Depolarization-Dependent Protein Phosphorylation in Rat Cortical Synaptosomes: Characterization of Active Protein Kinases by Phosphopeptide Analysis of Substrates

Depolarization of synaptosomes is known to cause a calcium-dependent increase in the phosphorylation of a number of proteins. It was the aim of this study to determine which protein kinases are activated on depolarization by analyzing the incorporation of 32Pi into synaptosomal phosphoproteins and phosphopeptides. The following well-characterized phosphoproteins were chosen for study: phosphoprotein "87K," synapsin Ia and Ib, phosphoproteins IIIa and IIIb, the catalytic subunits of calmodulin kinase II, and the B-50 protein. Each was initially identified as a phosphoprotein in lysed synaptosomes after incubation with [gamma-32P]ATP. Mobility on two-dimensional polyacrylamide gels and phosphorylation by specific protein kinases were the primary criteria used for identification. A technique was developed that allowed simultaneous analysis of the phosphopeptides derived from all of these proteins. Phosphopeptides were characterized in lysed synaptosomes after activating cyclic AMP-, calmodulin-, and phospholipid-stimulated protein kinases in the presence of [gamma-32P]ATP. Phosphoproteins labelled in intact synaptosomes after incubation with 32Pi were then compared with those seen after ATP-labelling of lysed synaptosomes. As expected from previous work, phosphoprotein "87K," and synapsin Ia and Ib were labelled, but for the first time, phosphoproteins IIIa, IIIb, and the B-50 protein were identified as being labelled in intact synaptosomes; the calmodulin kinase II subunits were hardly phosphorylated. From a comparison of the phosphopeptide profiles it was found that cyclic AMP-, calmodulin-, and phospholipid-stimulated protein kinases are all active in intact synaptosomes and their activity is dependent on extrasynaptosomal calcium. The activation of cyclic AMP-stimulated protein kinases in intact synaptosomes was confirmed by the addition of dibutyryl cyclic AMP and theophylline which specifically increased the labelling of phosphopeptides in synapsin Ia and Ib and in phosphoproteins IIIa and IIIb. On depolarization of intact synaptosomes, a number of phosphopeptides showed increased labelling and the pattern suggested that cyclic AMP-, calmodulin-, and phospholipid-stimulated protein kinases were all activated. No new peptides were phosphorylated, suggesting that depolarization simply increased the activity of already active protein kinases and that there was no depolarization-specific increase in protein phosphorylation.     

Tetanus Toxin Inhibits Depolarization-Stimulated Protein Phosphorylation in Rat Cortical Synaptosomes: Effect on Synapsin I Phosphorylation and Translocation

Synapsin I, a prominent phosphoprotein in nerve terminals, is proposed to modulate exocytosis by interaction with the cytoplasmic surface of small synaptic vesicles and cytoskeletal elements in a phosphorylation-dependent manner. Tetanus toxin (TeTx), a potent inhibitor of neurotransmitter release, attenuated the depolarization-stimulated increase in synapsin I phosphorylation in rat cortical particles and in synaptosomes. TeTx also markedly decreased the translocation of synapsin I from the small synaptic vesicles and the cytoskeleton into the cytosol, on depolarization of synaptosomes. The effect of TeTx on synapsin I phosphorylation was both time and TeTx concentration dependent and required active toxin. One- and two-dimensional peptide maps of synapsin I with V8 proteinase and trypsin, respectively, showed no differences in the relative phosphorylation of peptides for the control and TeTx-treated synaptosomes, suggesting that both the calmodulin- and the cyclic AMP-dependent kinases that label this protein are equally affected. Phosphorylation of synapsin IIb and the B-50 protein (GAP43), a known substrate of protein kinase C, was also inhibited by TeTx. TeTx affected only a limited number of phosphoproteins and the calcium-dependent decrease in dephosphin phosphorylation remained unaffected. In vitro phosphorylation of proteins in lysed synaptosomes was not influenced by prior TeTx treatment of the intact synaptosomes or by the addition of TeTx to lysates, suggesting that the effect of TeTx on protein phosphorylation was indirect. Our data demonstrate that TeTx inhibits neurotransmitter release, the phosphorylation of a select group of phosphoproteins in nerve terminals, and the translocation of synapsin I. These findings contribute to our understanding of the basic mechanism of TeTx action.     

