Properties of protein kinase C subspecies in human platelets

Protein kinase C (PKC) from human platelets was resolved into two fractions by hydroxyapatite column chromatography. One of the enzymes was indistinguishable from the brain type III PKC having alpha-sequence in its kinetic and immunological properties. The other enzyme was kinetically different from any of the brain PKC subspecies so far isolated, although it resembled the brain type II PKC having beta-sequence. With H1 histone as substrate, this platelet enzyme was not very sensitive to Ca2+, and activated partly by phosphatidylserine plus diacylglycerol or by free arachidonic acid. Both platelet enzymes could phosphorylate the P47 protein in vitro, but the enzyme physiologically responsible for the P47 protein phosphorylation in the activated platelets remains to be identified.     

80K-H interacts with inositol 1,4,5-trisphosphate (IP3) receptors and regulates IP3-induced calcium release activity

Inositol 1,4,5-trisphosphate receptors (IP3Rs) are intracellular channel proteins that mediate calcium (Ca2+) release from the endoplasmic reticulum, and they are involved in many biological processes (e.g. fertilization, secretion, and synaptic plasticity). Recent reports show that IP3R activity is strictly regulated by several interacting molecules (e.g. IP3R binding protein released with inositol 1,4,5-trisphosphate, huntingtin, presenilin, DANGER, and cytochrome c), and perturbation of this regulation causes intracellular Ca2+ elevation leading to several diseases (e.g. Huntington disease and Alzheimer disease). In this study, we identified protein kinase C substrate 80K-H (80K-H) to be a novel molecule interacting with the COOH-terminal tail of IP3Rs by yeast two-hybrid screening. 80K-H directly interacted with IP3R type 1 (IP3R1) in vitro and co-immunoprecipitated with IP3R1 in cell lysates. Immunocytochemical and immunohistochemical staining revealed that 80K-H colocalized with IP3R1 in COS-7 cells and in hippocampal neurons. We also showed that the purified recombinant 80K-H protein directly enhanced IP3-induced Ca2+ release activity by a Ca2+ release assay using mouse cerebellar microsomes. Furthermore 80K-H was found to regulate ATP-induced Ca2+ release in living cells. Thus, our findings suggest that 80K-H is a novel regulator of IP3R activity, and it may contribute to neuronal functions.     

Ca(2+)-independent, phospholipid-activated protein kinase in 3Y1 cells.

We obtained a Ca(2+)-independent but 12-O-tetradecanoyl phorbol ester (TPA).phospholipid-activated protein kinase from rat embryo fibroblast 3Y1 cells by succeeding steps of DEAE-cellulose, H-9 affinity, and hydroxylapatite chromatography. This kinase was separated chromatography. This kinase was separated from a conventional PKC (Type III), by H-9 affinity column chromatography. The major peak from H-9 affinity column was eluted at 0.4 M of arginine and on the following step of hydroxylapatite column chromatography, at the KPO4 concentration of 0.1 M. The enzyme could be stimulated by phospholipids and by the tumor promoter TPA, but did not respond to calcium. The Ca(2+)-independent, phospholipid-activated protein kinase activity was susceptible to the protein kinase C inhibitors H-7 and K252a, but showed a phospholipid dependency and substrate specificity distinct from the conventional types of PKC. This protein kinase did not react with monoclonal antibodies against Types I, II, and III PKC. The activity of this enzyme was specifically reduced by immunoprecipitation, depending on the concentration of the polyclonal antibody, PC-delta, which was raised against a peptide synthesized according to a sequence of rat brain nPKC delta. The enzyme had a Mr of 76,000 as estimated by Western blotting. These results provide evidence for a unique type of Ca(2+)-independent, phospholipid-activated kinase, as expressed in 3Y1 cells.     

Endogenous substrates of rat heart protein kinase C type I, II, and III isozymic forms in cardiac sarcolemma.

The endogenous substrate proteins of rat cardiac protein kinase C type I, II, and III isozymic forms were studied in rat cardiac sarcolemma. The 19-, 21-, 29-, 35-, and 95-kDa proteins were phosphorylated by both types II and III, but not type I. The extent of phosphorylation by individual protein kinase C isozymic forms was additive and equal to the extent of phosphorylation observed when a mixture of isozymic forms was employed. The extent of phosphorylation of the 21-kDa protein by type III was much higher than that by type II. These results suggest that the protein kinase C isozymes have preferences for specific endogenous substrate proteins. The phosphorylation of these endogenous substrate proteins by protein kinase C isozymes probably plays a role in cardiac cell functions.     

