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PubMed1993

Multi-site phosphorylation of the protein tyrosine phosphatase, PTP1B: identification of cell cycle regulated and phorbol ester stimulated sites of phosphorylation.

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.

Alanine↗
PubMed2001

Identification of a novel phosphorylation motif for CDPKs: phosphorylation of synthetic peptides lacking basic residues at P-3/P-4.

The Ca(2+)-dependent protein kinases (CDPKs) are members of a large subfamily of protein kinases in plants that have been implicated in the control of numerous aspects of plant growth and development. One known substrate of the CDPKs is the ER-located ACA2 calcium pump, which is regulated by phosphorylation of Ser(45). In the present study, a synthetic peptide based on the known regulatory phosphorylation site (RRFRFTANLS(45)KRYEA) was efficiently phosphorylated in vitro by CDPKs but not a plant SNF1-related protein kinase. Phosphorylation of the Ser(45)-ACA2 peptide was surprising because the sequence lacks basic residues at P-3/P-4 (relative to the phosphorylated Ser at position P) that are considered to be essential recognition elements for CDPKs. We demonstrate that phosphorylation of the Ser(45)-ACA2 peptide is dependent on the cluster of basic residues found N-terminal (P-6 to P-9) as well as C-terminal (P + 1/P + 2) to the phosphorylated Ser. The results establish a new general phosphorylation motif for CDPKs: [Basic-Basic-X-Basic]-phi-X(4)-S/T-X-Basic (where phi is a hydrophobic residue). The motif predicts a number of new phosphorylation sites in plant proteins. Evidence is presented that the novel motif may explain the phosphorylation by CDPKs of Ser271 in the aquaporin PM28A.

Amino Acid Motifs↗
PubMed2001

Frap-dependent serine phosphorylation of IRS-1 inhibits IRS-1 tyrosine phosphorylation.

We have previously shown that interferon-alpha (IFN alpha)-dependent tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1) is impaired by serine phosphorylation of IRS-1 due to the reduced ability of serine phosphorylated IRS-1 to serve as a substrate for Janus kinase 1 (JAK1). Here we report that FKBP12-rapamycin-associated protein (FRAP) is a physiologic IRS-1 kinase that blocks IFN alpha signaling by serine phosphorylating IRS-1. We found that both FRAP and insulin-activated p70 S6 kinase (p70(s6k)) serine phosphorylated IRS-1 between residues 511 and 772 (IRS-1(511-772)). Importantly, only FRAP-dependent IRS-1(511-772) serine phosphorylation inhibited by 50% subsequent JAK1-dependent tyrosine phosphorylation of IRS-1. Furthermore, treatment of U266 cells with the FRAP inhibitor rapamycin increased IFN alpha-dependent tyrosine phosphorylation by twofold while reducing constitutive IRS-1 serine phosphorylation within S/T-P motifs by 80%. Taken together, these data indicate that FRAP, but not p70(s6k), is a likely physiologic IRS-1 serine kinase that negatively regulates JAK1-dependent IRS-1 tyrosine phosphorylation and suggests that FRAP may modulate IRS-dependent cytokine signaling.

Amino Acid Sequence↗
PubMed1994

In vivo phosphorylation of 2',3'-cyclic nucleotide 3'-phosphohydrolase (CNP): CNP in brain myelin is phosphorylated by forskolin- and phorbol ester-sensitive protein kinases.

2',3'-cyclic nucleotide 3'-phosphohydrolase (CNP) was phosphorylated in vivo, in brain slices and in a cell free system. Phosphoamino acid analysis of immunoprecipitated CNP labeled in vivo and in brain slices revealed phosphorylation of phosphoserine (94%) and phosphothreonine (5%) residues. Phosphorylation of CNP increased by 3-fold after brain slices were incubated with forskolin. Similarly, incubation of isolated myelin with [gamma-32]ATP with cAMP (5 microM) and cAMP (5 microM)+catalytic unit of cAMP dependent protein kinase dramatically increased CNP2 phosphorylation by 4- and 6-fold, respectively. It is feasible that CNP2 was predominantly phosphorylated on serine and/or threonine residues of the amino terminal peptide of CNP2, and this phosphorylation was catalyzed by protein kinase A. Phosphorylation of CNP1 and CNP2 increased 2-fold by incubating brain slices with phorbol ester. Forskolin and phorbol ester increased the phosphorylation of single, but distinct, CNP peptides. We present the first biochemical evidence that CNP2, on a protein mass basis, is far more heavily phosphorylated than CNP1, suggesting there are more phosphorylation sites on CNP2 than CNP1 and that at least one site is located on the 20-amino acid terminus of CNP2 and that it is likely a PKA site.