Differential stimulation of protein kinase C activity by phorbol ester or calcium/phosphatidylserine in vitro and in intact synaptosomes.

Activation of protein kinase C (PKC) by phorbol 12-myristate 13-acetate (PMA) was compared with calcium/phosphatidylserine (Ca/PS). The substrate specificity of PKC was more limited with PS/PMA. Substrates could be divided into three overlapping groups according to their relative level of phosphorylation: C1, relatively preferred substrates with Ca/PS, included dephosphin, histone, and peptide GS1-10. C2, relatively preferred with PS/PMA, included myelin basic protein and MARCKS. C3, substrates independent of activators. PS/PMA altered the Vmax of PKC for substrate, and decreased the Km for Mg2+. Differential substrate phosphorylation by PS/PMA also occurred for PKC isozymes resolved by hydroxylapatite chromatography and was most dramatic for PKC-alpha, which could no longer phosphorylate histone or GS1-12. Differential activities of PKC were also observed in synaptosol and in intact synaptosomes where PMA stimulated phosphorylation of MARCKS, but not dephosphin. It was further shown that dephosphin was indeed a substrate of PKC in the intact synaptosomes by use of a repolarization-dependent dephosphin phosphorylation assay. The differential PKC activities could also be distinguished by inhibitors. H-7 was equipotent, palmitoylcarnitine did not inhibit in vitro C2 phosphorylation, but inhibited dephosphin in intact synaptosomes, and sphingosine did not inhibit C1 substrates and was without effect on dephosphin in intact synaptosomes. Therefore PS/PMA alters or limits the substrate specificity of PKC, leading to a differential substrate phosphorylation in vitro and in intact synaptosomes and differential inhibitor sensitivity. The pattern of protein phosphorylation observed after PKC activation in intact cells will therefore be dependent upon the activator.     

Major 56,000-dalton, soluble phosphoprotein present in bovine sperm is the regulatory subunit of a type II cAMP-dependent protein kinase

It has been shown that cAMP-dependent phosphorylation of a soluble sperm protein is important for the initiation of flagellar motion. The suggestion has been made that this motility initiation protein, named axokinin, is the major 56,000-dalton phosphoprotein present in both dog sperm and in other cells containing axokinin-like activity. Since the regulatory subunit of a type II cAMP-dependent protein kinase is a ubiquitous cAMP-dependent phosphoprotein of similar subunit molecular weight as reported for axokinin, we have addressed the question of how many soluble 56,000-dalton cAMP-dependent phosphoproteins are present in mammalian sperm. We report that in bovine sperm cytosol, the ratio of the type I to type II cAMP-dependent protein kinase is approximately 1:1. The type II regulatory subunit is related to the non-neural form of the enzyme and undergoes a phosphorylation-dependent electrophoretic mobility shift. The apparent subunit molecular weights of the phospho and dephospho forms are 56,000 and 54,000 daltons, respectively. When bovine sperm cytosol or detergent extracts are phosphorylated in the presence of catalytic subunits, two major proteins are phosphorylated and have subunit molecular weights of 56,000 and 40,000 daltons. If, however, the type II regulatory subunit (RII) is quantitatively removed from these extracts using either immobilized cAMP or an anti-RII monoclonal affinity column, the ability to phosphorylate the 56,000- but not 40,000-dalton polypeptide is lost. These data suggest that the major 56,000 dalton cAMP-dependent phosphoprotein present in bovine sperm is the regulatory subunit of a type II cAMP-dependent protein kinase and not the motility initiator protein, axokinin.     

