin
forms both in vitro and in vivo and revealed the existence of likely
structural interactions between the sites phosphorylated. In conclusion,
our results show that phosphorylation of serines 16, 25, 38, and 63
accounts for all of the major functional stathmin forms observed in vivo.
The present identification of these sites will foster a better
understanding of some intracellular mechanisms involved in the diverse
physiological regulation of the proliferation, differentiation, and
functions of cells, including the role of stathmin in these processes as a
relay integrating diverse signaling pathways.
SO - J Biol Chem 1993 Sep 25;268(27):20076-84.

PIR:A40936
FT - binding site | phosphate (Ser) (covalent) (by MAP and cdc2 kinases) | 25 (all)
TI - Multiple phosphorylation of stathmin. Identification of four sites
phosphorylated in intact cells and in vitro by cyclic AMP-dependent
protein kinase and p34cdc2.
AB - Stathmin is a ubiquitous, highly conserved phosphoprotein which most
likely acts as a relay integrating various intracellular pathways
regulating cell proliferation, differentiation, and functions. At least 14
molecular forms of stathmin have been identified so far, which migrate as
2 unphosphorylated and 12 increasingly phosphorylated spots (M(r) =
19,000-23,000; pI = 6.2-5.6) on two-dimensional electrophoretic gels, and
whose pattern may reflect the state of activation of cells. We found that
stathmin could be phosphorylated in vitro by at least three different
protein kinases: cAMP-dependent protein kinase, p34cdc2, and casein kinase
II, cAMP-dependent protein kinase catalyzed the phosphorylation of
stathmin on serines 16 (K-R-A-S) and 63 (R-R-K-S), whereas p34cdc2 induced
phosphorylation on serines 25 (I-L-S-P-R) and 38 (P-L-S-P-P-K-K-K).
Interestingly, phosphorylation by both kinases together yielded all of the
phosphoforms of stathmin identified so far. Two-dimensional phosphopeptide
analysis allowed us to demonstrate that the same four sites were
exclusively found to be phosphorylated in vivo, in brain tissue as well as
in control or nerve growth factor-stimulated PC12 cells. In this latter
case, the major site phosphorylated in response to nerve growth factor
being serine 25, it is likely that a kinase such as a mitogen-activated
protein kinase, known to be activated by growth factors, might directly
phosphorylate stathmin. The phosphopeptide map analysis allowed further
identification of the specific combinations among the four sites whose
phosphorylation is responsible for the characteristic two-dimensional
polyacrylamide gel electrophoresis migration of the resulting stathmin
forms both in vitro and in vivo and revealed the existence of likely
structural interactions between the sites phosphorylated. In conclusion,
our results show that phosphorylation of serines 16, 25, 38, and 63
accounts for all of the major functional stathmin forms observed in vivo.
The present identification of these sites will foster a better
understanding of some intracellular mechanisms involved in the diverse
physiological regulation of the proliferation, differentiation, and
functions of cells, including the role of stathmin in these processes as a
relay integrating diverse signaling pathways.
SO - J Biol Chem 1993 Sep 25;268(27):20076-84.

