The identification and purification of a mammalian-like protein kinase C in the yeast Saccharomyces cerevisiae.

We have purified a yeast protein kinase that is phospholipid-dependent and activated by Diacylglycerol (DAG) in the presence of Ca2+ or by the tumour-promoting agent tetradecanoyl-phorbol acetate (TPA). The properties of this enzyme are similar to those of the mammalian protein kinase C (PKC). The enzyme was purified using chromatography on DEAE-cellulose followed by hydroxylapatite. The latter chromatography separated the activity to three distinguishable sub-species, analogous to the mammalian PKC isoenzymes. The fractions enriched in PKC activity contain proteins that specifically bind TPA, are specifically phosphorylated in the presence of DAG and recognized by anti-mammalian PKC antibodies.     

Characterization of calcium-independent forms of protein kinase C-beta in phorbol ester-treated rabbit platelets

The subcellular distribution, size, and activation state of protein kinase C (PKC) were studied after short term exposure of rabbit platelets to a saturating dose of 12-O-tetradecanoylphorbol 13-acetate (TPA). Cytosolic and Nonidet P-40-solubilized particulate extracts prepared from TPA-treated platelets were subjected to analytical column chromatography on Mono Q, hydroxylapatite, and Superose 6/12. PKC activity was assayed according to the ability of the enzyme to phosphorylate (i) histone H1 in the presence of the activators calcium, diacylglycerol, and phosphatidylserine; (ii) histone H1 after proteolytic activation of PKC with trypsin; and (iii) protamine in the absence of calcium and lipid. Within 1 min of TPA treatment of platelets, greater than 95% of the PKC activity was particulate associated, as assessed by all three methods. The particulate PKC activity from 1-min TPA-treated cells eluted from Mono Q with approximately 0.35 M NaCl (peak I), and it was highly dependent upon Ca2+ and lipid for optimal histone H1 phosphorylation. With longer exposure times of platelets to TPA, the disappearance of the Mono Q peak I form of PKC was correlated with the production of new PKC species that were released from Mono Q with approximately 0.4 M NaCl (peak II), approximately 0.5 M NaCl (peak III), and approximately 0.6 M NaCl (peak IV). These last forms of PKC were still lipid activated but exhibited little Ca2+ dependence. The Mono Q peak III form displayed a particularly high level of histone H1 phosphorylating activity in the absence of lipid and Ca2+. All of these forms behaved as approximately 65-kDa proteins on Superose 6/12, but on sodium dodecyl sulfate-polyacrylamide gels, Western blotting with anti-PKC-beta antibodies revealed immunoreactive polypeptides of approximately 79 kDa (Mono Q peaks I, II, and IV) and approximately 100-kDa (Mono Q peak III). Hydroxylapatite column chromatography permitted partial resolution of the Mono Q peaks I and II forms, which were eluted within a concentration range of potassium phosphate (100-150 mM) which was typical of the beta isozyme of PKC. Treatment of the Mono Q peak III and IV PKC forms with alkaline phosphatase resulted in the production of the peak I form, which implicated protein phosphorylation in the interconversion of the various PKC forms.     

N-(2-Aminoethyl)-5-isoquinolinesulfonamide, a newly synthesized protein kinase inhibitor, functions as a ligand in affinity chromatography. Purification of Ca2+-activated, phospholipid-dependent and other protein kinases

