Phosphorylation by PKA of a site unique to B-Raf kinase

The Raf kinases serve as central intermediates to relay signals from Ras to ERK. Cell-specific effects of these signals on growth, differentiation and survival can be observed due to the recruitment of different isoenzymes of the Raf family. The in vitro phosphorylation of a site unique to B-Raf (Ser429) has been proposed to be responsible for the negative regulation of the isoenzyme by Akt. Using phosphopetide mapping and site-directed mutagenesis we showed that Ser429 is phosphorylated upon cAMP elevation in PC12 cells and proposed that PKA is a major kinase phosphorylating the B-Raf-specific site in vivo.     

Inhibition of fibronectin matrix assembly by the heparin-binding domain of vitronectin.

The deposition of fibronectin into the extracellular matrix is an integrin-dependent, multistep process that is tightly regulated in order to ensure controlled matrix deposition. Reduced fibronectin deposition has been associated with altered embryonic development, tumor cell invasion, and abnormal wound repair. In one of the initial steps of fibronectin matrix assembly, the amino-terminal region of fibronectin binds to cell surface receptors, termed matrix assembly sites. The present study was undertaken to investigate the role of extracellular signals in the regulation of fibronectin deposition. Our data indicate that the interaction of cells with the extracellular glycoprotein, vitronectin, specifically inhibits matrix assembly site expression and fibronectin deposition. The region of vitronectin responsible for the inhibition of fibronectin deposition was localized to the heparin-binding domain. Vitronectin's heparin-binding domain inhibited both beta(1) and non-beta(1) integrin-dependent matrix assembly site expression and could be overcome by treatment of cells with lysophosphatidic acid, an agent that promotes actin polymerization. The interaction of cells with the heparin-binding domain of vitronectin resulted in changes in actin microfilament organization and the subcellular distribution of the actin-associated proteins alpha-actinin and talin. These data suggest a mechanism whereby the heparin-binding domain of vitronectin regulates the deposition of fibronectin into the extracellular matrix through alterations in the organization of the actin cytoskeleton.     

Identification of the protein kinase C phosphorylation site in neuromodulin

Neuromodulin (P-57, GAP-43, B-50, F-1) is a neurospecific calmodulin binding protein that is phosphorylated by protein kinase C. Phosphorylation by protein kinase C has been shown to abolish the affinity of neuromodulin for calmodulin [Alexander, K. A., Cimler, B. M., Meier, K. E., & Storm, D. R. (1987) J. Biol. Chem. 262, 6108-6113], and we have proposed that the concentration of free CaM in neurons may be regulated by phosphorylation and dephosphorylation of neuromodulin. The purpose of this study was to identify the protein kinase C phosphorylation site(s) in neuromodulin using recombinant neuromodulin as a substrate. Toward this end, it was demonstrated that recombinant neuromodulin purified from Escherichia coli and bovine neuromodulin were phosphorylated with similar Km values and stoichiometries and that protein kinase C mediated phosphorylation of both proteins abolished binding to calmodulin-Sepharose. Recombinant neuromodulin was phosphorylated by using protein kinase C and [gamma-32P]ATP and digested with trypsin, and the resulting peptides were separated by HPLC. Only one 32P-labeled tryptic peptide was generated from phosphorylated neuromodulin. The sequence of this peptide was IQASFR. The serine in this peptide corresponds to position 41 of the entire protein, which is adjacent to or contained within the calmodulin binding domain of neuromodulin. A synthetic peptide, QASFRGHITRKKLKGEK, corresponding to the calmodulin binding domain with a few flanking residues, including serine-41, was also phosphorylated by protein kinase C. We conclude that serine-41 is the protein kinase C phosphorylation site of neuromodulin and that phosphorylation of this amino acid residue blocks binding of calmodulin to neuromodulin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Phosphorylation of smooth muscle caldesmon by calmodulin-dependent protein kinase II. Identification of the phosphorylation sites

