DIK, a novel protein kinase that interacts with protein kinase Cdelta. Cloning, characterization, and gene analysis.

A novel serine/threonine kinase, termed DIK, was cloned using the yeast two-hybrid system to screen a cDNA library from the human keratinocyte cell line HaCaT with the catalytic domain of rat protein kinase Cdelta (PKCdelta(cat)) cDNA as bait. The predicted 784-amino acid polypeptide with a calculated molecular mass of 86 kDa contains a catalytic kinase domain and a putative regulatory domain with ankyrin-like repeats and a nuclear localization signal. Expression of DIK at the mRNA and protein level could be demonstrated in several cell lines. The dik gene is located on chromosome 21q22.3 and possesses 8 exons and 7 introns. DIK was synthesized in an in vitro transcription/translation system and expressed as recombinant protein in bacteria, HEK, COS-7, and baculovirus-infected insect cells. In the in vitro system and in cells, but not in bacteria, various post-translationally modified forms of DIK were produced. DIK was shown to exhibit protein kinase activity toward autophosphorylation and substrate phosphorylation. The interaction of PKCdelta(cat) and PKCdelta with DIK was confirmed by coimmunoprecipitation of the proteins from HEK cells transiently transfected with PKCdelta(cat) or PKCdelta and DIK expression constructs.

Role of tyrosine phosphorylation in the regulation of the interaction of heterogenous nuclear ribonucleoprotein K protein with its protein and RNA partners.

The heterogeneous nuclear ribonucleoprotein K protein recruits a diversity of molecular partners and may act as a docking platform involved in such processes as transcription, RNA processing, and translation. We show that K protein is tyrosine-phosphorylated in vitro by Src and Lck. Treatment with H(2)O(2)/Na(3)VO(4), which induces oxidative stress, stimulated tyrosine phosphorylation of K protein in cultured cells and in intact livers. Tyrosine phosphorylation increased binding of Lck and the proto-oncoprotein Vav to K protein in vitro. Oxidative stress increased the association of K protein with Lck and Vav, suggesting that tyrosine phosphorylation regulates the ability of K protein to recruit these effectors in vivo. Translation-based assay showed that K protein is constitutively bound to many mRNAs in vivo. Native immunoprecipitated K protein-mRNA complexes were disrupted by tyrosine phosphorylation, suggesting that the in vivo binding of K protein to mRNA may be responsive to the extracellular signals that activate tyrosine kinases. This study shows that tyrosine phosphorylation of K protein regulates K protein-protein and K protein-RNA interactions. These data are consistent with a model in which functional interaction of K protein is responsive to changes in the extracellular environment. Acting as a docking platform, K protein may bridge signal transduction pathways to sites of nucleic acid-dependent process such as transcription, RNA processing, and translation.

Cloning, expression and characterization of an A6-related protein.

By interaction cloning (yeast two-hybrid system) using the catalytic domain of protein kinase Czeta (PKCzeta) as bait, we cloned a human full-length cDNA with 62% nucleotide homology to the A6 protein recently cloned and characterized by Beeler et al. [Beeler, J.F., LaRochelle, W.J., Chedid, M., Tronick, S.R. & Aaronson, S. A. (1994) Mol. Cell. Biol. 14, 982-988]. The deduced amino acid sequence (349 amino acids) of the A6-related protein (A6rp) contained potential actin-binding sites and ATP-binding sites. We also cloned the murine homolog of A6rp. Human A6rp was expressed in an in-vitro transcriptional/translational system with an apparent molecular mass of 40 kDa and as a glutathione S-transferase (GST) fusion protein in bacteria. A polyclonal anti-(A6rp) was raised in rabbits and used for the identification of A6rp by immunoblotting. A6rp was found to be expressed at the mRNA and the protein levels in all cells and tissues investigated. GST-A6rp was phosphorylated by PKCzeta but not significantly by other PKC isoenzymes. Moreover, it was phosphorylated by casein kinase 2 and most effectively by the tyrosine kinase Src. In contrast to GST-A6rp, GST-A6 was also phosphorylated by PKC isoforms other than PKCzeta and strongly by CK2, but just weakly by Src. In contrast to the results of Beeler et al. on beta-galactosidase-A6, we were unable to demonstrate autokinase activity or tyrosine phosphorylation of either GST-A6 or GST-A6rp. In accordance with the potential ATP-binding sites, both proteins were able to bind ATP.