Studies on protein kinase C tightly-bound to rat liver plasma membrane and its protease-activated form

1. Rat liver plasma membrane contained two types of protein kinase C which could be extracted by Ca2(+)-chelator and detergent, respectively. The activities of these two enzymes were nearly equivalent. 2. The detergent-extracted protein kinase C, tightly-bound to membrane, was separated into two subtypes by hydroxyapatite column chromatography. Based on the elution profile and the Ca2+/phospholipid requirement, the major and the minor components were identified as type III and type II protein kinase C, respectively. 3. The detergent-extracted protein kinase C was converted to an active fragment with Mr 45,000 by limited proteolysis with trypsin. Incubation under physiological level of ionic strength increased the stability of this active enzyme and protected it from further inactivation by trypsin. 4. Phosphorylation of H1 histone by the protease-activated kinase was stimulated 1.5-2-fold by phosphatidylserine. However, this enzyme phosphorylated multiple proteins in rat liver subcellular fractions in Ca2(+)- and phospholipid-independent manner. 5. These results suggest that the protein kinase C (mainly type III enzyme) tightly-bound to rat liver plasma membrane may have important role through protein phosphorylation by the native or the protease-activated kinase.     

Ca2+-phospholipid-dependent phosphorylation of vesicular preparations of myocardial sarcolemma

A Ca2+-phospholipid-dependent protein kinase C was isolated from the soluble fraction of bovine brain, using hydrophobic chromatography on phenyl-Sepharose CL-4B and high performance liquid chromatography on a Mono Q column. The enzyme had a specific activity of 822 nmol 32P/mg protein/min with histone H1 as a substrate. Phosphorylation of pig myocardium sarcolemma protein substrates was stimulated by Ca2+ and phosphatidylserine; the optimal concentrations of these compounds were 10(-4) M and 200 micrograms/ml, respectively. The value of Km(app) for Ca2+ was 3.10(-6) M. An addition of exogenous dioleine increased the enzyme affinity for Ca2+ which led to a decrease of Ca2+ concentration necessary for the maximal activation to occur. The optimal concentration of ATP needed for sarcolemmal preparation phosphorylation was 0.3-0.4 mM, which seems to be due to the high activity of sarcolemmal ATPases. The proteins phosphorylated in sarcolemmal preparations were identified, using SDS polyacrylamide gel electrophoresis with subsequent autoradiography. The 250, 140, 67, 58, 25 and 11 kD proteins appeared to be phosphorylated in the greatest degree. Since in myocardial sarcolemma protein kinase C predominantly phosphorylates the same proteins as does the cAMP-dependent protein kinase, it was assumed that protein kinase C can also play a role in the regulation of Ca2+-transporting systems of sarcolemma.     

Protein kinase C and an endogenous substrate associated with adenohypophyseal secretory granules.

Secretory granules isolated from anterior pituitary glands were examined for Ca2+/phospholipid-dependent protein kinase (protein kinase C) activity as well as the occurrence of granule-associated substrate proteins. Sheep adenohypophyses were fractionated by differential and sucrose-density-gradient centrifugation to yield a granule fraction enriched for luteinizing-hormone (lutropin)-containing secretory granules. Marker-enzyme analysis showed no detectable cytosolic contamination, although there were small amounts of plasma membranes (2-4%) and lysosomes (4-6%) associated with the preparation. As determined by histone-H1 phosphorylation after DEAE-cellulose DE-52 chromatography, protein kinase C activity with a marked dependence on Ca2+ and lipid (4-fold increase in their presence) was evident in the secretory-granule fraction. Phosphorylation in vitro of the secretory-granule fraction by endogenous and exogenous protein kinase C revealed a protein of Mr 36,000, which by two-dimensional SDS/polyacrylamide-gel electrophoresis showed multiple sites of phosphorylation. The Mr-36,000 protein was not found in cytosolic or plasma-membrane fractions and was not phosphorylated by the catalytic subunit of cyclic AMP-dependent protein kinase. Several secretory-granule proteins served as substrates for the catalytic subunit, the most prominent of which were of Mr 63,000, 23,000 and 21,000. From these data, we suggest that phosphorylation of secretory-granule-associated proteins by protein kinase C and by cyclic AMP-dependent protein kinase may be important in secretion regulation in the anterior pituitary gland.     