2',3'-Cyclic-Nucleotide Phosphodiesterases↗
PubMed1989

In vitro phosphorylation of phosphatidylethanolamine N-methyltransferase by cAMP-dependent protein kinase: lack of in vivo phosphorylation in response to N6-2'-O-dibutryladenosine 3',5'-cyclic monophosphate.

Phosphorylation of rat liver phosphatidylethanolamine (PE) N-methyltransferase by cAMP-dependent protein kinase was investigated. The 18 kDa methyltransferase was found to be phosphorylated in vitro by cAMP-dependent protein kinase on a serine residue. The stoichiometry of phosphate incorporation reached a maximum of 0.25 mol phosphate/mol methyltransferase at 30 min. Resolution of the phosphorylated methyltransferase by two-dimensional gel electrophoresis showed that two isoproteins were substrates. Phosphorylation of the purified PE N-methyltransferase for up to 1 h had no effect on the methylation of PE, PMME or PDME. To test for in vivo phosphorylation, isolated rate hepatocytes were exposed to 0.5 mM N6-2'-O-dibutryladenosine 3':5'-cyclic monophosphate (DiB-cAMP) and the phosphorylation state of microsomal proteins evaluated by two-dimensional gel electrophoresis, nitrocellulose blotting and autoradiography. The same nitrocellulose blots were probed with a rabbit anti-PE N-methyltransferase antibody, immunochemically stained and aligned with the autoradiogram. No phosphorylated proteins co-migrated with the methyltransferase under non-phosphorylating conditions, or when hepatocytes were exposed to the cAMP analogue for up to 2 h. Oddly, DiB-cAMP increased both PE- and PMME-dependent activity in isolated microsomes, but decreased PE to PC conversion measured in intact hepatocytes. The results indicated that PE N-methyltransferase is poorly phosphorylated by cAMP-dependent protein kinase in vitro, and is not phosphorylated in intact hepatocytes treated with a cAMP analogue.

Animals↗
PubMed1987

Protein phosphorylation in rat mast cell granules. Cyclic AMP dependent phosphorylation of a 44K protein associated with broken granules.

When rat mast cells are prelabeled with 32PO4 and exposed to non-immunologic or immunologic stimuli under conditions that lead to mediator release from granules, they show rapid increases in labeling of a number of high molecular weight proteins. To determine if granule membrane proteins are subject to protein phosphorylation and perhaps participate in this response, granules with intact or broken membranes were isolated from sonicated, purified rat serosal mast cells on a Percoll gradient. When the granules with broken membranes were incubated with [gamma-32P]ATP and Mg2+ in the absence of exogenous protein kinases, one major radioactive band was recovered in the 44K area after electrophoresis in sodium dodecyl sulfate/polyacrylamide gels. The phosphorylation reaction with ATP required Mg2+, was enhanced by 0.05 to 0.5 microM cyclic AMP, and was inhibited by Ca2+ (0.5 mM and higher). The initial reaction was rapid, and the maximal response was seen at 30 degrees. The 44K band was absent in granules with intact membranes incubated with [gamma-32P]ATP but present when intact granules were lysed with distilled water before adding the [gamma-32P]ATP. These observations indicate that granules have an endogenous phosphorylating system and that the phosphorylation response is on the inner surface of the granule membranes. The possibility was not excluded that a portion of the phosphorylating activity was derived from the cytosol and became firmly associated with broken granules when the intact cells were sonicated. Analysis for possible phosphorylated amino acids in the 44K band after acid hydrolysis showed both phosphoserine and phosphothreonine, indicating that the radioactivity was in a phosphorylated protein or glycoprotein. The 44K phosphorylated protein was made up of several components ranging in pI from approximately 7.6 to 6.6. While the identity of the phosphorylated 44K polypeptide is uncertain, one important possibility is that it is part of an autophosphorylated cAMP dependent protein kinase. The cyclic AMP dependent phosphorylating activity present in granules provides a mechanism by which the granules might respond rapidly to cyclic AMP during mediator release.