Smooth muscle caldesmon was phosphorylated by smooth muscle calmodulin-dependent protein kinase II. The extent of phosphorylation obtained was 5.65 mol of phosphate/mol of caldesmon. Phosphorylated protein was subjected to the complete trypsin proteolysis and the produced phosphopeptides were purified by C-8 reverse phase chromatography. Nine phosphopeptides were isolated and by amino acid sequence analysis, eight phosphorylation sites were identified. According to the published amino acid sequence of chicken gizzard caldesmon (Bryan, J., Imai, M., Lee, R., Moore, P., Cook, R. G., and Lin, W.-G. (1989) J. Biol. Chem. 264, 13873-13879), these sites were serine 26, serine 59, serine 73, threonine 469, serine 475, serine 587, serine 620, and serine 726. The time course of phosphorylation of these sites was also measured and it was concluded that the first site was serine 73, the second site was serine 26, the third site was serine 726, and the fourth site was serine 587. The preferred phosphorylation sites were located in the amino terminus myosin binding domain whereas slower phosphorylation occurred in the carboxyl terminus actin/calmodulin domain.     

Phosphorylation of histone H2A by protein kinase C and identification of the phosphorylation site.

In regenerating rat liver, nuclear protein histone H2A was shown to be phosphorylated on its amino-terminal serine residue [Sung et al. (1971) J. Biol. Chem. 246, 1358-1364], but the protein kinase which phosphorylates this residue has not been identified. To evaluate the possibility that protein kinase C can phosphorylate this residue, calf thymus histone H2A was 32P-labeled by incubation with [gamma-32P]ATP and highly purified protein kinase C from rat brain in the presence of calcium and phospholipid. About 1 mol of 32P was incorporated per mol of histone H2A and the Km and apparent Vmax of the reaction were calculated to be 2.1 microM and 0.35 mumol/min/mg, respectively. So histone H2A seemed to be a good substrate for protein kinase C. Further, the proteolytic phosphopeptides of 32P-labeled histone H2A were isolated by means of a series of column chromatographies and analyzed for their amino acid compositions. Comparison of the data with the known primary structure of histone H2A revealed their amino acid sequence as 1Ser-Gly-Arg. These data suggest that protein kinase C may be a candidate for the protein kinase which phosphorylates the amino-terminal serine residue of histone H2A during the regeneration of rat liver.     

Phosphorylation of muscle plasma membrane protein by a membrane-bound protein kinase

Protein kinase activity has been demonstrated in purified plasma membranes from rat diaphragm by measuring the incorporation of ³²P from [³²P]-ATP into endogenous membrane proteins and into histone, in vitro. Histone appears to be a better substrate than the endogenous membrane proteins; however, the properties of the enzyme are similar when phosphorylating endogenous or exogenous proteins. The activity of this membrane-associated protein kinase is not significantly affected by cyclic adenosine 3′,5′-monophosphate or by cyclic guanosine 3′,5′-monophosphate, but is inhibited by theophylline. The ³²P incorporated into membrane proteins is alkali-labile and is released from the membrane by protease digestion, but it is not removed by phospholipase C, by hydroxylamine, or by chloroform—methanoll extraction. Solubilization of ³²P-labeled membranes by sodium dodecylsulfate and fractionation by sodium dodecylsulfate polyacrylamide gel electrophoresis reveals that the radioactivity is predominantly associated with a single protein band with an apparent molecular weight of about 51 000. The phosphoprotein is a minor membrane component as judged by Coomassie blue staining.     

Phosphorylation of human CTP synthetase 1 by protein kinase A: identification of Thr455 as a major site of phosphorylation

