Protease-activated protein kinase C in rat liver

In regenerating rat liver, an elevated protein kinase activity was detected which phosphorylated ribosomal protein S6 and histones. The properties of this enzyme were closely similar with those of protease-activated protein kinase C with Mr 45,000. During the study of the mechanism of proteolytic activation, type III protein kinase C (encoding alpha-sequence) was shown to be subjected to limited proteolysis by trypsin-like protease and converted to protein kinase M in ionic strength- and pH-dependent manner. This reaction was stimulated in the presence of Ca2+ and phospholipid under slightly higher ionic strength condition than physiological level (greater than 140 mM NaCl) and alkaline pH (7.5-8.0). These results suggest that activation of Na+/H+ exchanger in plasma membrane may trigger this type of proteolytic activation of protein kinase C. In addition to protein kinase M, another type of protease-activated kinase with Mr 80,000 was detected when limited proteolysis of protein kinase C was performed on inactive form of this enzyme (in the absence of either Ca2+ or phospholipid or both activators) under lower ionic strength condition. The molecular mass of this active enzyme was slightly smaller (approximately 200) than that of native protein kinase C. However, it is not clear at this time whether this small fragment was released from amino-terminal or carboxy-terminal domain to make protein kinase C partially active in the absence of Ca2+ and phospholipid. Although it has been proposed that proteolytic degradation of protein kinase C is involved in down regulation of this enzyme, the physiological significance of these two types of protease-activated forms of protein kinases in liver has remained obscure.     

H1 histone stimulates limited proteolysis of protein kinase C subspecies by calpain II.

Limited proteolysis of protein kinase C (PKC) subspecies with Ca2(+)-dependent neutral protease II (calpain II) was remarkably stimulated by basic polypeptides, such as H1 histone and poly-L-lysine. This stimulatory effect was observed for proteolysis of the active form of PKC, which was associated with phospholipid and diacylglycerol. The inactive form of PKC was far less susceptible to proteolysis, both in the presence and absence of the basic polypeptides. The basic polypeptides did not appear to interact with calpain II, but made the PKC molecule more susceptible to proteolysis. The relative rates of cleavage of type I (gamma), II (beta), and III (alpha) PKC were 2:2:1. The available evidence suggests that, like calpain I, calpain II may also contribute to the down-regulation or depletion of PKC.     

Differential activation of protein kinase C isozymes by short chain phosphatidylserines and phosphatidylcholines

To investigate the importance of the physical state of phospholipids for activation of protein kinase C, we have used short chain phospholipids, which, depending on their concentration, can exist as either monomers or micelles. We previously reported that short chain phosphatidylcholines (PC) can activate protein kinase C at concentrations that correlate with the critical micelle concentration of the activating lipid (Walker, J. M., and Sando, J. J. (1988) J. Biol. Chem. 263, 4537-4540). We have now expanded this work to short chain phosphatidylserine (PS) systems in order to examine the role of Ca2(+)-phospholipid interactions in the activation process. Short chain PS were synthesized from corresponding PC and purified by reverse-phase high pressure liquid chromatography. Use of the short chain system has revealed significant differences in the activation of type II and type III protein kinase C isozymes. The type II isozyme required Ca2+ in the presence of long chain PS vesicles; in the presence of the short chain phospholipid micelles (PC or PS), most of the activity was Ca2+ independent. Addition of diacylglycerol caused a small increase in type II activity in all phospholipid systems. In contrast, type III protein kinase C was Ca(+)-dependent in all of the lipid systems. The concentration of Ca2+ required to activate type III protein kinase C was independent of the phospholipid type despite large differences in the ability of these lipids to bind Ca2+. This isozyme required diacylglycerol only in the PC micelle system or with vesicles composed of long chain saturated PS. The presence of short chain PS micelles or long chain PS with unsaturated fatty acyl chains rendered this Ca2(+)-dependent protein kinase C virtually diacylglycerol independent. These results are consistent with a model in which type II protein kinase C requires Ca2+ primarily for membrane association, a requirement which is bypassed with the micelle system, whereas type III protein kinase C has an additional Ca2+ requirement for activity that does not involve Ca2(+)-phospholipid interactions.     