Animals↗
PubMed1990

Phosphofructokinase from Dirofilaria immitis: effect of fructose 2,6-bisphosphate and AMP on the non-phosphorylated and phosphorylated forms of the enzyme.

The enzyme responsible for the synthesis of fructose 2,6-bisphosphate (Fru-2,6-P2), 6-phosphofructo-2-kinase, was shown to be present in the heart worm, Dirofilaria immitis. The level of Fru-2,6-P2 was determined to be 4 +/- 0.3 nmol(g wet weight)-1 in the tissues of the filariid. Fru-2,6-P2 stimulated the activity of both the non-phosphorylated and phosphorylated forms of the D. immitis phosphofructokinase (PFK). The Kact values for Fru-2,6-P2 were 378 +/- 18 nM and 65 +/- 6 nM for the non-phosphorylated and phosphorylated forms, respectively, at 1 mM fructose 6-phosphate (Fru-6-P) and 1 mM ATP at pH 6.8. AMP also stimulated the activity of both forms of the enzyme with Kact values of 230 +/- 10 microM and 37.3 +/- 6.1 microM for the non-phosphorylated and phosphorylated forms, respectively. In the absence of any effectors, the S0.5 values for Fru-6-P were 17.4 mM and 11.0 mM for the non-phosphorylated and phosphorylated forms, respectively, of the D. immitis PFK at 1 mM ATP, pH 6.8. These S0.5 values were lowered to 0.03 mM by the combined effects of saturating levels of Fru-2,6-P2 and AMP. A physiological assay was developed based on the level of metabolites in the parasite that influence the activity of PFK. This assay contained the known effectors of the PFK at concentrations approximating those found in the parasite. Under these conditions the KFru-6-P values were 153 microM and 60 microM for the non-phosphorylated and phosphorylated forms of the PFK, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Monophosphate↗
PubMed1996

Solid phase synthesis of pp60src-related phosphopeptides via 'global' phosphorylation and their use as substrates for enzymatic phosphorylation by casein kinase-2.

The seven phosphopeptide derivatives based on the native -NEYTA- sequence of the pp60src protein kinase family, Asn-Glu-Tyr(P)-Ser-Ala, Ala-Glu-Tyr(P)-Ser-Ala, Ala-Ser-Tyr(P)-Ser-Ala, Ala-Ser(P)-Tyr-Ser-Ala, Ala-Thr-Tyr(P)-Ser-Ala, Ala-Thr(P)-Tyr-Ser-Ala and Ala-Ser(P)-Tyr(P)-Ser-Ala, were prepared in good yield using the "global' "phosphite-triester' phosphorylation method. The peptide resins were assembled using the Fmoc mode of solid phase peptide synthesis (PyBOP coupling method) with specific Ser-, Thr-, or Tyr-residues incorporated as their side chain free Fmoc-derivatives. The final "global' phosphorylation of the peptide resins was accomplished using di-tert-butyl N, N-diethylphosphoramidite followed by m-chloroperoxybenzoic acid oxidation of the resultant di-t-butyl phosphite triester intermediate. Subsequent resin cleavage and deprotection of the phosphorylated peptide resins was effected by treatment with 5% anisole: TFA and gave the seven phosphopeptides in high yield and purity. The use of the seven synthetic phosphopeptides in enzymatic (casein kinase-2) phosphorylation studies showed that, (A) the change of the target Thr site to Ser resulted in markedly improved phosphorylation of the peptide substrates, (B) that the Tyr(P) residue in the - 1 position was significantly more important than the Ser(P)/Thr(P) residue in the - 2 position for efficient seryl phosphorylation, and (C) that an acidic residue in the - 2 position relative to the target site facilitated phosphorylation of the downstream seryl residue irrespective of the nature of the acidic residue in the -Xxx-Tyr(P)-Ser- and -Xxx-Tyr-Ser- sequences {Xxx = Ser(P), Thr(P), Glu}. In addition to the Tyr(P) residue directing phosphorylation to the +1 position, the good phosphorylation of both ASY(P)SA and ATY(P)SA by casein kinase-2 indicated that the Tyr(P) residue was also able to direct phosphorylation to a Ser/Thr in the - 1 position.

Amino Acid Sequence↗
PubMed1998

Tau proteins-enhanced Ca2+/calmodulin (CaM)-dependent phosphorylation by the brain supernatant of diisopropyl phosphorofluoridate (DFP)-treated hen: tau mutants indicate phosphorylation of more amino acids in tau by CaM kinase II.