CTP synthetase is an essential enzyme that generates the CTP required for the synthesis of nucleic acids and membrane phospholipids. In this study, we examined the phosphorylation of the human CTPS1-encoded CTP synthetase 1 by protein kinase A. CTP synthetase 1 was expressed and purified from a Saccharomyces cerevisiae ura7Delta ura8Delta double mutant that lacks CTP synthetase activity. Using purified CTP synthetase 1 as a substrate, protein kinase A activity was time- and dose-dependent. The phosphorylation, which primarily occurred on a threonine residue, was accompanied by a 50% decrease in CTP synthetase 1 activity. The synthetic peptide LGKRRTLFQT that contains the protein kinase A motif for Thr(455) was a substrate for protein kinase A. A Thr(455) to Ala (T455A) mutation in CTP synthetase 1 was constructed by site-directed mutagenesis and was expressed and purified from the S. cerevisiae ura7Delta ura8Delta mutant. The T455A mutation caused a 78% decrease in protein kinase A phosphorylation and the loss of the phosphothreonine residue and a major phosphopeptide that were present in the purified wild type enzyme phosphorylated by protein kinase A. The CTP synthetase 1 activity of the T455A mutant enzyme was 2-fold higher than the wild type enzyme. In addition, the T455A mutation caused a 44% decrease in the amount of human CTP synthetase 1 that was phosphorylated in S. cerevisiae cells, and this was accompanied by a 2.5-fold increase in the cellular concentration of CTP and a 1.5-fold increase in the choline-dependent synthesis of phosphatidylcholine.     

Phosphorylation of plant actin-depolymerising factor by calmodulin-like domain protein kinase.

The actin-depolymerising factor (ADF)/cofilin group of proteins are stimulus-responsive actin-severing proteins, members of which are regulated by reversible phosphorylation. The phosphorylation site on the maize ADF, ZmADF3, is Ser-6 but the kinase responsible is unknown [Smertenko et al., Plant J. 14 (1998) 187-193]. We have partially purified the ADF kinase(s) and found it to be calcium-regulated and inhibited by N-(6-aminohexyl)-[(3)H]5-chloro-1-naphthalenesulphonamide. Immunoblotting reveals that calmodulin-like domain protein kinase(s) (CDPK) are enriched in the purified preparation and addition of anti-CDPK to in vitro phosphorylation assays results in the inhibition of ADF phosphorylation. These data strongly suggest that plant ADF is phosphorylated by CDPK(s), a class of protein kinases unique to plants and protozoa.     

Identification of a novel phosphorylation site in c-jun directly targeted in vitro by protein kinase D

Protein kinase D (PKD) phosphorylates the c-jun amino-terminal in vitro at site(s) distinct from JNK [C. Hurd, R.T. Waldron, E. Rozengurt, Protein kinase D complexes with c-jun N-terminal kinase via activation loop phosphorylation and phosphorylates the c-jun N-terminus, Oncogene 21 (2002) 2154-2160], but the sites have not been identified. Here, metabolic (32)P-labeling of c-jun protein in COS-7 cells indicated that PKD phosphorylates c-jun in vivo at a site(s) between aa 43-93, a region containing important functional elements. On this basis, the PKD-mediated phosphorylation site(s) was further characterized in vitro using GST-c-jun fusion proteins. PKD did not incorporate phosphate into Ser63 and Ser73, the JNK sites in GST-c-jun(1-89). Rather, PKD and JNK could sequentially phosphorylate distinct site(s) simultaneously. By mass spectrometry of tryptic phosphopeptides, Ser58 interposed between the JNK-binding portion of the delta domain and the adjacent TAD1 was identified as a prominent site phosphorylated in vitro by PKD. These data were further supported by kinase reactions using truncations or point-mutations of GST-c-jun. Together, these data suggest that PKD-mediated phosphorylation modulates c-jun at the level of its N-terminal functional domains.     

Phosphorylation of Xenopus elongation factor-1 gamma by cdc2 protein kinase: identification of the phosphorylation site.

The cdc2 protein kinase phosphorylates elongation factor-1 gamma (EF-1 gamma) during meiotic maturation of Xenopus oocytes. A synthetic peptide P2: PKKETPKKEKPA matching the cDNA-deduced sequence of EF-1 gamma was an in vitro substrate for cdc2 protein kinase and inhibited phosphorylation of EF-1 gamma. Tryptic hydrolysis of EF-1 gamma and the P2 peptide, both phosphorylated by cdc2 protein kinase, resulted in multiple partial digestion products generated by the presence of barely hydrolysable bonds. The two peptides obtained from the hydrolysis of EF-1 gamma comigrated exactly in two-dimensional separation with two of the P2 peptide hydrolysates. EF-1 gamma therefore contains one unique phosphoacceptor for cdc2 protein kinase, identified as threonine-230.     