Protein kinase C from chicken gizzard: characterization and detection of an inhibitor and endogenous substrates

Protein kinase C was purified from the cytosolic fraction of chicken gizzard by Ca2+ -dependent hydrophobic interaction chromatography, anion-exchange chromatography, and hydrophobic chromatography. The molecular weight was estimated as 61,500 by gel filtration and 80,000 by denaturing gel electrophoresis, indicating that the native enzyme is a monomer. Using the mixed micellar assay, with histone III-S as the substrate, protein kinase C required Ca2+, phospholipid, and diacylglycerol for activity, with half-maximal activation at approximately 5 x 10(-7) M Ca2+ in the presence of L-alpha-phosphatidyl-L-serine and 1,2-diolein. No activation by Ca2+ was observed in the absence of diacylglycerol. Protein kinase C requires free Mg2+, in addition to the MgATP2- substrate, for activity. The Km for ATP was determined to be 20 microM. Activity was sensitive to ionic strength, with half-maximal inhibition at 70 mM NaCl. Using the liposomal assay, phosphorylation of platelet P47 protein and smooth muscle vinculin was more strongly dependent on Ca2+ and lipids than was histone phosphorylation. Partial digestion of protein kinase C with trypsin yielded a constitutively active fragment. A heat-stable inhibitor and three major endogenous protein substrates of protein kinase C were also detected in chicken gizzard smooth muscle.     

Protease-activated form of protein kinase C with Mr 80,000 generated from rat liver plasma membrane by trypsin-like protease

New type of protease-activated form of protein kinase C was generated from rat liver plasma membrane by action of endogenous trypsin-like protease. The molecular mass was estimated to be about 80,000 by immunoblot analysis which was slightly smaller (approximately 2,000) than that of native protein kinase C. The protein kinase activity was 2-times stimulated by Ca2+ and phospholipid and inhibited by the synthetic peptide derived from the pseudosubstrate region of protein kinase C. This type of activated kinase was produced in purified enzyme system in the absence of either Ca2+ or phospholipid or both. These results suggest that limited proteolysis generating the active form of Mr 80,000 may occur on the inactive form of protein kinase C.     

Different requirements for protein kinase C activation and Ca2+-independent insulin secretion in response to guanine nucleotides. Endogenously generated diacylglycerol requires elevated Ca2+ for kinase C insertion into membranes

Electrically permeabilized RINm5F cells were used to assess the factors required for activation of protein kinase C (PKC) and insulin secretion. PKC was activated either by phorbol 12-myristate 13-acetate (PMA) or by the generation of endogenous diacylglycerol in response to the nonhydrolyzable guanine nucleotide analog guanosine 5'-O-(thiotriphosphate) (GTP gamma S). As shown previously, both PMA and GTP gamma S elicit Ca2+-independent insulin secretion. This effect was mimicked by guanyl-5'-yl imidodiphosphate (Gpp(NH)p) but not by guanosine 5'-O-(3-fluorotriphosphate) and guanosine 5'-O-(3-phenyltriphosphate) possessing only one negative charge in the gamma-phosphate group. The action of PMA was mediated by PKC, since the agent caused both phosphorylation of specific protein substrates and association of the enzyme with cellular membranes. This translocation was independent of the Ca2+ concentration employed. In contrast, GTP gamma S only promoted association of PKC with membranes at 10(-6) and 10(-5) M Ca2+ and failed to alter significantly protein phosphorylation in the absence of Ca2+. Neither Gpp(NH)p, which stimulates insulin release, nor the other two GTP analogs, increased the proportion of PKC associated with membranes. To verify that the Ca2+-dependent effect of GTP gamma S on PKC is due to activation of phospholipase C, we measured the generation of diacylglycerol. GTP gamma S indeed stimulated diacylglycerol production in the leaky cells by about 50% at Ca2+ concentrations between 10(-7) and 10(-5) M, an effect which was almost abolished in the absence of Ca2+. Thus, at 10(-7) M Ca2+, the concentration found in resting intact cells, the generated diacylglycerol was not sufficient to cause PKC insertion into the membrane, demonstrating that both elevated Ca2+ and diacylglycerol are necessary for translocation to occur. It is concluded that while PKC activation by PMA elicits Ca2+-independent insulin secretion, the kinase seems not to mediate the stimulatory action of GTP analogs in the absence of Ca2+.     