We designed a simple procedure for the purification of Ca2+-activated, phospholipid-dependent protein kinase (protein kinase C) from rabbit brain, using affinity chromatography with a new affinity adsorbent. The adsorbent was synthesized by attaching the amino residue of N-(2-aminoethyl)-5-isoquinolinesulfonamide (H-9) to cyanogen bromide-activated Sepharose. H-9 is a potent competitive inhibitor of protein kinase C, cGMP-, and cAMP-dependent protein kinase with respect to ATP and exhibits inhibition constants of 18, 0.87, and 1.9 microM, respectively (Hidaka, H., Inagaki, M., Kawamoto, S., and Sasaki, Y. (1984) Biochemistry, 23, 5036). A 960-fold purification was achieved in the two-step procedure, which entailed DEAE-cellulose and the affinity chromatography. The resultant preparation was essentially homogeneous, as indicated by polyacrylamide gel electrophoresis under conditions of denaturation with sodium dodecyl sulfate. The affinity of protein kinase C for the H-9-Sepharose was high, and the enzyme could not be eluted either by a high concentration of sodium chloride or by 40% glycerol. The protein kinase C could be eluted from H-9-Sepharose by the buffer containing both 0.2 M NaCl and 20% glycerol, thereby suggesting that the binding between protein kinase C and H-9-Sepharose was due to both hydrophobic and electrostatic interactions. H-9 coupled to Sepharose retained both cyclic nucleotide-dependent protein kinases and protein kinase C, and these enzymes could be eluted separately by the buffer containing L-arginine, a potent inhibitor of these three kinases. The novel aspects of these three multifunctional protein kinases can thus be investigated using isoquinolinesulfonamide derivatives.     

Biochemical and immunological studies of protein kinase C from Trypanosoma cruzi.

Phorbol ester binding was studied in protein kinase C-containing extracts obtained from Trypanosoma cruzi epimastigote forms. Specific 12-O-tetradecanoyl phorbol 13-acetate, [3H]PMA, or 12,13-O-dibutyryl phorbol, [3H]PDBu, binding activities, determined in T. cruzi epimastigote membranes, were dependent on ester concentration with a Kd of 9x10(-8) M and 11.3x10(-8) M, respectively. The soluble form of T. cruzi protein kinase C was purified through DEAE-cellulose chromatography. Both protein kinase C and phorbol ester binding activities co-eluted in a single peak. The DEAE-cellulose fraction was further purified into three subtypes by hydroxylapatite chromatography. These kinase activity peaks were dependent on Ca2+ and phospholipids and eluted at 40 mM (PKC I), 90 mM (PKC II) and 150 mM (PKC III) phosphate buffer, respectively. Western blot analysis of the DEAE-cellulose fractions, using antibodies against different isoforms of mammalian protein kinase C enzymes, revealed that the parasite expresses high levels of the alpha-PKC isoform. Immunoaffinity purified T. cruzi protein kinase C, isolated with an anti-protein kinase C antibody-sepharose column, were subjected to phosphorylation in the absence of exogenous phosphate acceptor. A phosphorylated 80 kDa band was observed in the presence of Ca2+, phosphatidylserine and diacylglycerol.     

Purification and characterization of a calcium-unresponsive, phorbol ester/phospholipid-activated protein kinase from porcine spleen

A calcium-unresponsive, phorbol ester/phospholipid-activated protein kinase was purified to apparent homogeneity from a Triton X-100 extract of an EGTA/EDTA-preextracted particulate fraction of porcine spleen by chromatography on S-Sepharose Fast Flow, phenyl-Sepharose Fast Flow, protamine-agarose, and Superdex 200. The enzyme had a Mr of 76,000, as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (p76-kinase). A similar value (78,000) was obtained by gel filtration. The purified p76-kinase proved to be much more stable than the enzyme in crude preparations. Storage in a buffer containing 50 mM mercaptoethanol and 20% glycerol at -20 degrees C for at least 4 months caused less than 20% loss in enzyme activity. The enzyme exhibited a pH optimum of 8.3. The affinity of the novel enzyme for substrates and cofactors differed to some extent from that of conventional alpha, beta, gamma protein kinase C (PKC). p76-kinase did not respond to calcium, had a lower requirement for magnesium, and a higher affinity for histone III-S than PKC. Both the p76-kinase-catalyzed phosphorylation of histone III-S and the autophosphorylation of the enzyme could be activated by the phorbol ester TPA (or diacylglycerol) plus phosphatidyl serine, but not by calcium plus phosphatidyl serine. The stoichiometry of autophosphorylation suggested that fully phosphorylated p76-kinase contained two phosphoserine residues and one phosphothreonine residue. Like PKC, p76-kinase bound TPA with high affinity (KD = 9.6 nM). In the absence of TPA, various unsaturated fatty acids, particularly arachidonic acid, were more potent as activators of the enzyme than phosphatidyl serine. The p76-kinase was recognized by an antiserum raised against a delta PKC-specific peptide, but not by an alpha, beta, gamma PKC-specific antiserum. The previously described p82-kinase of mouse epidermis and spleen exhibiting the same properties as the p76-kinase did also react with the p76-kinase-specific antiserum.     