Rapid purification of protein kinase C from rat brain. A novel method employing protamine-agarose affinity column chromatography

We describe a rapid purification of protein kinase C from rat brain cytosol employing a specific substrate, protamine-coupled to agarose. Sequential chromatography on DEAE-Sephacel, phenyl-Sepharose CL-4B, and protamine-agarose columns resulted in a 1,500-fold purification of protein kinase C. SDS-PAGE analysis of the purified enzyme resolved a doublet protein of 77-80 kDa. This doublet was recognized by a polyclonal antiserum against protein kinase C. Proteolytic digestion of each protein band generated similar peptide fragments. The underlying principle of the protamine sulfate purification method was also clarified. Protamine can serve as a Ca2+/phospholipid-independent substrate. We demonstrate phosphorylation of protamine on the column; phosphorylated protamine did not bind the enzyme with the same affinity and this covalent modification was most probably responsible for releasing the bound enzyme from the column after addition of Mg2+ and ATP. The C kinase inhibitor, H7, inhibits protamine phosphorylation in a dose-dependent fashion but does not prevent binding of the enzyme to a protamine-agarose column. We therefore conclude that protamine interacts with the active center of the enzyme enabling it to be phosphorylated, upon which it loses its binding affinity for C kinase.     

The 38 kDa Ca2+/membrane-binding protein of pig granulocytes needs a high Ca2+ concentration to be phosphorylated by protein kinase C

The 38 kDa Ca2+/membrane-binding protein reported to be the dominant substrate of protein kinase C in the extracts of pig neutrophil granulocytes was purified partially and its phosphorylation was investigated. In pig granulocytes type II protein kinase C was the major isoform, while type III isoenzyme was present only as a minor activity. Phosphorylation of the 38 kDa protein was performed with rat brain protein kinase C. Each of the three isoenzymes purified from rat brain was able to phosphorylate this protein, though on the conditions used in our experiments it was phosphorylated most intensively by type II protein kinase C. A phospholipid-dependent, but Ca2(+)-independent, form of protein kinase C was demonstrated with the aid of a synthetic oligopeptide substrate. Phosphorylation of the 38 kDa protein by the Ca2(+)-independent enzyme proceeded exclusively in the presence of Ca2+. The Ca2+ concentration necessary for the phosphorylation of the 38 kDa by either form of protein kinase C was by orders of magnitude higher than that required for the activation of protein kinase C.     

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.     

Molecular cloning and chromosomal mapping of a cDNA encoding human 80K-L protein: major substrate for protein kinase C.

We have isolated and sequenced complementary DNA (cDNA) for the human 80K-L protein, a major substrate for protein kinase C and the human homologue of an 80- to 87-kDa bovine protein named MARCKS (myristoylated alanine-rich C kinase substrate). The human 80K-L cDNA encodes a protein of 332 amino acids with a calculated molecular weight of 31,534. Homology comparisons of the nucleotide sequences of the cDNAs indicated that their 3'-untranslated regions are more homologous than the coding regions. Spot blot hybridization using flow-sorted human chromosomes indicated that the gene encoding the 80K-L protein, designated MACS, maps to the q15----qter region of human chromosome 6, and it also suggested that a genomic region with a sequence homologous to the 3'-untranslated region of the 80K-L mRNA exists on chromosome 21.     

Fibroblast growth factor stimulates protein kinase C in quiescent 3T3 cells without Ca2+ mobilization or inositol phosphate accumulation

To elucidate the transmembrane signalling processes initiated by fibroblast growth factor (FGF), we have studied the effect of recombinant basic FGF (bFGF) on various early events associated with mitogenesis in Swiss 3T3 fibroblasts. bFGF, at mitogenic concentrations, neither induced Ca2+ mobilization from intracellular stores nor increased the accumulation of inositol phosphates. In contrast, bFGF stimulated the phosphorylation of the Mr 80,000 (80K) cellular protein which is a major substrate of protein kinase C. This effect was potentiated by the diacylglycerol kinase inhibitor R59022. Two-dimensional polyacrylamide gel electrophoresis and phosphopeptide mapping showed that the 80K phosphoproteins generated in response to bFGF, bombesin, and phorbol 12,13-dibutyrate were indistinguishable. Down-regulation of protein kinase C prevented bFGF stimulation of 80K phosphorylation. Other protein kinase C-dependent early events such as transmodulation of the epidermal growth factor receptor, cytoplasmic alkalinization, inhibition of vasopressin induced increase in cytosolic [Ca2+], and enhancement of cAMP accumulation in response to forskolin were also induced by bFGF. Similar results were obtained when bFGF was added to quiescent cultures of tertiary mouse embryo fibroblasts. We conclude that bFGF stimulates protein kinase C through a signal transduction pathway distinct from inositol phospholipid turnover and Ca2+ mobilization.     