Effect of Zinc on Calmodulin-Stimulated Protein Kinase II and Protein Phosphorylation in Rat Cerebral Cortex

The effect of increasing concentrations of Zn2+ (1 microM-5 mM) on protein phosphorylation was investigated in cytosol (S3) and crude synaptic plasma membrane (P2-M) fractions from rat cerebral cortex and purified calmodulin-stimulated protein kinase II (CMK II). Zn2+ was found to be a potent inhibitor of both protein kinase and protein phosphatase activities, with highly specific effects on CMK II. Only one phosphoprotein band (40 kDa in P2-M phosphorylated under basal conditions) was unaffected by addition of Zn2+. The vast majority of phosphoprotein bands in both basal and calcium/calmodulin-stimulated conditions showed a dose-dependent inhibition of phosphorylation, which varied with individual phosphoproteins. Two basal phosphoprotein bands (58 and 66 kDa in S3) showed a significant stimulation of phosphorylation at 100 microM Zn2+ with decreased stimulation at higher concentrations, which was absent by 5 mM Zn2+. A few Ca2+/calmodulin-stimulated phosphoproteins in P2-M and S3 showed biphasic behavior; inhibition at less than 100 microM Zn2+ and stimulation by millimolar concentrations of Zn2+ in the presence or absence of added Ca2+/calmodulin. The two major phosphoproteins in this group were identified as the alpha and beta subunits of CMK II. Using purified enzyme, Zn2+ was shown to have two direct effects on CMK II: an inhibition of Ca2+/calmodulin-stimulated autophosphorylation and substrate phosphorylation activity at low concentrations and the creation of a new Zn(2+)-stimulated, Ca2+/calmodulin-independent activity at concentrations of greater than 100 microM that produces a redistribution of activity biased toward autophosphorylation and an alpha subunit with an altered mobility on sodium dodecyl sulfate-containing gels.     

Caldesmon phosphorylation in intact human platelets by cAMP-dependent protein kinase and protein kinase C

Caldesmon is a calmodulin- and actin-binding protein present in both smooth and non-muscle tissue. The present study demonstrates that platelet caldesmon is a substrate for cAMP-dependent protein kinase (protein kinase A). Purified platelet caldesmon has an apparent molecular mass of 82 kDa on sodium dodecyl sulfate-polyacrylamide gels and can be phosphorylated in vitro by the catalytic subunit of protein kinase A to a level of 2 mol of phosphate/mol of caldesmon. Phosphorylation of caldesmon by protein kinase A results in a shift in the apparent molecular mass of the protein to 86 kDa. When caldesmon was immunoprecipitated from intact platelets treated with prostacyclin (PGI2) the same shift in apparent molecular mass of caldesmon was observed. Comparison of two-dimensional tryptic phosphopeptide maps of caldesmon phosphorylated in vitro by protein kinase A with caldesmon immunoprecipitated from intact platelets verified that protein kinase A was responsible for the observed increase in caldesmon phosphorylation in PGI2-treated platelets. The present study demonstrates that although caldesmon is basally phosphorylated in the intact platelet, activation of protein kinase A by PGI2 results in the significant incorporation of phosphate into two new sites. In addition, the effects of phorbol ester, collagen, and thrombin on caldesmon phosphorylation were also examined. Although phorbol ester treatment results in a significant increase in caldesmon phosphorylation apparently by protein kinase C, treatment of intact platelets with thrombin or collagen does not result in an increase in caldesmon phosphorylation.     