Differential down-regulation of protein kinase C subspecies in KM3 cells

The down-regulation of protein kinase C (PKC) subspecies in KM3 cells (a pre-B, pre-T cell line) has been examined. The PKC from KM3 cells was resolved into two subspecies, type II (mainly beta II) and type III (alpha), upon hydroxyapatite column chromatography. Biochemical and immunocytochemical analysis revealed that, when these cells were treated with 12-O-tetradecanoylphorbol 13-acetate (TPA), the time course of down-regulation of the PKC subspecies was different; type II PKC was translocated and depleted from the cell more quickly than type III enzyme. The results suggest that each PKC subspecies plays a different role in the cellular response to TPA and probably to other external stimuli.     

Translocation of protein kinase C is not required to inhibit the antigen-induced increase of cytosolic calcium in a mast cell line

Cross-linking of receptor bound IgE antibodies by multivalent antigen (DNP8-BSA) on PB-3c cells leads to an increase of cytosolic calcium ((Ca2+)i). Active tumor promoting phorbol esters and teleocidin which specifically activate the phospholipid Ca2+-sensitive protein kinase (PKC), inhibited the antigen-mediated rise in (Ca2+)i and induced a time and dose-dependent translocation of cytosolic PKC to membranes of the PB-3c cells as determined by enzyme activity or immunoblotting using a polyclonal anti-PKC antibody. This TPA concentration did not affect the subcellular distribution of PKC, although 1 nM of 12-O-tetradecanoylphorbol-13-acetate (TPA) inhibited to 50% the antigen-mediated increase in (Ca2+)i. The concentration of TPA required to induce a half-maximal subcellular redistribution of immunodetectable PKC activity was an order of magnitude greater than the half-maximal dose required to inhibit the antigen-mediated increase in (Ca2+)i. These data demonstrate that the TPA-dependent activation of PKC is not directly coupled to its translocation to membranes.     

Ca(2+)-dependent protein kinase from the halotolerant green alga Dunaliella tertiolecta: partial purification and Ca(2+)-dependent association of the enzyme to the microsomes.

Ca(2+)-dependent protein kinase (CDPK) was purified 900-fold from the soluble fraction of Dunaliella tertiolecta cells by ammonium sulfate precipitation, DEAE-Toyopearl, phenyl-Sepharose, and hydroxylapatite column chromatography. The CDPK was activated by micromolar concentration of Ca2+ and required neither calmodulin nor phospholipids for its activation. The enzyme phosphorylated casein, myosin light chain, and histone type III-S (histone H-1), but did not phosphorylate protamine and phosvitin. The Km values for ATP and casein were 11 microM and 300 micrograms/ml, respectively. Phosphorylation of casein was inhibited by calmodulin antagonists, calmidazolium, trifluoperazine, and compound 48/80, but not affected by calmodulin. CDPK bound to phenyl-Sepharose in the presence of Ca2+ and was eluted by ethylene glycol bis(beta-aminoethyl ether) N,N'-tetraacetic acid (EGTA). This suggests that hydrophobicity of the enzyme was increased by Ca2+. CDPK was also bound to the microsomes isolated from Dunaliella cells in the presence of micromolar concentration of Ca2+ and released in the presence of EGTA, suggesting the possibility of in vivo Ca(2+)-dependent association of the enzyme. The enzyme phosphorylated many proteins in the microsomes but few in the cytosol, if at all.     