Diisopropyl phosphorofluoridate (DFP) produces organophosphorus ester-induced delayed neurotoxicity (OPIDN) in hen, human, and other sensitive species. A single dose of DFP (1.7 mg/kg, s.c.) produces mild ataxia in 7-14 days in hens, followed by progression to severe ataxia or paralysis. We studied the effect of DFP administration on Ca2+/calmodulin-dependent phosphorylation of tau proteins by the brain supernatants of control and DFP-treated hens. Brain supernatants from DFP-treated hens showed enhanced in vitro phosphorylation of htau40 and its various mutants, but no change in the two-dimensional phosphopeptide pattern, when compared to control hen brain supernatants. Analysis of tau mutants phosphorylated by brain supernatant and recombinant CaM kinase II alpha-subunit showed that (1) brain supernatant CaM kinase II is mainly responsible for the phosphorylation of Ser416, (2) Ser356, but probably not Ser262, is phosphorylated by CaM kinase II, (3) no amino acid between Lys395-Ala437 except Ser416 is phosphorylated by CaM kinase II, (4) a number of amino acids in the tau molecule, which are phosphorylated by the brain supernatant in the absence of Ca2+/calmodulin are also mildly phosphorylated by CaM kinase II. The enhanced Ca2+/calmodulin-dependent phosphorylation of tau proteins by brain supernatant of DFP-treated hens that includes phosphorylation of a number of amino acids is likely to alter the functional properties of tau proteins in OPIDN. The hyperphosphorylated tau may destabilize microtubules, alter axonal transport, and result in degeneration of axons in OPIDN.

Amino Acid Substitution↗
PubMed1998

Csk-mediated phosphorylation of substrates is regulated by substrate tyrosine phosphorylation.

Csk is a cellular protein tyrosine kinase (PTK) that has been shown to specifically regulate the activity of Src kinase family members by phosphorylation of a carboxy-terminal tyrosine residue. The molecular mechanisms controlling Csk regulation and its substrate specificity have not been elucidated. Here we report a novel type of overlay kinase assay that allows to probe for Csk-mediated phosphorylation of cellular substrates separated by polyacrylamide gel electrophoresis and transferred to nitrocellulose filters. Most of the cell lines analyzed with this method revealed only a few potential Csk substrates. However, an increased number of Csk substrates was detected in NIH3T3 cells expressing a constitutively activated form of the Src kinase Lck or in PC12 and NIH3T3 cells that had been treated with pervanadate. These cells all display an increased level of cellular protein tyrosine phosphorylation which led to the conclusion that Csk preferentially phosphorylates tyrosine-phosphorylated proteins. To verify this hypothesis we analyzed Csk-mediated phosphorylation of recombinant Lck, a known Csk substrate. Results demonstrated that autophosphorylation of Lck (at Tyr394) facilitates Csk-mediated phosphorylation of Lck at its regulatory site (Tyr505). Subsequent peptide binding studies revealed that Csk can bind to a peptide corresponding to the Lck-autophosphorylation site only when it is phosphorylated. These findings suggest that autophosphorylation of Lck at Tyr394 triggers an interaction with Csk and thereby facilitates subsequent phosphorylation and inactivation of Lck. The phosphorylation of other cellular Csk substrates may be regulated by a similar mechanism.

3T3 Cells↗
PubMed2001

Phosphorylation of the leucocyte NADPH oxidase subunit p47(phox) by casein kinase 2: conformation-dependent phosphorylation and modulation of oxidase activity.

The leucocyte NADPH oxidase of neutrophils is a membrane-bound enzyme that catalyses the reduction of oxygen to O(-)(2) at the expense of NADPH. The enzyme is dormant in resting neutrophils but becomes active when the cells are exposed to the appropriate stimuli. During oxidase activation, the highly basic cytosolic oxidase component p47(phox) becomes phosphorylated on several serines and migrates to the plasma membrane. Protein kinase CK2 is an essential serine/threonine kinase present in all eukaryotic organisms. The leucocyte NADPH oxidase subunit p47(phox) has several putative CK2 phosphorylation sites. In the present study, we report that CK2 is able to catalyse the phosphorylation of p47(phox) in vitro. Phosphoamino acid analysis of phosphorylated p47(phox) by CK2 indicated that the phosphorylation occurs on serine residues. CNBr mapping and phosphorylation of peptides containing the putative site of CK2 indicated that the main phosphorylated residues are Ser-208 and Ser-283 in the Src homology 3 (SH3) domains, and Ser-348 in the C-terminal domain of p47(phox). Dependence of phosphorylation on the conformation of p47(phox) is supported by the finding that p47(phox) undergoes better phosphorylation by CK2 in the presence of arachidonic acid, a known activator of NADPH oxidase which induces conformational changes in p47(phox). In addition, 5,6-dichloro-1-beta-o-ribofuranosyl benzimidazole, a CK2 inhibitor, potentiates formyl-Met-Leu-Phe-induced NADPH oxidase activity in DMSO-differentiated HL-60 cells. Taken together, we propose that CK2 is the p47(phox) kinase, and that phosphorylation of p47(phox) by CK2 regulates the deactivation of NADPH oxidase.