Presence in many mammalian tissues of an identical major cytosolic substrate (Mr 100 000) for calmodulin-dependent protein kinase

Incubation of cytosol fractions from a variety of mammalian tissues (heart, liver, lung, adrenal, spleen and skeletal muscle) with Ca2+ (0.5 mM) in the presence of gamma-[32P]ATP resulted in the phosphorylation of a prominent substrate of Mr approximately 100 000 (100 kDa). One-dimensional peptide maps and two-dimensional tryptic fingerprints of the phosphoprotein from these sources were identical. A single major phosphopeptide was generated by trypsin and was determined to contain exclusively phosphothreonine. The 100 kDa substrate could be distinguished from glycogen phosphorylase (Mr approximately 97 000) by a number of criteria including phosphopeptide mapping and by its failure to bind either to glycogen or to a specific antiphosphorylase antibody. The Ca2+-dependent protein kinase responsible for phosphorylation of the 100 kDa protein appeared to be a calmodulin (CaM)-requiring enzyme in that it could be inhibited in cytosol extracts by trifluoperazine (IC50 6-16 microM) and that exogenous CaM was necessary for 100 kDa phosphorylation in CaM-depleted cytosol. These results suggest that a rise in intracellular Ca2+ resulting in an activation of CaM-dependent protein kinase leads to the phosphorylation of a common 100 kDa substrate in many tissues.     

Phosphorylation of Animal Virus Proteins by a Virion Protein Kinase

Compared with several other enveloped viruses, purified virions of frog virus 3 contained a relatively high activity of a protein kinase which catalyzed the phosphorylation of endogenous polypeptides or added substrate proteins. Virions also contained a phosphoprotein phosphatase activity which released phosphate covalently linked to proteins. It was possible to select reaction conditions where turnover of protein phosphoesters was minimal, as the phosphatase required Mn(2+) ions for activity whereas the protein kinase was active in the presence of Mg(2+) ions. Electrophoretic studies in polyacrylamide gels containing sodium dodecyl sulfate indicated that at least 10 of the virion polypeptides were phosphorylated in the in vitro protein kinase reaction. Characterization of these phosphoproteins demonstrated that the phosphate was incorporated predominantly in a phosphoester linkage with serine residues. The protein kinase was solubilized by disrupting purified virions with a nonionic detergent in a high-ionic-strength buffer and was separated from many of the virion substrate proteins by zonal centrifugation in glycerol gradients. The partially purified protein kinase would phosphorylate polypeptides of many different animal viruses, and maximal activity was not dependent on added cyclic nucleotides. These properties distinguished the virion protein kinase from a well characterized cyclic AMP-dependent protein kinase which phosphorylated viral proteins only to a small extent.     

Phosphorylation of an alpha 1-like subunit of an omega-conotoxin-sensitive brain calcium channel by cAMP-dependent protein kinase and protein kinase.

Antibodies that recognize the alpha 2 delta and alpha 1 subunits of skeletal muscle L-type calcium channels have been used to investigate the subunit components and phosphorylation of omega-conotoxin (omega-CgTx)-sensitive N-type calcium channels from rabbit brain. Photolabeling of the N-type channel with a photoreactive derivative of 125I-omega-CgTx results in the identification of a single polypeptide of 240 kDa. MANC-1, a monoclonal antibody recognizing alpha 2 delta subunits of L-type calcium channels from skeletal muscle, immunoprecipitates the omega-CgTx-labeled 240-kDa polypeptide and approximately 6% of the digitonin-solubilized 125I-omega-CgTx-labeled N-type channels. MANC-1 also immunoprecipitates a phosphoprotein of 240 kDa that comigrates with 125I-omega-CgTx-labeled N-type calcium channels, but not with L-type calcium channels, in sucrose gradients. Both cAMP-dependent protein kinase and protein kinase C are effective in the phosphorylation of this polypeptide. Similar to the alpha 1 subunits of skeletal muscle L-type calcium channels, the immunoprecipitation of the 240-kDa phosphoprotein by MANC-1 is prevented by the detergent Triton X-100. Anti-CP-(1382-1400), an antipeptide antibody against a highly conserved segment of the alpha 1 subunits of calcium channels, immunoprecipitates the 240-kDa phosphopeptide in Triton X-100. The 240-kDa protein is phosphorylated to a stoichiometry of approximately 1 mol of phosphate/mol of omega-CgTx-binding N-type calcium channels by both cAMP-dependent protein kinase and protein kinase C. Our results show that the 240-kDa polypeptide is an alpha 1-like subunit of an omega-CgTx-sensitive N-type calcium channel. The N-type calcium channels containing this subunit are phosphorylated by cAMP-dependent protein kinase and protein kinase C and contain noncovalently associated alpha 1-like and alpha 2 delta-like subunits as part of their oligomeric structure.     