Identification of isoenzymic forms of hepatic Ca2+-activated-phospholipid-dependent protein kinase in various animal models

We have examined the protein kinase C that are present in mouse, rat, guinea pig and rabbit liver. Initial subcellular fractionation analysis indicated that the majority (75-85%) of the activity was associated with particulate fraction of the liver. The bound protein kinase C was dissociated by homogenization of livers in buffer containing EGTA, EDTA and various proteolytic inhibitors and the solubilized extract was used to resolve multiple forms of the enzyme. The fractionation procedure, sequentially utilized (NH4)2SO4 precipitation, ion exchange chromatography, gel permeation chromatography, and hydroxylapatite column chromatography. With hydroxylapatite, protein kinase C was resolved into three isoenzymic forms designated C-I, C-II and C-III. In each case, the predominant activity consisted of C-II and C-III and together they represented about 80-88% of the total activity. All three isoenzymes from each source demonstrated an absolute requirement for PS + Ca2+ (approximately 25-50 fold stimulation over basal activity); for maximal activity the isoenzymes also required the presence of divalent metal ion, Mg2+ (5-10 mM) and lysine rich histone (H1). Both diolein and TPA decreased the Ca2+ and PS requirement of the enzyme and directly stimulated enzyme activity in the presence of suboptimal concentrations of Ca2+ and PS. In conclusion, the present studies suggest that protein kinase C in mammalian liver exists in isoenzymic forms.     

Involvement of different protein kinases and phospholipases A2 in phorbol ester (TPA)-induced arachidonic acid liberation in bovine platelets

The effect of various phospholipase A2 and protein kinase inhibitors on the arachidonic acid liberation in bovine platelets induced by the protein kinase activator 12-O-tetradecanoylphorbol-13-acetate (TPA) was studied. TPA stimulates arachidonic acid release mainly by activating group IV cytosolic PLA2 (cPLA2), since inhibitors of this enzyme markedly inhibited arachidonic acid formation. However, group VI Ca2+-independent PLA2 (iPLA2) seems to contribute to the arachidonic acid liberation too, since the relatively specific iPLA2 inhibitor bromoenol lactone (BEL) decreased arachidonic acid generation in part. The pronounced inhibition of the TPA-induced arachidonic acid release by the protein kinase C (PKC) inhibitors GF 109203X and Ro 31-82220, respectively, and by the p38 MAP kinase inhibitor SB 202190 suggests that the activation of the PLA2s by TPA is mediated via PKC and p38 MAP kinase.     

zeta-Related protein kinase C in nuclei of nerve cells

To determine whether or not PKC is present in the nuclei of nerve tissue we made use of biochemical and immunocytochemical techniques. A 219-fold purification of rabbit brain nuclear protein kinase C was achieved by sequential steps of Triton X-100 extraction of isolated nuclei, DEAE-cellulose, Butyl-toyopearl and hydroxylapatite chromatography. The major peak of protein kinase C activity was eluted from the hydroxylapatite column at the KPO4 concentration of 0.3 M. Both Ca2+ and Ptd Ser were required for stimulation of the enzyme. Immunoblot analysis revealed that the kinase fraction was immunoreactive with a polyclonal antibody, PC-zeta, that had been raised against a peptide synthesized according to the deduced sequence of rat zeta protein kinase C. Light-microscopy revealed strong immunoreactivity in the nuclei of Purkinje cells in cerebellum and pyramidal cells in the rat cerebral cortex. These observations suggest that a zeta-related protein kinase C is present in the nuclei of nerve cells.     