The multifunctional Ca2+/calmodulin-dependent protein kinase mediates Ca2+-dependent phosphorylation of tyrosine hydroxylase

Stimulation of rat pheochromocytoma PC12 cells with ionophore A23187, carbachol, or high K+ medium, agents which increase intracellular Ca2+, results in the phosphorylation and activation of tyrosine hydroxylase (Nose, P., Griffith, L. C., and Schulman, H. (1985) J. Cell Biol. 101, 1182-1190). We have identified three major protein kinases in PC12 cells and investigated their roles in the Ca2+-dependent phosphorylation of tyrosine hydroxylase and other cytosolic proteins. A set of PC12 proteins were phosphorylated in response to both elevation of intracellular Ca2+ and to protein kinase C (Ca2+/phospholipid-dependent protein kinase) activators. In addition, distinct sets of proteins responded to either one or the other stimulus. The three major regulatory kinases, the multifunctional Ca2+/calmodulin-dependent protein kinase, the cAMP-dependent protein kinase, and protein kinase C all phosphorylate tyrosine hydroxylase in vitro. Neither the agents which increase Ca2+ nor the agents which directly activate kinase C (12-O-tetradecanoylphorbol-13-acetate or 1-oleyl-2-acetylglycerol) increase cAMP or activate the cAMP-dependent protein kinase, thereby excluding this pathway as a mediator of these stimuli. The role of protein kinase C was assessed by long term treatment of PC12 cells with 12-O-tetradecanoylphorbol-13-acetate, which causes its "desensitization." In cells pretreated in this manner, agents which increase Ca2+ influx continue to stimulate tyrosine hydroxylase phosphorylation maximally, while protein kinase C activators are completely ineffective. Comparison of tryptic peptide maps of tyrosine hydroxylase phosphorylated by the three protein kinases in vitro with phosphopeptide maps generated from tyrosine hydroxylase phosphorylated in vivo indicates that phosphorylation by the Ca2+/calmodulin-dependent kinase most closely mirrors the in vivo phosphorylation pattern. These results indicate that the multifunctional Ca2+/calmodulin-dependent protein kinase mediates phosphorylation of tyrosine hydroxylase by hormonal and electrical stimuli which elevate intracellular Ca2+ in PC12 cells.     

Amblyomma americanum (L.): protein kinase C-independent fluid secretion by isolated salivary glands.

Protein kinase C activity was partially purified from tick salivary glands by fast protein liquid chromatography anion-exchange chromatography. Enzyme activity was stimulated by Ca2+, phosphatidylserine, and diacylglycerol with the highest activity observed in the presence of all three modulators. Enzyme activity was inhibited by a synthetic pseudosubstrate peptide with an amino acid sequence resembling the protein kinase C substrate phosphorylation site. The protein kinase C activator, 1-oleoyl-2-acetyl-sn-glycerol (OAG), when added to whole in vitro salivary glands previously prelabeled with 32P, stimulated the phosphorylation of salivary gland proteins. Activators of protein kinase C (phorbol ester or OAG) did not stimulate fluid secretion by isolated tick salivary glands. OAG and phorbol ester had only minimal affects on the ability of dopamine to stimulate secretion by isolated salivary glands and dopamine's ability to increase salivary gland cyclic AMP.     

Comparison of substrate recognition by protein kinase C (type III) between rat liver cytosolic and particulate fractions

1. Phosphorylation of rat liver endogenous substrates by protein kinase C (type III) was compared between cytosolic and particulate (mitochondria, microsomes and plasma membrane) fractions. 2. The rate and the maximum level of protein phosphorylation were several-fold higher in particulate fractions than in cytosolic fraction. 3. Protein phosphorylation in cytosolic fraction was dependent on both Ca2+ and phospholipid, but only Ca2+ was necessary in phosphorylation of particulate fractions. 4. These results suggest that protein kinase C (type III) has much more target proteins in particulate fractions rather than in cytosolic fraction and Ca2+ was important regulator in particulate protein phosphorylation.     