Characterization of phosphoproteins and protein kinase activity of virions, noninfectious enveloped particles, and dense bodies of human cytomegalovirus.

Phosphorylation of the proteins of human cytomegalovirus (CMV) virions, noninfectious enveloped particles (NIEPs), and dense bodies was investigated. Analyses of particles phosphorylated in vivo showed the following. Virions contain three predominant phosphoproteins (i.e., basic phosphoprotein and upper and lower matrix proteins) and at least nine minor phosphorylated species. NIEPs contain all of these and one additional major species, the assembly protein. Dense bodies contain only one (i.e., lower matrix) of the predominant and four of the minor virion phosphoproteins. Two-dimensional (charge-size) separations in denaturing polyacrylamide gels showed that the relative net charges of the predominant phosphorylated species ranged from the basic phosphoprotein to the more neutral upper matrix protein. In vitro assays showed that purified virions of human CMV have an associated protein kinase activity. The activity was detected only after disrupting the envelope; it had a pH optimum of approximately 9 to 9.5 and required a divalent cation, preferring magnesium to manganese. In vitro, this activity catalyzed phosphorylation of the virion proteins observed to be phosphorylated in vivo. Peptide comparisons indicated that the sites phosphorylated in vitro are a subset of those phosphorylated in vivo, underscoring the probable biological relevance of the kinase activity. Casein, phosvitin, and to a minor extent lysine-rich histones served as exogenous phosphate acceptors. Arginine-rich and lysine-rich histones and protamine sulfate, as well as the polyamines spermine and spermidine, stimulated incorporation of phosphate into the endogenous viral proteins. Virions of all human and simian CMV strains tested showed this activity. Analyses of other virus particles, including three intracellular capsid forms (i.e., A, B, and C capsids), NIEPs, and dense bodies, indicated that the active enzyme was not present in the capsid. Rate-velocity sedimentation of disrupted virions separated the protein kinase activity into two fractions: one that phosphorylated exogenous casein and another that phosphorylated primarily the endogenous virion proteins.     

Effects of synthetic peptides on thylakoid phosphoprotein phosphatase reactions

A synthetic peptide analogue of the phosphorylation site of LHC II, when phosphorylated by thylakoid membranes, served as a substrate for the thylakoid phosphoprotein phosphatase. The phosphopeptide became dephosphorylated at a low rate, comparable to that of the 9 kDa phosphoprotein. Phospho-LHC II itself became dephosphorylated much more rapidly, at a rate unaffected by endogenous phosphorylation of the peptide. Endogenous phosphorylation of the peptide was also without effect on other thylakoid protein phosphorylation and dephosphorylation reactions. In contrast, dephosphorylation of many thylakoid phosphoproteins was inhibited by addition of a pure, chemically-synthesised phosphopeptide analogue of phospho-LHC II. This result suggests that at least one thylakoid phosphoprotein phosphatase exhibits a broad substrate specificity. The results indicate that any one of a number of amino acid sequences can give a phosphoprotein configuration that is recognised by a single phosphatase.     