Protein kinase C activation by platelet-activating factor is independent of enzyme translocation

The subcellular distribution and activation state of protein kinase C (PKC) was studied after short-term exposure of rabbit platelets to platelet-activating factor (PAF). Cytosolic and nonidet P-40-solubilized particulate extracts prepared from treated platelets were subjected to analytical column chromatography on MonoQ, hydroxylapatite and Superose 6/12. PKC activity was assayed by the ability of the enzyme to phosphorylate the following substrates: (i) histone H1 in the presence of the activators calcium, diacylglycerol and phosphatidylserine; (ii) histone H1 following proteolytic activation of PKC with 0.5 micrograms trypsin/ml; and (iii) protamine in the absence of calcium and lipid. PAF treatment for 1-20 min elicited a rapid 2-4-fold activation of both cytosolic and particulate-derived PKC as assessed by all three methods. On the other hand, there were no significant PAF-induced changes in the level of [3H]phorbol-12,13-dibutyrate binding by soluble and particulate-associated PKC. Hydroxyapatite column chromatography revealed that in non-treated rabbit platelets the type II (beta) form of PKC predominated, but PAF appeared to induce a shift in the elution profile from this resin. The stability of the PAF activation of PKC to column chromatography and the altered binding affinity to hydroxylapatite indicated that the stimulation might be a consequence of covalent modification, albeit minor, since PKC still eluted as an 80 kDa protein from Superose 6/12. As the PAF-induced increases in the kinase activity of PKC were preserved even after proteolytic activation with trypsin, but were without effect on the phorbol ester binding activity, such a putative modification may have occurred within or near the catalytic domain of PKC. These findings imply that PAF may directly modulate the activity of preexisting membrane-associated PKC by a novel mechanism, rather than by eliciting its recruitment from the cytoplasm.     

Estrogen modulates Ca(2+)-independent lipid-stimulated kinase in the rabbit corpus luteum of pseudopregnancy. Identification of luteal estrogen-modulated lipid-stimulated kinase as protein kinase C delta.

Rabbit corpora lutea were tested for the presence of phosphorylative responses sensitive to estrogen. Luteal Ca(2+)-independent lipid-stimulated kinase activity was detected by phosphorylation of the endogenous substrate, p76. Estrogen treatment, by way of estradiol-17 beta implant, increased levels of the lipid-stimulated phosphoprotein 2-3-fold throughout pseudopregnancy. Midpseudopregnant rabbit luteal extracts were further evaluated to determine the identity of the lipid-stimulated kinase. Results of low pH-activated phosphorylation were consistent with the identification of p76 as an autophosphorylated member of the protein kinase C (PKC) family. Partial purification of the luteal lipid-stimulated kinase was performed using sequential DEAE-cellulose/hydroxylapatite chromatographies and using gel filtration. Western immunoblot with type-specific anti-PKC delta antiserum showed coelution of kinase p76 activity with immunoreactive PKC delta. Immunoblot analysis confirmed that luteal levels of PKC delta were increased by estrogen treatment.     

Mechanism of diacylglycerol-induced membrane targeting and activation of protein kinase Ctheta

Protein kinase C (PKC) is a novel PKC that plays a key role in T lymphocyte activation. PKC has been shown to be specifically recruited to the immunological synapse in response to T cell receptor activation. To understand the basis of its unique subcellular localization properties, we investigated the mechanism of in vitro and cellular sn-1,2-diacylglycerol (DAG)-mediated membrane binding of PKC. PKC showed phosphatidylserine selectivity in membrane binding and kinase action, which contributes to its translocation to the phosphatidylserine-rich plasma membrane in HEK293 cells. Unlike any other PKCs characterized so far, the isolated C1B domain of PKC had much higher affinity for DAG-containing membranes than the C1A domain. Also, the mutational analysis indicates that the C1B domain plays a predominant role in the DAG-induced membrane binding and activation of PKC. Furthermore, the Ca(2+)-independent C2 domain of PKC has significant affinity for anionic membranes, and the truncation of the C2 domain greatly enhanced the membrane affinity and enzyme activity of PKC. In addition, membrane binding properties of Y90E and Y90F mutants indicate that phosphorylation of Tyr(90) of the C2 domain enhances the affinity of PKC for model and cell membranes. Collectively, these results show that PKC has a unique membrane binding and activation mechanism that may account for its subcellular targeting properties.     