Comparison of calcium-activated, cyclic nucleotide-independent protein kinase and adenosine 3':5'-monophosphate-dependent protein kinase as regards the ability to stimulate glycogen breakdown in vitro.

A cyclic nucleotide-independent protein kinase, which was produced from its proenzyme upon limited proteolysis by a Ca2+-dependent protease (Takai, Y., Yamamoto, M., Inoue, M., Kishimoto, A., & Nishizuka , Y. (1977) Biochem. Biophys. Res. Commun. 77, 542-550), showed an ability to phosphorylate not only muscle glycogen phosphorylase kinase but also glycogen synthase, resulting in activation and inactivation of the respective enzymes, although the protein kinase was less active than adenosine 3':5'-monophosphate (cyclic AMP)-dependent protein kinase toward glycogen synthase. Available evidence indicates that this new protein kinase shows pleiotropic functions apparently similar to those described for cyclic AMP-dependent protein kinase. Nevertheless, these protein kinases were clearly distinguishable from each other in their response to cyclic nucleotides and susceptibility to protein inhibitor.     

Calcium-dependent activation of a multifunctional protein kinase by membrane phospholipids.

The proenzyme of a Ca2+-dependent protease-activated protein kinase previously obtained from mammalian tissues (Inoue, M., Kishimoto, A., Takai, Y., and Nishizuka, Y. (1977) J. Biol. Chem. 252, 7610-7616) was enzymatically fully active without limited proteolysis when Ca2+ and a membrane-associated factor were simultaneously present in the reaction mixture. The activation process was reversed by removing Ca2+ with ethylene glycol bis(beta-aminoethyl ether)N,N,N',N'-tetraacetic acid. An apparent Ka value for Ca2+ was less than 5 x 10(-5) M. Other divalent cations were inactive except for Sr2+, which was 5% as active as Ca2+. The factor was almost exclusively localized in membrane fractions of various tissues including brain, liver, kidney, skeletal muscle, blood cells, and adipose tissue. It was easily extractable with chloroform/methanol (2:1), and was recovered in the phospholipid fraction. In fact, this membrane factor could be replaced by chromatographically pure phosphatidylinositol, phosphatidylserine, phosphatidic acid, or diphosphatidylglycerol. Phosphatidylethanolamine, phosphatidylcholine, and sphingomyelin were far less effective under the comparable conditions. Ca2+-dependent modulator protein was unable to support enzymatic activity. The enzyme thus activated showed an ability to phosphorylate five histone fractions and muscle phosphorylase kinase, and appeared to possess multifunctional catalytic activities.     

Mechanism of proteolytic activation of rat liver protein kinase C generating Ca2(+)-phospholipid-independent form with apparent molecular mass of 80,000

1. New Ca2(+)-phospholipid-independent form of protein kinase C was produced by limited proteolysis with trypsin. 2. The molecular mass of this active enzyme was slightly smaller than that of original protein kinase C. 3. The active enzyme cross-reacted with antibody against the pseudosubstrate region on amino-terminal end of protein kinase C. 4. The active enzyme was inhibited by the peptide inhibitor derived from the pseudosubstrate region. 5. These results suggest that the limited proteolysis at or near the pseudosubstrate region made protein kinase C active without Ca2+ and phospholipid.     

Protein kinase C activation by cis-fatty acid in the absence of Ca2+ and phospholipids