Phosphorylation of the human erythrocyte glucose transporter by protein kinase C: localization of the site of in vivo and in vitro phosphorylation

1. The human erythrocyte glucose transporter was phosphorylated in vitro by protein kinase C. 2. Tryptic cleavage of phosphorylated native transporter produced two major unphosphorylated membrane-embedded fragments weighing 23 and 19 kDa and released numerous water-soluble peptides. 3. Ion-exchange FPLC of the soluble tryptic peptides resolved the mixture into two phosphopeptide peaks. 4. Tryptic digestion of glucose transporter that was phosphorylated in vivo in response to phorbol esters produced soluble phosphopeptides that eluted at identical salt concentrations. 5. Proteolytic digestion and peptide mapping of the transporter revealed that the site(s) of phosphorylation lie within the large cytoplasmic domain that bisects the molecule.     

The phosphorylation of the two-chain form of vitronectin by protein kinase A is heparin dependent.

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.     

Neutralization and binding of heparin by S protein/vitronectin in the inhibition of factor Xa by antithrombin III. Involvement of an inducible heparin-binding domain of S protein/vitronectin

The interference of the heparin-neutralizing plasma component S protein (vitronectin) (Mr = 78,000) with heparin-catalyzed inhibition of coagulation factor Xa by antithrombin III was investigated in plasma and in a purified system. In plasma, S protein effectively counteracted the anticoagulant activity of heparin, since factor Xa inhibition was markedly reduced in comparison to heparinized plasma deficient in S protein. Using purified components in the presence of heparin, S protein induced a concentration-dependent reduction of the inhibition rate of factor Xa by antithrombin III. This resulted in a decrease of the apparent pseudo-first order rate constant by more than 10-fold at a physiological ratio of antithrombin III to S protein. S protein not only counteracted the anticoagulant activity of commercial heparin but also of low molecular weight forms of heparin (mean Mr of 4,500). The heparin-neutralizing activity of S protein was found to be mainly expressed in the range 0.2-10 micrograms/ml of high Mr as well as low Mr heparin. S protein and high affinity heparin reacted with apparent 1:1 stoichiometry to form a complex with a dissociation constant KD = 1 X 10(-8) M as determined by a functional assay. As deduced from dot-blot analysis, direct interaction of radiolabeled heparin with S protein revealed a dissociation constant KD = 4 X 10(-8) M. Heparin binding as well as heparin neutralization by S protein increased significantly when reduced/carboxymethylated or guanidine-treated S protein was employed indicating the existence of a partly buried heparin-binding domain in native S protein. Radiolabeled heparin bound to the native protein molecule as well as to a BrCN fragment (Mr = 12,000) containing the heparin-binding domain as demonstrated by direct binding on nitrocellulose replicas of sodium dodecyl sulfate-polyacrylamide gels. Kinetic analysis revealed that the heparin neutralization activity of S protein in the inhibition of factor Xa by antithrombin III could be mimicked by a synthetic tridecapeptide from the amino-terminal portion of the heparin-binding domain. These data provide evidence that the heparin-binding domain of S protein appears to be unique in binding to heparin and thereby neutralizing its anticoagulant activity in the inhibition of coagulation factors by antithrombin III. The induction of heparin binding and neutralization may be considered a possible physiological mechanism initiated by conformational alteration of the S protein molecule.(ABSTRACT TRUNCATED AT 400 WORDS)     

Phosphorylation of basic fibroblast growth factor by purified protein kinase C and the identification of a cryptic site of phosphorylation