Involvement of protein kinase C, tyrosine kinases, and Rho kinase in Ca(2+) handling of human small arteries

The mechanisms of Ca(2+) handling and sensitization were investigated in human small omental arteries exposed to norepinephrine (NE) and to the thromboxane A(2) analog U-46619. Contractions elicited by NE and U-46619 were associated with an increase in intracellular Ca(2+) concentration ([Ca(2+)](i)), an increase in Ca(2+)-independent signaling pathways, or an enhancement of the sensitivity of the myofilaments to Ca(2+). The two latter pathways were abolished by protein kinase C (PKC), tyrosine kinase (TK), and Rho-associated protein kinase (ROK) inhibitors. In Ca(2+)-free medium, both NE and U-46619 elicited an increase in tension that was greatly reduced by PKC inhibitors and abolished by caffeine or ryanodine. After depletion of Ca(2+) stores with NE and U-46619 in Ca(2+)-free medium, addition of CaCl(2) in the continuous presence of the agonists produced increases in [Ca(2+)](i) and contractions that were inhibited by nitrendipine and TK inhibitors but not affected by PKC inhibitors. NE and U-46619 induced tyrosine phosphorylation of a 42- or a 58-kDa protein, respectively. These results indicate that the mechanisms leading to contraction elicited by NE and U-46619 in human small omental arteries are composed of Ca(2+) release from ryanodine-sensitive stores, Ca(2+) influx through nitrendipine-sensitive channels, and Ca(2+) sensitization and/or Ca(2+)-independent pathways. They also show that the TK pathway is involved in the tonic contraction associated with Ca(2+) entry, whereas TK, PKC, and ROK mechanisms regulate Ca(2+)-independent signaling pathways or Ca(2+) sensitization.     

Activation of Protein Kinase C Suppresses the γ-Aminobutyric AcidB Receptor-Mediated Inhibition of the Vesicular Release of Noradrenaline and Acetylcholine

Modulation of the gamma-aminobutyric acidB (GABAB) receptor-mediated response by protein kinase C (PKC) was examined with regard to inhibition by stimulation of the GABAB receptor of stimulation-evoked release of noradrenaline (NA) from slices of cerebellar cortex and of acetylcholine (ACh) from strips of ileum. 12-O-Tetradecanoylphorbol 13-acetate (TPA) potentiated the high K(+)-evoked Ca2+-dependent release of NA and ACh, but not the ouabain-evoked release, even in the presence of external Ca2+. The potentiating effect was antagonized by sphingosine, thereby suggesting that PKC participates in the exocytotic-vesicular release of neurotransmitters, but does not do so in case of a nonvesicular release. GABA inhibited the high K(+)-evoked release of NA and ACh, but not the ouabain-evoked Ca(2+)-independent release. The effect of GABA was mimicked by baclofen and was antagonized by phaclofen, thereby suggesting that stimulation of the GABAB receptor inhibits the vesicular but not the nonvesicular release of neurotransmitters. TPA suppressed the GABAB receptor-mediated inhibition of high K(+)-evoked release of NA and ACh. The effect of TPA was antagonized by sphingosine. These results indicate that stimulation of the GABAB receptor inhibits the stimulation-evoked Ca(2+)-dependent release of neurotransmitters and that activation of PKC suppresses the GABAB receptor-mediated response.     

Evidence for the presence of protein kinases which stimulate phosphorylation of c-erb A protein in rat kidney nuclei.