Possible identity of a membrane-bound with a soluble cyclic AMP-independent erythrocyte protein kinase that phosphorylates spectrin

A soluble casein kinase isolated and purified to homogeneity from the human erythrocyte cytosol by phosphocellulose and Sephadex G-200 chromatographies is indistinguishable from the membrane-bound casein (spectrin) kinase according to physical and site-specificity criteria. The soluble enzyme shows an Mr of about 30000 by gel filtration and comigrates with the purified membrane spectrin kinase as a single polypeptide of 32000 Da on sodium dodecyl sulfate polyacrylamide gels. The soluble kinase phosphorylates spectrin in situ in spectrin kinase-depleted ghosts and catalyzes the in vitro phosphorylation of partially dephosphorylated spectrin with saturation kinetics identical to those displayed by the membrane spectrin kinase. When component 2 of spectrin that had been phosphorylated with [gamma-32P]ATP by either the soluble or the membrane kinases was subjected to limited proteolysis, the same 21500 Da papain-generated phosphopeptide was found to have been produced by the two enzymes. The same 21500 Da phosphopeptide was identified after papain digestion of spectrin isolated from intact cells that had been incubated with 32Pi. However, this particular peptide was not labeled in spectrin that had been phosphorylated in vitro by the catalytic subunit of cyclic AMP-dependent protein kinase. Identical phosphopeptide patterns were obtained by gel filtration and two-dimensional peptide maps of trypsin-cleaved component 2 of spectrin that had been labeled in situ, in intact ghosts or in spectrin kinase-depleted ghosts supplemented with the soluble kinase. These findings indicate a possible identity of the soluble with the membrane-bound casein (spectrin) kinase.     

Studies in pig heart tissue on various 60,000 Da phosphoproteins

Pig heart tissue have been shown to contain 3 different 60,000 Da phosphoproteins. Different purification procedures were used in order to separate them, suggesting that the 3 phosphoproteins differ in their environmental parameters. The 2 major ones appear essentially as peripheral phosphoproteins that are associated with cellular membranes through ionic forces, whereas the third minor phosphoprotein behaves as an integral plasma membrane protein. The three phosphoproteins also differ in their relative amount of phosphorylated serine, threonine and tyrosine residues after in vitro protein kinase assay. Evidence that the 3 phosphoproteins are related arises from the similarity between their respective phosphopeptide maps after partial hydrolysis with proteases, an experiment that also points out relatedness in primary structure between them and the transforming protein of Rous sarcoma virus, pp60v-src. The 3 phosphoproteins, however, do not appear to be immunologically related to pp60v-src since none of them is immunoprecipitated by sera that precipitate pp60v-src. The possibility that the three 60,000 Da phosphoproteins under study represent 3 differentially localized and phosphorylated products of c-src and/or c-src related genes is discussed.     

A Protein Modulator Stimulates C Kinase-Dependent Phosphorylation of a 90K Substrate in Synaptic Membranes

We have identified and partially purified an acidic, heat-stable, noncalmodulin protein from bovine brain cytosol that stimulates Ca2+-dependent phosphorylation of an Mr 90K substrate in crude rat brain synaptic membranes. We show that this modulator of phosphorylation (MOP) enhances Ca2+- and phospholipid-dependent protein kinase (C kinase) phosphorylation of this 90K substrate. The 90K substrate is a higher Mr form of an 87K substrate that is a major C kinase substrate in rat brain. The Ca2+-dependent phosphorylation of both substrates is inhibited by the Ca2+-binding proteins S-100 and calmodulin. Both substrates yield phosphopeptide fragments of Mr 9K and 13K after limited proteolysis with V8 protease. Two-dimensional polyacrylamide gel electrophoresis reveals that they have similar acidic isoelectric points (pI 5.0). MOP enhances Ca2+-dependent phosphorylation of the 90K substrate whereas the phosphorylation of 87K is diminished. This reciprocal relationship suggests that the mobility of the 87K substrate in sodium dodecyl sulfate-polyacrylamide gels is decreased to 90K with increasing phosphorylation. MOP may be a novel protein modulator of C kinase-mediated phosphorylation in the nervous system.     