We have further characterized the protein kinase C (PK-C) dependent phosphorylation of basic fibroblast growth factor (FGF). Intact recombinant basic FGF and a series of ten peptide fragments of basic FGF were phosphorylated by PK-C and the products were analyzed by SDS-PAGE and autoradiography. As expected, peptide fragments containing the known site of phosphorylation (Ser64) are substrates for phosphorylation. Surprisingly however, peptides containing the receptor binding domain of the mitogen [basic FGF(106-115)] are also phosphorylated. An examination of this sequence reveals the presence of a consensus sequence (Ser108-Ala109-Lys110) that mediates the reaction. Accordingly, all peptides that contain the core amino acids basic FGF(106-111) are substrates for phosphorylation. Peptide mapping of basic FGF confirms that Ser64 is the primary site of phosphorylation, suggesting that Ser108 is a cryptic consensus sequence. Because basic FGF is metabolized to sequence specific fragments after its binding and internalization into target cells, this cryptic site may in fact be phosphorylated in vivo.     

Identification of a general anesthetic binding site in the diacylglycerol-binding domain of protein kinase Cdelta

Protein kinase C (PKC) is an important signal transduction protein that has been proposed to interact with general anesthetics at its cysteine-rich diacylglycerol/phorbol ester-binding domain C1, a tandem repeat of C1A and C1B subdomains. To test this hypothesis, we expressed, purified, and characterized the high affinity phorbol-binding subdomain, C1B, of mouse protein kinase Cdelta, and studied its interaction with general anesthetic alcohols. When the fluorescent phorbol ester, sapintoxin-D, bound to PKCdelta C1B in buffer at a molar ratio of 1:2, its fluorescence emission maximum, lambda(max), shifted from 437 to 425 nm. The general anesthetic alcohols, butanol and octanol, further shifted lambda(max) of the PKCdelta C1B-bound sapintoxin-D in a concentration-dependent, saturable manner to approximately 415 nm, suggesting that alcohols interact at a discrete allosteric binding site. To identify this site, PKCdelta C1B was photolabeled with three photo-activable diazirine alcohol analogs, 3-azioctanol, 7-azioctanol, and 3-azibutanol. Mass spectrometry showed photoincorporation of all three alcohols in PKCdelta C1B at a stoichiometry of 1:1 in the labeled fraction. The photolabeled PKCdelta C1B was subjected to tryptic digest, the fragments were separated by online chromatography and sequenced by mass spectrometry. Each azialcohol photoincorporated at Tyr-236. Inspection of the known structure of PKCdelta C1B shows that this residue is situated adjacent to the phorbol ester binding pocket, and within approximately 10 A of the bound phorbol ester. The present results provide direct evidence for an allosteric anesthetic site on protein kinase C.     

Regulation of calcineurin by phosphorylation. Identification of the regulatory site phosphorylated by Ca2+/calmodulin-dependent protein kinase II and protein kinase C

The site in calcineurin, the Ca2+/calmodulin (CaM)-dependent protein phosphatase, which is phosphorylated by Ca2+/CaM-dependent protein kinase II (CaM-kinase II) has been identified. Analyses of 32P release from tryptic and cyanogen bromide peptides derived from [32P]calcineurin plus direct sequence determination established the site as -Arg-Val-Phe-Ser(PO4)-Val-Leu-Arg-, which conformed to the consensus phosphorylation sequence for CaM-kinase II (Arg-X-X-Ser/Thr-). This phosphorylation site is located at the C-terminal boundary of the putative CaM-binding domain in calcinerin (Kincaid, R. L., Nightingale, M. S., and Martin, B. M. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 8983-8987), thereby accounting for the observed inhibition of this phosphorylation when Ca2+/CaM is bound to calcineurin. Since the phosphorylation site sequence also contains elements of the specificity determinants for Ca2+/phospholipid-dependent protein kinase (protein kinase C) (basic residues both N-terminal and C-terminal to Ser/Thr), we tested calcineurin as a substrate for protein kinase C. Protein kinase C catalyzed rapid stoichiometric phosphorylation, and the characteristics of the reaction were the same as with CaM-kinase II: 1) the phosphorylation was blocked by binding of Ca2+/CaM to calcineurin; 2) phosphorylation partially inactivated calcineurin by increasing the Km (from 9.9 +/- 1.1 to 17.5 +/- 1.1 microM 32P-labeled myosin light chain); and 3) [32P]calcineurin exhibited very slow autodephosphorylation but was rapidly dephosphorylated by protein phosphatase IIA. Tryptic and thermolytic 32P-peptide mapping and sequential phosphoamino acid sequence analysis confirmed that protein kinase C and CaM-kinase II phosphorylated the same site.     