Protein kinase C has been shown to be a phospholipid/Ca2+-dependent enzyme activated by diacylglycerol (Nishizuka, Y. (1984) Nature 308, 693-697; Nishizuka, Y. (1984) Science 225, 1365-1370). We have reported that unsaturated fatty acids (oleic acid and arachidonic acid) can activate protein kinase C independently of Ca2+ and phospholipid (Murakami, K., and Routtenberg, A. (1985) FEBS Lett. 192, 189-193). This study shows that other cis-fatty acids such as linoleic acid also fully activate protein kinase C in the same manner. None of the saturated fatty acids (C:4 to C:18) nor the detergents (sodium dodecyl sulfate and Triton X-100) tested here were as effective as oleic acid. Unlike oleic acid, these detergents strongly inhibited protein kinase C activity induced by Ca2+/phosphatidylserine (PS) and diacylglycerol. Lowering the critical micelle concentration of oleic acid by increasing ionic strength also strongly inhibited oleic acid activation of protein kinase C activity. Dioleoylphosphatidylserine activated protein kinase C effectively (Ka = 7.2 microM). On the other hand, dimyristoylphosphatidylserine, which contains saturated fatty acids at both acyl positions, failed to activate protein kinase C even in the presence of Ca2+. These observations suggest that: protein kinase C activation by free fatty acid is specific to the cis-form and is not due to their detergent-like action, cis-fatty acid activation is due to the direct interaction of protein kinase C with the monomeric form of cis-fatty acids and not with the micelles of fatty acids, and cis-fatty acids at acyl positions in PS are also important for Ca2+/PS activation of protein kinase C.     

Dual calcium-dependent protein phosphorylation systems in pancreas and their differential regulation by polymyxin B1

Both phospholipid/calcium (PL/Ca2+) activated and calmodulin/Ca2+ (CaM/Ca2+)activated protein kinase systems were found in rat pancreatic extracts treated with Sephadex G-25. At least four substrate proteins for PL/Ca2+-activated kinase and one for a CaM/Ca2+-activated kinase were noted. Polymyxin B, an amphipathic antibiotic, was over 100-fold more potent as an inhibitor of PL/Ca2+-dependent protein phosphorylation than of the CaM/Ca2+-dependent system (Ki = app. 7 microM v. 950 microM). Fluphenazine inhibited both PL/Ca2+- and CaM/Ca2+-dependent protein kinases with equal potency, as did dibucaine. Inhibition by polymyxin B of PL/Ca2+-dependent phosphorylation could be overcome by increased amounts of phosphatidylserine. Low concentrations (10(-5)M) of polymyxin B completely inhibited carbachol-stimulated amylase release from intact pancreatic acini. These results indicate that polymyxin B may be useful in delineating the relative roles of PL/Ca2+-dependent and CaM/Ca2+-dependent protein phosphorylation in biological systems and suggest a potential role for the PL/Ca2+-activated kinase in regulation of pancreatic exocrine function.     

Protein kinase C as a possible receptor protein of tumor-promoting phorbol esters

A tumor-promoting phorbol ester, [3H]phorbol-12,13-dibutyrate, may bind to a homogeneous preparation of Ca2+-activated, phospholipid-dependent protein kinase (protein kinase C) in the simultaneous presence of Ca2+ and phospholipid. This tumor promoter does not bind simply to phospholipid nor to the enzyme per se irrespective of the presence and absence of Ca2+. All four components mentioned above appear to be bound together, and the quaternary complex thus produced is enzymatically fully active for protein phosphorylation. Phosphatidylserine is most effective. Various other phorbol derivatives which are active in tumor promotion compete with [3H]phorbol-12,13-dibutyrate for the binding, and an apparent dissociation binding constant of the tumor promoter is 8 nM. This value is identical with the activation constant for protein kinase C and remarkably similar to the dissociation binding constant that is described for intact cell surface receptors. The binding of the phorbol ester is prevented specifically by the addition of diacylglycerol, which serves as activator of protein kinase C under physiological conditions. Scatchard analysis suggests that one molecule of the tumor promoter may bind to every molecule of protein kinase C in the presence of Ca2+ and excess phospholipid. It is suggestive that protein kinase C is a phorbol ester-receptive protein, and the results presented seem to provide clues for clarifying the mechanism of tumor promotion.     

Properties and distribution of the protein inhibitor (Mr 17,000) of protein kinase C.