Possible identity of a membrane-bound with a soluble cyclic AMP-independent erythorocyte protein kinase that phosphorylates spectrin

A soluble casein kinase isolated and purified to homogeneity from the human erythrocyte cytosol by phosphocellulose and Sephadex G-200 chromatographies is indistinguishable from the membrane-bound casein (spectrin_kinase according and site-specificity criteria. The soluble enzyme shows an M_r of about 30 000 by gel filtration and comigrates with the purified membrane spectrin kinase as a single polypeptide of 32 000 Da on sodium dodecyl sulfate polyacrylamide gels. The soluble kinase phosphorylates spectrin in situ in spectrin kinase-depleted ghosts and catalyzes the in vitro phosphorylation of partially dephosphorylated spectrin with saturation kinetics identical to those displayed by the membrane spectrin kinase. When component 2 of spectrin that has been phosphorylated with [γ-³²P]ATP by either the soluble or the membrane kinases was subjected to limited proteolysis, the same 21500 Da papain-generated phosphopeptide was found to have been produced by the two enzymes. The same 21 500 Da phosphopeptide was identified after papain digestion of spectrin isolated from intact cells that had been incubated with ³²P_i. However, this particular peptide was not labeled in spectrin that had been phosphorylated in vitro by the catalytic subunit of cyclic AMP-dependent protein kinase. Identical phosphopeptide patterns were obtained by gel filtration and two-dimensional peptide maps of trypsin-cleaved component 2 of spectrin that had been labeled in situ, in intact ghosts or in spectrin kinase-depleted ghosts supplemented with the soluble kinase. These findings indicate a possible identity of the soluble with the membrane-bound casein (spectrin) kinase.     

Ribosomal phosphoproteins in Microsporum canis

Ribosomal phosphoproteins of Microsporum canis labelled in vivo were characterised by two-dimensional and SDS polyacrylamide gel electrophoresis. A small subunit protein, S6, was the only phosphoprotein identified in 40S and 80S in basic-acidic two-dimensional gels. Three different forms of phosphorylated S6 were also observed in 40S subunit. On SDS gels five phosphoproteins were identified in 80S; of these three were present in 40S and two in 60S. S6 was the only basic phosphoprotein, while the other four were acidic.     

Comparison of two phosphoproteins in chicken bone and their similarities to the mammalian bone proteins, osteopontin and bone sialoprotein II.

Two phosphorylated proteins of approximately 66 kDa and approximately 60 kDa mass with different DEAE-Sephacel elution patterns were isolated from chicken bone and were shown to be genetically distinct by both biochemical and immunological analysis. A tryptic peptide from the 60 kDa protein was identified that was similar to a sequence of the rat bone sialoprotein II. Both proteins showed RGD inhibited cell-attachment with the MG-63 osteosarcoma cell, and the approximately 66 kDa phosphoprotein appeared to promote cell adhesion better than human vitronectin. The two phosphoproteins appear to share functional and biochemical characteristics and to be homologous to the mammalian bone phosphoproteins, osteopontin and bone sialoprotein II.     

Purification of two distinct proteins of approximate Mr 80,000 from human epithelial cells and identification as proper substrates for protein kinase C.

A Mr-80,000 acidic phosphoprotein ('80K protein') is a specific substrate for protein kinase C. We attempted to purify the 80K protein from a human squamous-cell carcinoma cell line, Ca9-22, by the sequential use of heat treatment, (NH4)2SO4 precipitation, Mono Q column chromatography, proRPC column chromatography and gel filtration. The 80K protein was assayed by phosphorylation in vitro by using partially purified human type III protein kinase C, and was fractionated into two distinct molecular species with slightly different Mr values, designated 80K-L and 80K-H proteins. Phosphorylation occurred mainly at serine residues of these proteins. Two-dimensional phosphopeptide maps after trypsin digestion and kinetic profiles of phosphorylation were different from each other. Ca2(+)- and phospholipid-dependency of the phosphorylation in vitro confirmed that both 80K-L and 80K-H proteins are true substrates for three subtypes of protein kinase C. The 80K-L protein was a preferential substrate for type III protein kinase C, and the 80K-H protein was phosphorylated more effectively by type I and type II protein kinase C. The possible roles of these two distinct 80K proteins in signal transduction are discussed.     