An enzymatic assay for vitronectin based on its selective phosphorylation by protein kinase A.

The catalytic subunit (C) of cAMP-dependent protein kinase selectively phosphorylates vitronectin, a plasma protein that promotes cell adhesion and platelet aggregation, inhibits the inactivation of thrombin by antithrombin III, and participates in complement function. This specific phosphorylation is used here (a) to develop an enzymatic assay for vitronectin (with C and [gamma-32P]ATP) which can be used to identify the vitronectin-containing fractions at each stage of its purification; (b) to radioactively label vitronectin and differentiate between the intact and the nicked form of this protein in structure-function studies; and (c) to identify possible vitronectin-related proteins in the plasma of other animal species.     

Phosphorylation of the yeast choline kinase by protein kinase C. Identification of Ser25 and Ser30 as major sites of phosphorylation

The Saccharomyces cerevisiae CKI1-encoded choline kinase catalyzes the committed step in phosphatidylcholine synthesis via the Kennedy pathway. The enzyme is phosphorylated on multiple serine residues, and some of this phosphorylation is mediated by protein kinase A. In this work we examined the hypothesis that choline kinase is also phosphorylated by protein kinase C. Using choline kinase as a substrate, protein kinase C activity was dose- and time-dependent and dependent on the concentrations of choline kinase (K(m) = 27 microg/ml) and ATP (K(m) = 15 microM). This phosphorylation, which occurred on a serine residue, was accompanied by a 1.6-fold stimulation of choline kinase activity. The synthetic peptide SRSSSQRRHS (V5max/K(m) = 17.5 mm(-1) micromol min(-1) mg(-1)) that contains the protein kinase C motif for Ser25 was a substrate for protein kinase C. A Ser25 to Ala (S25A) mutation in choline kinase resulted in a 60% decrease in protein kinase C phosphorylation of the enzyme. Phosphopeptide mapping analysis of the S25A mutant enzyme confirmed that Ser25 was a protein kinase C target site. In vivo the S25A mutation correlated with a decrease (55%) in phosphatidylcholine synthesis via the Kennedy pathway, whereas an S25D phosphorylation site mimic correlated with an increase (44%) in phosphatidylcholine synthesis. Although the S25A (protein kinase C site) mutation did not affect the phosphorylation of choline kinase by protein kinase A, the S30A (protein kinase A site) mutation caused a 46% reduction in enzyme phosphorylation by protein kinase C. A choline kinase synthetic peptide (SQRRHSLTRQ) containing Ser30 was a substrate (V(max)/K(m) = 3.0 mm(-1) micromol min(-1) mg(-1)) for protein kinase C. Comparison of phosphopeptide maps of the wild type and S30A mutant choline kinase enzymes phosphorylated by protein kinase C confirmed that Ser30 was also a target site for protein kinase C.     

Identification of a human Akt3 (protein kinase B gamma) which contains the regulatory serine phosphorylation site

The family of protein kinases called Akt, protein kinase B (PKB), or related to A and C kinase (RAC) have been implicated in numerous biological processes including adipocyte and muscle differentiation, glycogen synthesis, glucose uptake, apoptosis and cellular proliferation. There are 3 known isoforms of this enzyme in mammalian cells (1/alpha, 2/beta and 3/gamma). Akt1 and 2 contain a key regulatory serine phosphorylation site in the carboxy-terminal region of the protein. However, the reported sequence of the rat Akt3 protein differed significantly from this in that it lacked 25 amino acids in the C-terminal region, including this key regulatory serine phosphorylation site (Biochem. Biophys. Res. Commun. 216, 526-534). In the present studies we show that the deduced sequence of human Akt3 contains this serine and that it is phosphorylated in response to insulin. These results indicate that human Akt3 is regulated similarly to Akt1 and Akt2.