Ca2+-dependent hydrophobic-interaction chromatography is a powerful tool for the identification and isolation of a variety of Ca2+-binding proteins which expose a hydrophobic site(s) in the presence of Ca2+ [Gopalakrishna & Anderson (1982) Biochem. Biophys. Res. Commun. 104, 830-836; Walsh, Valentine, Ngai, Carruthers & Hollenberg (1984) Biochem. J. 224, 117-127; McDonald & Walsh (1985) Biochem. J. 232, 559-567]. Using this approach, we isolated two potent and specific protein inhibitors of protein kinase C, of 17 kDa [McDonald & Walsh (1985) Biochem. J. 232, 559-567] and 12 kDa [McDonald & Walsh (1986) Biochem. Soc. Trans. 14, 585-586]. Although these inhibitors were purified by Ca2+-dependent hydrophobic-interaction chromatography and exhibit properties similar to those of calmodulin and related Ca2+-binding proteins, we were unable to demonstrate high-affinity Ca2+ binding to these inhibitors, using equilibrium dialysis. Protein kinase C exhibited half-maximal activity at 0.6 microM-Ca2+ in the presence of phospholipid and diacylglycerol, and complete inhibition by both inhibitors was observed over the range of Ca2+ concentrations examined (10 nM-10 microM). These observations suggest that the inhibitory action of these proteins does not require Ca2+. The inclusion of proteinase inhibitors during isolation of the kinase C inhibitors, as well as two-dimensional peptide mapping and amino acid analysis of the isolated proteins, suggested that the 12 kDa inhibitor is a proteolytic fragment of the 17 kDa protein which is generated during purification. Antibodies raised in rabbits against the bovine brain 17 kDa inhibitor were shown to be specific by Western immunoblotting and the competitive enzyme-linked immunosorbent assay method and were used to study the tissue and species distribution of this protein. The inhibitor was found to be present in several bovine, murine, avian and human tissues, consistent with a role in the regulation of a variety of physiological functions involving the widely distributed protein kinase C.     

Evidence for the calcium-dependent activation of phospholipase D in thrombin-stimulated human erythroleukaemia cells.

Human erythroleukaemia (HEL) cells were exposed to thrombin and other platelet-activating stimuli, and changes in radiolabelled phospholipid metabolism were measured. Thrombin caused a transient fall in PtdInsP and PtdInsP2 levels, accompanied by a rise in diacylglycerol and phosphatidic acid, indicative of a classical phospholipase C/diacylglycerol kinase pathway. However, the rise in phosphatidic acid preceded that of diacylglycerol, which is inconsistent with phospholipase C/diacylglycerol kinase being the sole source of phosphatidic acid. In the presence of ethanol, thrombin and other agonists (platelet-activating factor, adrenaline and ADP, as well as fetal-calf serum) stimulated the appearance of phosphatidylethanol, an indicator of phospholipase D activity. The Ca2+ ionophore A23187 and the protein kinase C activator phorbol myristate acetate (PMA) also elicited phosphatidylethanol formation, although A23187 was at least 5-fold more effective than PMA. Phosphatidylethanol production stimulated by agonists or A23187 was Ca2(+)-dependent, whereas that with PMA was not. These result suggest that phosphatidic acid is generated in agonist-stimulated HEL cells by two routes: phospholipase C/diacylglycerol kinase and phospholipase D. Activation of the HEL-cell phospholipase D in response to agonists may be mediated by a rise in intracellular Ca2+.     

Phorbol esters inhibit phosphate uptake in opossum kidney cells: a model of proximal renal tubular cells

The effects of phorbol esters and diacylglycerol on phosphate uptake in opossum kidney (OK) cells were investigated to assess the possible role of Ca2+-activated, phospholipid dependent protein kinase (protein kinase C) on renal phosphate handling. OK cells are widely used as a model of proximal renal tubular cells and are reported to possess a Na+-dependent phosphate transport system. Phorbol-12,13-dibutyrate (PDBu) inhibited phosphate uptake. This inhibitory effect was synergistically enhanced with A23187. 4 beta-phorbol 12,13-didecanoate inhibited phosphate uptake, while 4 alpha-phorbol 12,13-didecanoate did not. 1-oleoyl-2-acetyl-glycerol (OAG), a synthetic diacylglycerol, also exhibited an inhibitory effect on phosphate uptake. These data suggest the possible involvement of protein kinase C in proximal renal tubular phosphate transport.     