Amino Acid Sequence↗
PubMed1991

Analysis of insulin receptor phosphorylation sites in intact rat liver cells by two-dimensional phosphopeptide mapping. Predominance of the tris-phosphorylated form of the kinase domain after stimulation by insulin.

1. Insulin receptors were partially purified from rat liver by chromatography on wheat-germ-lectin-Sepharose. Incubation with [gamma-32P]ATP in the presence of insulin resulted in increased phosphorylation of the beta-subunit on both tyrosine and serine residues. Two-dimensional mapping of tryptic peptides showed that, in agreement with previous studies using preparations of receptors from other sources, the tyrosine residues involved were the three tyrosines in the kinase domain (corresponding to tyrosines 1158, 1162 and 1163 of the human receptor) plus two tyrosines close to the C-terminus (corresponding to tyrosines 1328 and 1334). 2. The effects of insulin on the phosphorylation of receptors within intact rat liver cells were determined by incubating cells in the presence of [32P]Pi for 50 min and then with or without insulin for a further 10 min. The labelled receptors were then rapidly isolated by sequential use of wheat-germ-lectin-Sepharose chromatography and immuno-isolation using a monoclonal antibody to the C-terminal end of the beta-subunit. 3. Insulin was found to increase overall phosphorylation of the receptor nearly 3-fold. Two-dimensional mapping was then carried out in combination with phosphoamino acid analysis. This revealed that the pattern of phosphorylation of the receptors in cells incubated in the absence and presence of insulin exhibited a number of marked differences from that observed in previous studies on intact cells, which had been restricted to cells expressing very high levels of insulin receptors such as certain hepatoma-derived cells or cells transfected with insulin receptor cDNA. The differences in the effects of insulin included a larger increase in the proportion of receptors being phosphorylated on the three tyrosine residues of the kinase domain, no apparent phosphorylation of the two tyrosine residues close to the C-terminus and no increase in either threonine or overall serine phosphorylation. 4. The receptors appeared to be phosphorylated on a number of different serine residues in cells incubated in the absence of insulin. Evidence for both increases and decreases in the phosphorylation of specific serine residues on addition of insulin was obtained. 5. It is concluded that care should be taken when extrapolating findings on the phosphorylation of the insulin receptor within cultured cells to more physiological situations.

Amino Acid Sequence↗
PubMed2002

Phosphorylation of the protein kinase C-theta activation loop and hydrophobic motif regulates its kinase activity, but only activation loop phosphorylation is critical to in vivo nuclear-factor-kappaB induction.

Protein kinase C (PKC)-theta, a member of the 'novel' subfamily of PKC isoforms, is of singular importance in transducing signals in T-lymphocytes. Since understanding of regulatory phosphorylation of novel PKCs is fragmentary and inconsistent with findings for 'classical' PKC isoforms, we investigated three potential phosphorylation sites on PKC-theta; in the activation loop (Thr(538)), turn motif (Ser(676)) and hydrophobic motif (Ser(695)). Combined evidence from phospho-specific antisera and MS demonstrates phosphorylation at all three sites. Unlike its closest paralogue, PKC-delta, lack of negative charge in the activation loop of PKC-theta results in a profound catalytic defect (>100-fold reduction in the T538A mutant); the high sequence similarity between PKC-theta and -delta assists in the formulation of structural hypotheses to account for this major difference. In contrast with mechanisms proposed for other PKC isoforms, phosphorylation at the other two sites does not reconstitute catalytic activity. Activation loop phosphorylation is critical in vivo, since the T538A mutant completely lost its capacity to mediate T-cell receptor-stimulation of nuclear factor kappaB (NF-kappaB) activation in Jurkat T-cells. Hydrophobic motif phosphorylation also substantially influences PKC-theta catalytic activity (5-fold reduction in the S695A mutant), but does not impair NF-kappaB activation in Jurkat T-cells. Its mechanism is independent of secondary effects on activation loop phosphorylation and cannot be explained by thermal instability. Turn motif phosphorylation has a limited effect on kinase activity, but negatively regulates other aspects of PKC-theta function, since the S676A mutant is more efficient than wild-type in inducing NF-kappaB activation in Jurkat T-cells. These findings expand our understanding of the roles of phosphorylation in novel PKCs, and indicate that PKC-theta is a constitutively competent kinase as a consequence of constitutive phosphorylation of its activation loop.