Regulated interaction of protein kinase Cdelta with the heterogeneous nuclear ribonucleoprotein K protein.

The heterogeneous nuclear ribonucleoprotein (hnRNP) K protein recruits a diversity of molecular partners that are involved in signal transduction, transcription, RNA processing, and translation. K protein is phosphorylated in vivo and in vitro by inducible kinase(s) and contains several potential sites for protein kinase C (PKC) phosphorylation. In this study we show that K protein is phosphorylated in vitro by PKCdelta and by other PKCs. Deletion analysis and site-directed mutagenesis revealed that Ser302 is a major K protein site phosphorylated by PKCdelta in vitro. This residue is located in the middle of a short amino acid fragment that divides the two clusters of SH3-binding domains. Mutation of Ser302 decreased the level of phosphorylation of exogenously expressed K protein in phorbol 12-myristate 13-acetate-treated COS cells, suggesting that Ser302 is also a site for PKC-mediated phosphorylation in vivo. In vitro, PKCdelta binds K protein via the highly interactive KI domain, an interaction that is blocked by poly(C) RNA. Mutation of Ser302 did not alter the K protein-PKCdelta interaction in vitro, suggesting that phosphorylation of this residue alone is not sufficient to alter this interaction. Instead, binding of PKCdelta to K protein in vitro and in vivo was greatly increased by K protein phosphorylation on tyrosine residues. The ability of PKCdelta to bind and phosphorylate K protein may serve not only to alter the activity of K protein itself, but K protein may also bridge PKCdelta to other K protein molecular partners and thus facilitate molecular cross-talk. The regulated nature of the PKCdelta-K protein interaction may serve to meet cellular needs at sites of active transcription, RNA processing and translation in response to changing extracellular environment.

Requirements of protein kinase cdelta for catalytic function. Role of glutamic acid 500 and autophosphorylation on serine 643.

Recently, we reported that, in contrast to protein kinase C (PKC)alpha and betaII, PKCdelta does not require phosphorylation of a specific threonine (Thr505) in the activation loop for catalytic competence (Stempka et al. (1997) J. Biol. Chem. 272, 6805-6811). Here, we show that the acidic residue glutamic acid 500 (Glu500) in the activation loop is important for the catalytic function of PKCdelta. A Glu500 to valine mutant shows 76 and 73% reduced kinase activity toward autophosphorylation and substrate phosphorylation, respectively. With regard to thermal stability and inhibition by the inhibitors Gö6976 and Gö6983 the mutant does not differ from the wild type, indicating that the general conformation of the molecule is not altered by the site-directed mutagenesis. Thus, Glu500 in the activation loop of PKCdelta might take over at least part of the role of the phosphate groups on Thr497 and Thr500 of PKCalpha and betaII, respectively. Accordingly, PKCdelta exhibits kinase activity and is able to autophosphorylate probably without posttranslational modification. Autophosphorylation of PKCdelta in vitro occurs on Ser643, as demonstrated by matrix-assisted laser desorption ionization mass spectrometry of tryptic peptides of autophosphorylated PKCdelta wild type and mutants. A peptide containing this site is phosphorylated also in vivo, i.e. in recombinant PKCdelta purified from baculovirus-infected insect cells. A Ser643 to alanine mutation indicates that autophosphorylation of Ser643 is not essential for the kinase activity of PKCdelta. Probably additional (auto)phosphorylation site(s) exist that have not yet been identified.

Protein kinase C delta.

The protein kinase C (PKC) family consists of 11 isoenzymes that, due to structural and enzymatic differences, can be subdivided into three groups: The Ca(2+)-dependent, diacylglycerol (DAG)-activated cPKCs (conventional PKCs: alpha, beta 1, beta 2, gamma); the Ca(2+)-independent, DAG-activated nPKCs (novel PKCs: delta, epsilon, eta, theta, mu), and the Ca(2+)-dependent, DAG non-responsive aPKCs (atypical PKCs: zeta, lambda/iota). PKC mu is a novel PKC, but with some special structural and enzymatic properties.

Amyloid beta protein (25-35) phosphorylates MARCKS through tyrosine kinase-activated protein kinase C signaling pathway in microglia.