PIR:A40936
FT - binding site | phosphate (Ser) (covalent) (by cdc2 kinase) | 38 (all)
TI - Multiple phosphorylation of stathmin. Identification of four sites
phosphorylated in intact cells and in vitro by cyclic AMP-dependent
protein kinase and p34cdc2.
AB - Stathmin is a ubiquitous, highly conserved phosphoprotein which most
likely acts as a relay integrating various intracellular pathways
regulating cell proliferation, differentiation, and functions. At least 14
molecular forms of stathmin have been identified so far, which migrate as
2 unphosphorylated and 12 increasingly phosphorylated spots (M(r) =
19,000-23,000; pI = 6.2-5.6) on two-dimensional electrophoretic gels, and
whose pattern may reflect the state of activation of cells. We found that
stathmin could be phosphorylated in vitro by at least three different
protein kinases: cAMP-dependent protein kinase, p34cdc2, and casein kinase
II, cAMP-dependent protein kinase catalyzed the phosphorylation of
stathmin on serines 16 (K-R-A-S) and 63 (R-R-K-S), whereas p34cdc2 induced
phosphorylation on serines 25 (I-L-S-P-R) and 38 (P-L-S-P-P-K-K-K).
Interestingly, phosphorylation by both kinases together yielded all of the
phosphoforms of stathmin identified so far. Two-dimensional phosphopeptide
analysis allowed us to demonstrate that the same four sites were
exclusively found to be phosphorylated in vivo, in brain tissue as well as
in control or nerve growth factor-stimulated PC12 cells. In this latter
case, the major site phosphorylated in response to nerve growth factor
being serine 25, it is likely that a kinase such as a mitogen-activated
protein kinase, known to be activated by growth factors, might directly
phosphorylate stathmin. The phosphopeptide map analysis allowed further
identification of the specific combinations among the four sites whose
phosphorylation is responsible for the characteristic two-dimensional
polyacrylamide gel electrophoresis migration of the resulting stathmin
forms both in vitro and in vivo and revealed the existence of likely
structural interactions between the sites phosphorylated. In conclusion,
our results show that phosphorylation of serines 16, 25, 38, and 63
accounts for all of the major functional stathmin forms observed in vivo.
The present identification of these sites will foster a better
understanding of some intracellular mechanisms involved in the diverse
physiological regulation of the proliferation, differentiation, and
functions of cells, including the role of stathmin in these processes as a
relay integrating diverse signaling pathways.
SO - J Biol Chem 1993 Sep 25;268(27):20076-84.

56. PMID:838735
PIR:SBHUP
FT - binding site | phosphate (Ser) (covalent) | 21,22 (all)
TI - Complete covalent structure of statherin, a tyrosine-rich acidic peptide
which inhibits calcium phosphate precipitation from human parotid saliva.
AB - The complete amino acid sequence of human salivary statherin, a peptide
which strongly inhibits precipitation from supersaturated calcium
phosphate solutions, and therefore stabilizes supersaturated saliva, has
been determined. The NH2-terminal half of this Mr=5380 (43 amino acids)
polypeptide was determined by automated Edman degradations (liquid phase)
on native statherin. The peptide was digested separately with trypsin,
chymotrypsin, and Staphylococcus aureus protease, and the resulting
peptides were purified by gel filtration. Manual Edman degradations on
purified peptide fragments yielded peptides that completed the amino acid
sequence through the penultimate COOH-terminal residue. These analyses,
together with carboxypeptidase digestion of native statherin and of
peptide fragments of statherin, established the complete sequence of the
molecule. The 2 serine residues (positions 2 and 3) in statherin were
identified as phosphoserine. The amino acid sequence of human salivary
statherin is striking in a number of ways. The NH2-terminal one-third is
highly polar and includes three polar dipeptides:
H2PO3-Ser-Ser-H2PO3-Arg-Arg-, and Glu-Glu-. The COOH-terminal two-thirds
of the molecule is hydrophobic, containing several repeating dipeptides:
four of -Gn-Pro-, three of -Tyr-Gln-, two of -Gly-Tyr-, two of-Gln-Tyr-,
and two of the tetrapeptide sequence -Pro-Tyr-Gln-Pro-. Unusual cleavage
sites in the statherin sequence obtained with chymotrypsin and S. aureus
protease were also noted.
SO - J Biol Chem 1977 Mar 10;252(5):1689-95.