Amino Acid Motifs↗
PubMed1984

Hormonal regulation of hormone-sensitive lipase in intact adipocytes: identification of phosphorylated sites and effects on the phosphorylation by lipolytic hormones and insulin.

In isolated adipocytes, fast-acting lipolytic hormones and insulin have been shown previously to control lipolysis by regulating the activity of hormone-sensitive lipase, the rate-limiting enzyme, through an increase or decrease, respectively, of the extent of phosphorylation of the enzyme. Here, we demonstrate that exposure to lipolytic hormones (corticotropin, noradrenaline) led to phosphorylation at two sites on the Mr 84,000 lipase subunit. One, designated "basal site," was phosphorylated also in the absence of any hormonal stimulation, its phosphorylation apparently not being influenced by hormones. The second, designated "regulatory site," was identical to that phosphorylated by cyclic AMP-dependent protein kinase on the isolated lipase. The regulatory site was not appreciably phosphorylated in the absence of hormones, but exposure of the cells to noradrenaline increased its phosphorylation extent to that of the basal site. Insulin or the beta-adrenergic antagonist propranolol decreased the extent of phosphorylation of the regulatory site to the low level before stimulation, apparently without effect on the basal site. Phosphoserine was the only phosphorylated amino acid residue at both sites. Limited proteolytic digestion indicated that the two sites were separated by less than about 170 amino acid residues. Thus, control of adipose tissue lipolysis by fast-acting lipolytic hormones and by insulin is exerted through the regulation of the phosphorylation state of a single phosphoserine residue in the hormone-sensitive lipase.

Adipose Tissue↗
PubMed1989

Basic fibroblast growth factor is a substrate for protein phosphorylation and is phosphorylated by capillary endothelial cells in culture.

A phosphorylated basic fibroblast growth factor (FGF) can be detected in extracts of bovine capillary endothelial cells and human hepatoma cells. Accordingly, human basic FGF contains consensus sequences that account for its phosphorylation on Thr-112 by the catalytic subunit of the cAMP-dependent protein kinase A (PK-A) and on Ser-64 by the calcium- and phospholipid-dependent protein kinase C (PK-C). A kinetic analysis of both of these reactions revealed that basic FGF is among the better substrates for these enzymes. Although the kinase responsible for the phosphorylation in vivo has not yet been identified, we examined the effects of phosphorylation on the biological activity, heparin-binding capacity, and receptor-binding capacity of phosphorylated basic FGF. No effects of phosphorylation were observed when the mitogen was phosphorylated by PK-C. In contrast, when basic FGF was phosphorylated in the receptor-binding domain with PK-A, the growth factor was 3-8 times better at displacing radiolabeled basic FGF in the radioreceptor assay. No effects were seen on the binding of this FGF to immobilized heparin or cell-associated glycosaminoglycans, suggesting that this phosphorylation modifies the affinity of basic FGF for its receptor. Biological assays for basic FGF failed to identify differences between the phosphorylated and unphosphorylated forms of recombinant basic FGFs presumably because of the presence of ectophosphatases and the experimental conditions of proliferation and mitogenic assays (37 degrees C, 24-96 hr). Because the relative affinity of basic FGF for its receptor and cell-associated glycosaminoglycans may regulate its activity, the identification of a modified form of basic FGF may be of particular importance in understanding the mechanisms that regulate its biological activity, bioavailability, and processing to and from the extracellular matrix.

Animals↗
PubMed1996

Immunodetection of phosphorylation sites gives new insights into the mechanisms underlying phospholamban phosphorylation in the intact heart.