Calcium-dependent phosphorylation of the amphibian oocyte plasma membrane: an early event in initiating the meiotic divisions

Plasma membranes isolated from Rana oocytes showed a 7-10 fold increase in the Ca2+-dependent phosphorylation of endogenous protein following exposure to meiotic stimuli (progesterone, insulin) either in vivo or in-vitro. Exogenous phosphatidylmonomethylethanolamine (PME) was effective in stimulating Ca2+-dependent membrane phosphorylation and also induced meiosis. Induction of phosphorylation was blocked by the protease inhibitor leupeptin, as are all other responses to meiotic stimuli. Phosphatidylserine was inactive when added to intact oocytes, but stimulated membrane phosphorylation nearly 15-fold when added to isolated membranes. The results indicate a link between phospholipid methylation and protein kinase C activation.     

Down regulation of phorbol diester receptors by proteolytic degradation of protein kinase C in a cultured cell line of fetal rat skin keratinocytes

Down regulation of phorbol diester receptors was studied with respect to proteolysis of protein kinase C, which is activated by Ca2+, phospholipids, and diacylglycerols and which binds to phorbol diesters. We used FRSK cells, a cell line derived from fetal rat skin keratinocytes, because in these cells specific binding of phorbol 12,13-dibutyrate decreased rapidly (50% decrease in 30 min). This decrease (down regulation) was inhibited by some protease inhibitors, such as N-tosyl-L-phenylalanine chloromethyl ketone (TPCK), N-p-tosyl-L-lysine chloromethyl ketone (TLCK), and leupeptin, but not by inhibitors of lysosomal hydrolases. On treatment with 12-O-tetradecanoylphorbol 13-acetate, protein kinase C was rapidly translocated from the cytosol to the membranes and then decreased. This decrease in protein kinase C was also inhibited by TPCK, TLCK, and leupeptin. The decrease in membrane activity of protein kinase C was associated with increase in cytosolic activity of a protein kinase that was smaller in molecular weight (Mr 40,000-60,000) than protein kinase C, did not depend on Ca2+/phosphatidylserine/diacylglycerol, and did not bind to phorbol 12,13-dibutyrate. These results indicate that down regulation of phorbol diester receptors is probably caused by nonlysosomal proteolysis of protein kinase C. The kinase formed by cleavage may be an active catalytic site of protein kinase C.     

Regulation of protein kinase C activity by gangliosides

The activity of protein kinase C (Ca2+/phospholipid-dependent enzyme) in the presence of phosphatidylserine and its physiological regulator, diacylglycerol, could be suppressed by a mixture of brain gangliosides. Half-maximal inhibition was observed at 30 microM and was nearly complete at 100 microM. Inhibition was observed at all concentrations of Ca2+ between 10(-8) and 10(-4) M. Inhibition of protein kinase C activity could not be reversed by increasing the concentration of diacylglycerol or the substrate, histone. Inhibition was also observed when myelin basic protein or a synthetic myelin basic protein peptide was used as substrate. Among the individual gangliosides, the rank order of potency was GT1b greater than GD1a = GD1b greater than GM3 = GM1. Our results suggest that gangliosides may regulate the responsiveness of protein kinase C to diacylglycerol.     

Differential activation of two protease-activated protein kinases from reticulocytes by a Ca2+-stimulated protease and identification of phosphorylated translational components

Two protein kinases have been partially purified from rabbit reticulocytes and shown to be activated by limited proteolysis with trypsin [S.M. Tahara and J.A. Traugh (1981) J. Biol. Chem. 256, 11558-11564; P.T. Tuazon, W.C. Merrick, and J.A. Traugh (1980) J. Biol. Chem. 255, 10954-10958]. Reticulocyte lysate was examined for protease activities which might be involved in activation of the protein kinases in vivo. Two neutral proteases, differentially activated by Fe2+ and Ca2+, were identified and partially purified. The Ca2+-stimulated protease specifically activated protease-activated kinase II; no effect was observed on protease-activated kinase I. The Fe2+-stimulated protease was not active on either protein kinase. The protease-activated kinases were examined using initiation factors (eIF) and 40-S ribosomal subunits as substrate. Protease-activated kinase I phosphorylated one subunit of eIF-3 (Mr 130000), eIF-4B and 40-S ribosomal protein S10. Protease-activated kinase II modified the beta subunit of eIF-2 (Mr 53000) and 40-S ribosomal protein S6. The substrate specificities are unique when compared with other cAMP-dependent and cAMP-independent protein kinases from reticulocytes.     

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.