57. PMID:8491187
PIR:TPHUN1
FT - binding site | phosphate (Ser) (covalent) (by protein kinase C) | 378 (all)
TI - Multi-site phosphorylation of the protein tyrosine phosphatase, PTP1B:
identification of cell cycle regulated and phorbol ester stimulated sites
of phosphorylation.
AB - The non-transmembrane protein tyrosine phosphatase, PTP1B, comprises 435
amino acids, of which the C-terminal 114 residues have been implicated in
controlling both localization and function of this enzyme. Inspection of
the sequence of the C-terminal segment reveals a number of potential sites
of phosphorylation. We show that PTP1B is phosphorylated on seryl residues
in vivo. Increased phosphorylation of PTP1B is seen to accompany the
transition from G2 to M phase of the cell cycle. Two major tryptic
phosphopeptides appear in two-dimensional maps of PTP1B from mitotic
cells. One of these comigrates with the peptide generated following
phosphorylation of PTP1B in vitro at Ser386 by the mitotic protein Ser/Thr
kinase p34cdc2:cyclin B. The site of phosphorylation that is responsible
for the pronounced retardation in the electrophoretic mobility of PTP1B
from mitotic cells has been identified by site directed mutagenesis as
Ser352. The identify of the kinase responsible for this modification is
presently unknown. We also show that stimulation of HeLa cells with the
phorbol ester TPA enhances phosphorylation of PTP1B. Two dimensional
phosphopeptide mapping reveals that the bulk of the phosphate is in a
single tryptic peptide. The site, identified as Ser378, is also the site
of phosphorylation by protein kinase C (PKC) in vitro. Thus the
TPA-stimulated phosphorylation of PTP1B in vivo appears to result directly
from phosphorylation by PKC. The effect of phosphorylation on the activity
of PTP1B has been examined in immunoprecipitates from TPA-treated and
nocodazole-arrested cells. TPA treatment does not appear to affect
activity directly, whereas the activity of PTP1B from nocodazole-arrested
cells is only 70% of that from asynchronous populations.
SO - EMBO J 1993 May;12(5):1937-46.

PIR:TPHUN1
FT - binding site | phosphate (Ser) (covalent) (by cdc2 kinase) | 386 (all)
TI - Multi-site phosphorylation of the protein tyrosine phosphatase, PTP1B:
identification of cell cycle regulated and phorbol ester stimulated sites
of phosphorylation.
AB - The non-transmembrane protein tyrosine phosphatase, PTP1B, comprises 435
amino acids, of which the C-terminal 114 residues have been implicated in
controlling both localization and function of this enzyme. Inspection of
the sequence of the C-terminal segment reveals a number of potential sites
of phosphorylation. We show that PTP1B is phosphorylated on seryl residues
in vivo. Increased phosphorylation of PTP1B is seen to accompany the
transition from G2 to M phase of the cell cycle. Two major tryptic
phosphopeptides appear in two-dimensional maps of PTP1B from mitotic
cells. One of these comigrates with the peptide generated following
phosphorylation of PTP1B in vitro at Ser386 by the mitotic protein Ser/Thr
kinase p34cdc2:cyclin B. The site of phosphorylation that is responsible
for the pronounced retardation in the electrophoretic mobility of PTP1B
from mitotic cells has been identified by site directed mutagenesis as
Ser352. The identify of the kinase responsible for this modification is
presently unknown. We also show that stimulation of HeLa cells with the
phorbol ester TPA enhances phosphorylation of PTP1B. Two dimensional
phosphopeptide mapping reveals that the bulk of the phosphate is in a
single tryptic peptide. The site, identified as Ser378, is also the site
of phosphorylation by protein kinase C (PKC) in vitro. Thus the
TPA-stimulated phosphorylation of PTP1B in vivo appears to result directly
from phosphorylation by PKC. The effect of phosphorylation on the activity
of PTP1B has been examined in immunoprecipitates from TPA-treated and
nocodazole-arrested cells. TPA treatment does not appear to affect
activity directly, whereas the activity of PTP1B from nocodazole-arrested
cells is only 70% of that from asynchronous populations.
SO - EMBO J 1993 May;12(5):1937-46.