Phosphorylation site-specific antibodies, quantification of 32P incorporation into phospholamban, and simultaneous measurements of mechanical activity were used in Langendorff-perfused rat hearts to provide further insights into the underlying mechanisms of phospholamban phosphorylation. Immunological detection of phospholamban phosphorylation sites showed that the isoproterenol concentration-dependent increase in phospholamban phosphorylation was due to increases in phosphorylation of both Ser16 and Thr17 residues. When isoproterenol concentration was increased at extremely low Ca2+ supply to the myocardium, phosphorylation of Thr17 was virtually absent. Under these conditions, 32P incorporation into phospholamban, due to Ser16, decreased by 50%. Changes in Ca2+ supply to the myocardium either at constant beta-adrenergic stimulation or in the presence of okadaic acid, a phosphatase inhibitor, exclusively modified Thr17 phosphorylation. Changes in phospholamban phosphorylation due to either Ser16 and/or Thr17 were paralleled by changes in myocardial relaxation. The results indicate that cAMP- (Ser16) and Ca2+-calmodulin (Thr17)-dependent pathways of phospholamban phosphorylation can occur independently of each other. However, in the absence of beta-adrenergic stimulation, phosphorylation of Thr17 could only be detected after simultaneous activation of Ca2+-calmodulin-dependent protein kinase and inactivation of phosphatase. It is suggested that under physiological conditions, this requisite is only filled by cAMP-dependent mechanisms.

Adenosine Triphosphatases↗
PubMed1997

Characterization of protein kinase A and protein kinase C phosphorylation of the N-methyl-D-aspartate receptor NR1 subunit using phosphorylation site-specific antibodies.

Modulation of N-methyl-D-aspartate receptors in the brain by protein phosphorylation may play a central role in the regulation of synaptic plasticity. To examine the phosphorylation of the NR1 subunit of N-methyl-D-aspartate receptors in situ, we have generated several polyclonal antibodies that recognize the NR1 subunit only when specific serine residues are phosphorylated. Using these antibodies, we demonstrate that protein kinase C (PKC) phosphorylates serine residues 890 and 896 and cAMP-dependent protein kinase (PKA) phosphorylates serine residue 897 of the NR1 subunit. Activation of PKC and PKA together lead to the simultaneous phosphorylation of neighboring serine residues 896 and 897. Phosphorylation of serine 890 by PKC results in the dispersion of surface-associated clusters of the NR1 subunit expressed in fibroblasts, while phosphorylation of serine 896 and 897 has no effect on the subcellular distribution of NR1. The PKC-induced redistribution of the NR1 subunit in cells occurs within minutes of serine 890 phosphorylation and reverses upon dephosphorylation. These results demonstrate that PKA and PKC phosphorylate distinct residues within a small region of the NR1 subunit and differentially affect the subcellular distribution of the NR1 subunit.

Amino Acid Sequence↗
PubMed1999

Identification and mutation of phosphorylation sites in a linker histone. Phosphorylation of macronuclear H1 is not essential for viability in tetrahymena.

Linker histone phosphorylation has been suggested to play roles in both chromosome condensation and transcriptional regulation. In the ciliated protozoan Tetrahymena, in contrast to many eukaryotes, histone H1 of macronuclei is highly phosphorylated during interphase. Macronuclei divide amitotically without overt chromosome condensation in this organism, suggesting that requirements for phosphorylation of macronuclear H1 may be limited to transcriptional regulation. Here we report the major sites of phosphorylation of macronuclear H1 in Tetrahymena thermophila. Five phosphorylation sites, present in a single cluster, were identified by sequencing 32P-labeled peptides isolated from tryptic peptide maps. Phosphothreonine was detected within two TPVK motifs and one TPTK motif that resemble established p34(cdc2) kinase consensus sequences. Phosphoserine was detected at two non-proline-directed sites that do not resemble known kinase consensus sequences. Phosphorylation at the two noncanonical sites appears to be hierarchical because it was observed only when a nearby p34(cdc2) site was also phosphorylated. Cells expressing macronuclear H1 containing alanine substitutions at all five of these phosphorylation sites were viable even though macronuclear H1 phosphorylation was abolished. These data suggest that the five sites identified comprise the entire collection of sites utilized by Tetrahymena and demonstrate that phosphorylation of macronuclear H1, like the protein itself, is not essential for viability in Tetrahymena.

Amino Acid Sequence↗