Depolarisation-Dependent Protein Phosphorylation and Dephosphorylation in Rat Cortical Synaptosomes Is Modulated by Calcium

The effect of calcium on protein phosphorylation was investigated using intact synaptosomes isolated from rat cerebral cortex and prelabelled with 32Pi. For nondepolarised synaptosomes a group of calcium-sensitive phosphoproteins were maximally labelled in the presence of 0.1 mM calcium. The phosphorylation of these proteins was slightly decreased in the presence of strontium and absent in the presence of barium, consistent with the decreased ability of these cations to activate calcium-stimulated protein kinases. Addition of calcium alone to synaptosomes prelabelled in its absence increased phosphorylation of a number of proteins. On depolarisation in the presence of calcium certain of the calcium-sensitive phosphoproteins were further increased in labelling above nondepolarised levels. These increases were maximal and most sustained after prelabelling at 0.1 mM calcium. On prolonged depolarisation at this calcium concentration a slow decrease in labelling was observed for most phosphoproteins, whereas a greater rate and extent of decrease occurred at higher calcium concentrations. At 2.5 mM calcium a rapid and then a subsequent slow dephosphorylation was observed, indicating two distinct phases of dephosphorylation. Of all the phosphoproteins normally stimulated by depolarisation, only phosphoprotein 59 did not exhibit the rapid phase of dephosphorylation at high calcium concentrations. Replacing calcium with strontium markedly decreased the extent of change observed on depolarisation whereas barium decreased phosphorylation changes even further. Taken together these data suggest that an influx of calcium into synaptosomes initially activates protein phosphorylation, but as the levels of intrasynaptosomal calcium rise protein dephosphorylation predominates. Other phosphoproteins were dephosphorylated immediately on depolarisation in the presence of calcium. The fine control of protein phosphorylation levels exerted by calcium supports the idea that the synaptosomal phosphoproteins could play a role in modulating events such as neurotransmitter release in the nerve terminal.     

Ca2+/calmodulin-dependent protein phosphorylation associated with the cytoskeleton of quiescent rat fibroblast (3Y1) cells.

Endogenous phosphorylation of the crude membrane fraction of cultured 3Y1 fibroblast cells was enhanced by the addition of Ca2+/calmodulin. Both Ca2+/calmodulin-dependent protein kinase activity and its substrate were present in a cytoskeletal fraction, obtained as a pellet after washing of the membrane fraction with 2 mM EGTA, 0.6 M NaCl, and 1% Triton X-100. The phosphorylatable protein in the Triton X-insoluble fraction was identified by immunoblotting as vimentin. This endogenous phosphorylation induced by calmodulin was inhibited by the addition of KN-62, a specific Ca2+/calmodulin-dependent protein kinase II inhibitor, in a dose-dependent manner. However, phosphorylation of the 59 kDa protein (vimentin) in this fraction was not stimulated by adding both phosphatidyl serine and cAMP, thereby suggesting the absence of protein kinase C or of cAMP-dependent protein kinase in this fraction. The protein kinase associated with the Triton X-insoluble fraction phosphorylated the Ca2+/calmodulin-dependent protein kinase II-specific site of synapsin I from the bovine cortex. Two-dimensional phosphopeptide maps of vimentin indicated that a major phosphopeptide phosphorylated by the endogenous calmodulin-dependent kinase also appears to be the same as a major phosphopeptide phosphorylated by the exogenous Ca2+/calmodulin-dependent protein kinase II. Our results suggest that cytoskeleton-associated Ca2+/calmodulin-dependent protein kinase II regulates dynamic cellular functions through the phosphorylation of cytoskeletal elements in non-neural cells.