Protein kinases were separated from rat kidney nuclear extract by hydroxylapatite column chromatography. Five (I-V) different protein kinases were isolated when histone was used as a substrate. Two (I and III) of them stimulated phosphorylation of c-erb A-beta protein (50 kDa) expressed in Escherichia coli. The c-erb A product has an activity of high affinity T3 binding. One (I) of the kinases was dependent on cyclic adenosine 3',5'-monophosphate (cyclic AMP). The other kinase (III) was not dependent on cyclic nucleotides. The latter kinase was eluted from hydroxylapatite column with 0.05 M PO4 at pH 7.4. The sedimentation coefficient(s) estimated by continuous sucrose density gradient centrifugation was approximately 6.0 Km values for ATP were estimated by double reciprocal analyses, which gave 110.0 microM in the protein kinase I (in the presence of 10(-6) M cyclic AMP) and 25 microM in the protein kinase III, respectively. The data showed that 1.0 mol phosphate was incorporated into 80 mol of c-erb A protein (50 kDa) either in the presence of protein kinase I (with 10(-6) M cyclic AMP) or in the presence of protein kinase III. These results suggested that there are protein kinases for c-erb A protein, whose functional properties are similar to those of nuclear T3 receptor, in rat kidney nuclei.     

A brain-specific Ca2+/calmodulin-dependent protein kinase (CaM kinase-Gr) is regulated by autophosphorylation. Relevance to neuronal Ca2+ signaling.

A neuronal Ca2+/calmodulin-dependent protein kinase (CaM kinase-Gr) undergoes autophosphorylation on a serine residue(s) in response to Ca2+ and calmodulin. Phosphate incorporation leads to the formation of a Ca(2+)-independent (autonomous) activity state, as well as potentiation of the Ca2+/calmodulin-dependent response. The autonomous enzyme activity of the phosphorylated enzyme approximately equals the Ca2+/calmodulin-stimulated activity of the unphosphorylated enzyme, but displays diminished affinity toward ATP and the synthetic substrate, syntide-2. The Km(app) for ATP and syntide-2 increased 4.3- and 1.7-fold, respectively. Further activation of the autonomous enzyme by Ca2+/calmodulin yields a marked increase in the affinity for ATP and peptide substrate such that the Km(app) for ATP and syntide-2 decreased by 14- and 8-fold, respectively. Both autophosphorylation and the addition of Ca2+/calmodulin are required to produce the maximum level of enzyme activation and to increase substrate affinity. Unlike Ca2+/calmodulin-dependent protein kinase type II that is dephosphorylated by the Mg(2+)-independent phosphoprotein phosphatases 1 and 2A, CaM kinase-Gr is dephosphorylated by a Mg(2+)-dependent phosphoprotein phosphatase that may be related to the type 2C enzyme. Dephosphorylation of CaM kinase-Gr reverses the effects of autophosphorylation on enzyme activity. A comparison between the autophosphorylation and dephosphorylation reactions of CaM kinase-Gr and Ca2+/calmodulin-dependent protein kinase type II provides useful insights into the operation of Ca(2+)-sensitive molecular switches.     

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.     

Retention of specific protein kinase C isozymes following chronic phorbol ester treatment in BC3H-1 myocytes

Since insulin effects on glucose transport persist in phorbol ester "desensitized" or "down-regulated" BC3H-1 myocytes, we reexamined the evidence for protein kinase C (PKC) depletion. After 24 hrs of 5 microM 12-0-tetradecanoyl phorbol-13-acetate (TPA) treatment, PKC-directed histone phosphorylation and acute TPA effects on glucose transport were lost, but PKC-dependent vinculin phosphorylation was still evident. Hydroxylapatite (HAP) chromatography revealed loss of a type III, but not a type II, PKC-dependent vinculin phosphorylation. Immunoblots of cytosolic preparations of PKC-"depleted" myocytes confirmed the retention of PKC. Our findings indicate that TPA "down-regulated" BC3H-1 myocytes contain immunoreactive and functionally active PKC. The latter may explain the continued effectiveness of both insulin and diacylglycerol (DiC8) for stimulating glucose transport in "down-regulated" cells.