A calmodulin-dependent protein kinase in Rous sarcoma virus-transformed rat cells and normal liver

A calmodulin-dependent protein kinase has been purified extensively from a Rous sarcoma virus-transformed rat cell line (RR1022) and from normal rat liver. The calmodulin-dependent protein kinase activity was manifested by in vitro phosphorylation of a single Mr 57 000 endogenous phosphoprotein (pp57) present in both the virally transformed cells and normal rat liver. The calmodulin-dependent protein kinase from transformed cells fractionated with the viral src gene product, pp60v-src, through a 650-fold purification of the oncogene product. However, purification of the calmodulin-dependent protein kinase from normal liver demonstrated that the calmodulin-dependent kinase was distinct from pp60v-src. Phosphorylation of pp57 by the kinase purified from the transformed cell line required Ca2+ and calmodulin, was inhibited by EDTA and was unaffected by cAMP or the heat- and acid-stable protein inhibitor of cAMP-dependent protein kinase. Troponin C did not substitute for calmodulin. A virtually identical calmodulin-dependent protein kinase activity was purified from rat liver by affinity chromatography on calmodulin-Sepharose. Phosphorylation of pp57 by the affinity-purified liver protein kinase was also observed, and required Ca2+ and calmodulin. EGTA and trifluoroperazine inhibited pp57 phosphorylation. The calmodulin-dependent protein kinase reported here did not phosphorylate substrates of known calmodulin-dependent protein kinases in vitro (myosin light chain, phosphorylase b, glycogen synthase, microtubule-associated proteins, tubulin, alpha-casein). Because none of these proteins served as substrates in vitro and pp57 was the only endogenous substrate found, the properties of this enzyme appear to be different from any previously described calmodulin-dependent protein kinase.     

Association of a myristoylated protein with a biological membrane and its increased phosphorylation by protein kinase C

A hydrophilic enzyme, lysozyme, was myristoylated in vitro by the N-hydroxysuccinimide ester of myristic acid, and the monomyristoylated lysozyme was isolated by CM-cellulose cation-exchange column chromatography. The monomyristoylated lysozyme associated with phospholipid vesicles, whereas the association of native lysozyme was negligible. The membrane-associated monomyristoylated lysozyme was phosphorylated with partially purified rat brain Ca2+- and phospholipid-dependent protein kinase (protein kinase C) in the presence of Ca2+, phosphatidylserine and phorbolmyristate acetate. Thus, the myristoylated lysozyme became a substrate of protein kinase C through its hydrophobic association with the membrane. The present results suggest that the myristoylation of cytoplasmic proteins may have an important role in signal transduction.     

Protein phosphorylation and secretion in digitonin-permeabilized adrenal chromaffin cells. Effects of micromolar Ca2+, phorbol esters, and diacylglycerol

The effects of phorbol esters, dioctanoylglycerol (DiC8), and micromolar Ca2+ on protein phosphorylation and catecholamine secretion in digitonin-treated chromaffin cells were investigated. [gamma-32P]ATP was used as a substrate for phosphorylation in the permeabilized cells. 12-O-Tetradecanoylphorbol-13-acetate (TPA) enhanced Ca2+-dependent catecholamine secretion from digitonin-permeabilized cells. The enhancement required MgATP. Only those phorbol esters which activate protein kinase C in vitro enhanced both catecholamine secretion and protein phosphorylation. DiC8, which activates protein kinase C in vitro and mimics phorbol ester effects in situ, also enhanced both catecholamine secretion and protein phosphorylation. Preincubation of intact cells with TPA or DiC8 was necessary for maximal effects on both catecholamine secretion and protein phosphorylation in subsequently digitonin-treated chromaffin cells. The TPA-induced enhancement of protein phosphorylation was almost entirely Ca2+-independent, whereas DiC8-induced enhancement of protein phosphorylation was mainly Ca2+-dependent. Micromolar Ca2+ alone also enhanced the phosphorylation of a large number of proteins. Most of the proteins phosphorylated in response to TPA or potentiated by DiC8 in combination with Ca2+ were also phosphorylated by micromolar Ca2+ in the absence of exogenous protein kinase C activators. In intact cells, 1,1-dimethyl-4-phenylpiperazinium (DMPP) induced Ca2+-dependent phosphorylation of at least 17 proteins which were detected by two-dimensional gel electrophoresis. All of the proteins phosphorylated upon incubation with 1,1-dimethyl-4-phenylpiperazinium were phosphorylated upon incubation with micromolar Ca2+ in digitonin-treated cells. These results demonstrate that TPA- or DiC8-enhanced Ca2+-dependent catecholamine secretion is associated with enhanced protein phosphorylation which is probably mediated by protein kinase C and that activation of protein kinase C modulates catecholamine secretion from digitonin-treated chromaffin cells.