PIR:TPHUN1
FT - binding site | phosphate (Ser) (covalent) (by unidentified kinase) | 352 (all)
TI - Multi-site phosphorylation of the protein tyrosine phosphatase, PTP1B:
identification of cell cycle regulated and phorbol ester stimulated sites
of phosphorylation.
AB - The non-transmembrane protein tyrosine phosphatase, PTP1B, comprises 435
amino acids, of which the C-terminal 114 residues have been implicated in
controlling both localization and function of this enzyme. Inspection of
the sequence of the C-terminal segment reveals a number of potential sites
of phosphorylation. We show that PTP1B is phosphorylated on seryl residues
in vivo. Increased phosphorylation of PTP1B is seen to accompany the
transition from G2 to M phase of the cell cycle. Two major tryptic
phosphopeptides appear in two-dimensional maps of PTP1B from mitotic
cells. One of these comigrates with the peptide generated following
phosphorylation of PTP1B in vitro at Ser386 by the mitotic protein Ser/Thr
kinase p34cdc2:cyclin B. The site of phosphorylation that is responsible
for the pronounced retardation in the electrophoretic mobility of PTP1B
from mitotic cells has been identified by site directed mutagenesis as
Ser352. The identify of the kinase responsible for this modification is
presently unknown. We also show that stimulation of HeLa cells with the
phorbol ester TPA enhances phosphorylation of PTP1B. Two dimensional
phosphopeptide mapping reveals that the bulk of the phosphate is in a
single tryptic peptide. The site, identified as Ser378, is also the site
of phosphorylation by protein kinase C (PKC) in vitro. Thus the
TPA-stimulated phosphorylation of PTP1B in vivo appears to result directly
from phosphorylation by PKC. The effect of phosphorylation on the activity
of PTP1B has been examined in immunoprecipitates from TPA-treated and
nocodazole-arrested cells. TPA treatment does not appear to affect
activity directly, whereas the activity of PTP1B from nocodazole-arrested
cells is only 70% of that from asynchronous populations.
SO - EMBO J 1993 May;12(5):1937-46.

58. PMID:8524294
PIR:T45138
FT - binding site | phosphate (Ser) (covalent) | 335 (all)
TI - Schizosaccharomyces pombe skp1+ encodes a protein kinase related to
mammalian glycogen synthase kinase 3 and complements a cdc14 cytokinesis
mutant.
AB - We report the cloning of the skp1+ gene, a Schizosaccharomyces pombe
homolog of the glycogen synthase kinase 3 (GSK-3) family whose members in
higher eukaryotes are involved in cell fate determination, nuclear
signalling, and hormonal regulation. skp1 is 67% identical to mammalian
GSK-3 beta and displays similar biochemical properties in vitro. Like
GSK-3 beta, skp1 is phosphorylated on a conserved tyrosine residue, and
this phosphorylation is required for efficient activity. skp1 is also
phosphorylated at a serine which has been identified as S-335.
Phosphorylation at this site is likely to inhibit its function. Unlike the
mammalian enzyme, skp1 both tyrosine autophosphorylates in yeast cells and
can phosphorylate other proteins on tyrosine in bacteria. The skp1+ gene
is not essential. However, cells with deletions in skp1+ are sensitive to
heat shock and exhibit defects in sporulation. Overexpression of wild-type
skp1+ specifically complements cdc14-118, one of several mutations causing
a defect in cytokinesis. In addition, certain phosphorylation site mutants
induce a delay or block in cytokinesis when overexpressed. Together, these
data identify novel interactions of a fission yeast GSK-3 homolog with
elements of the cytokinesis machinery.
SO - Mol Cell Biol 1996 Jan;16(1):179-91.

59. PMID:8635594
PIR:C1HURB
FT - binding site | phosphate (Ser) (covalent) (by casein kinase II) | 206 (all)
TI - Identification of a cryptic protein kinase CK2 phosphorylation site in
human complement protease Clr, and its use to probe intramolecular
interaction.
AB - Treatment of human (activated)C1r by CK2 resulted in the incorporation of
[32P]phosphate into the N-terminal alpha region of its non-catalytic A
chain. Fragmentation of 32P-labelled (activated)C1r followed by N-terminal
sequence and mass spectrometry analyses allowed identification of Ser189
as the phosphorylation site. Accessibility of Ser189 was low in intact
C1r, due in part to the presence of one of the oligosaccharides borne by
the alpha region, further reduced in the presence of calcium, and
abolished when C1r was incorporated into the C1s-C1r-C1r-C1s tetramer or
the C1 complex. In contrast, phosphorylation was enhanced in the isolated
alpha fragment and insensitive to calcium. Taken together, these data
provide support for the occurrence of a (Ca2+)-dependent interaction
between the alpha region and the remainder of the C1r molecule.
SO - FEBS Lett 1996 May 13;386(1):15-20.