Myristoylated alanine-rich C kinase substrate (MARCKS) is a widely distributed specific protein kinase C (PKC) substrate and has been implicated in membrane trafficking, cell motility, secretion, cell cycle, and transformation. We found that amyloid beta protein (A beta) (25-35) and A beta (1-40) phosphorylate MARCKS in primary cultured rat microglia. Treatment of microglia with A beta (25-35) at 10 nM or 12-O-tetradecanoylphorbol 13-acetate (1.6 nM) led to phosphorylation of MARCKS, an event inhibited by PKC inhibitors, staurosporine, calphostin C, and chelerythrine. The A beta (25-35)-induced phosphorylation of MARCKS was inhibited by pretreatment with the tyrosine kinase inhibitors genistein and herbimycin A, but not with pertussis toxin. PKC isoforms alpha, delta, and epsilon were identified in microglia by immunocytochemistry and western blots using isoform-specific antibodies. PKC-delta was tyrosine-phosphorylated by the treatment of microglia for 10 min with A beta (25-35) at 10 nM. Other PKC isoforms alpha and epsilon were tyrosine-phosphorylated by A beta (25-35), but only to a small extent. We propose that a tyrosine kinase-activated PKC pathway is involved in the A beta (25-35)-induced phosphorylation of MARCKS in rat microglia.

Proteolytic cleavage of protein kinase Cmu upon induction of apoptosis in U937 cells.

Treatment of U937 cells with various apoptosis-inducing agents, such as TNFalpha and beta-D-arabinofuranosylcytosine (ara-C) alone or in combination with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA), bryostatin 1 or cycloheximide, causes proteolytic cleavage of protein kinase Cmu (PKCmu) between the regulatory and catalytic domain, generating a 62 kDa catalytic fragment of the kinase. The formation of this fragment is effectively suppressed by the caspase-3 inhibitor Z-DEVD-FMK. In accordance with these in vivo data, treatment of recombinant PKCmu with caspase-3 in vitro results also in the generation of a 62 kDa fragment (p62). Treatment of several aspartic acid to alanine mutants of PKCmu with caspase-3 resulted in an unexpected finding. PKCmu is not cleaved at one of the typical cleavage sites containing the motif DXXD but at the atypical site CQND378/S379. The respective fragment (amino acids 379-912) was expressed in bacteria as a GST fusion protein (GST-p62) and partially purified. In contrast to the intact kinase, the fragment does not respond to the activating cofactors TPA and phosphatidylserine and is thus unable to phosphorylate substrates effectively.

Colon carcinoma cells use different mechanisms to escape CD95-mediated apoptosis.

CD95(APO-1/Fas) is a cell surface receptor that, when oligomerized by natural ligand, CD95L, or antibody, confers an apoptotic signal to apoptosis-sensitive cells. Whereas CD95 is expressed in every colonocyte of normal colon mucosa, CD95 is down-regulated or lost in the majority of colon carcinomas. To investigate the sensitivity to CD95-mediated apoptosis of normal and neoplastic colonocytes, we applied cross-linking CD95(anti-APO-1) monoclonal antibody to freshly isolated colon crypts and colon carcinoma cell lines and monitored apoptosis by DNA fragmentation and morphology. Normal colonocytes were constitutively sensitive to CD95-mediated apoptosis. All carcinoma lines were constitutively resistant but were sensitized upon pretreatment with IFN-gamma. Transcription blocking, protein synthesis, and export in carcinoma cells indicated that even low surface levels of CD95 were sufficient to efficiently transmit the signal. Despite low CD95 surface levels of non-IFNgamma-treated cells, actinomycin D, cycloheximide, and brefeldin A each sensitized all cell lines, but at different rates and kinetics. In this context, it was observed that a greatly delayed apoptotic response of SW620 cells was associated with the absence of antibody-induced CD95 capping. Phorbol 12-myristate 13-acetate inhibited CD95-mediated apoptosis by counteracting the IFNgamma-, actinomycin D-, and cycloheximide-mediated but not the brefeldin A-mediated sensitization. This phorbol 12-myristate 13-acetate-induced protection against apoptosis was completely abolished by staurosporine and by a selective protein kinase C inhibitor, Goe 6983. We conclude that, during malignant transformation, colonocytes acquire different mechanisms to escape CD95-mediated apoptosis. These include abrogation of CD95, inhibition of CD95 capping, and activation of antiapoptotic programs, both governed by and independent of protein kinase C.

Differential effects of suramin on protein kinase C isoenzymes. A novel tool for discriminating protein kinase C activities.

Suramin, a hexasulfonated naphthylurea, is known to induce differentiation and inhibit proliferation, angiogenesis, and development of tumors. It has also been shown to suppress the activity of the protein kinase C (PKC) isoenzymes alpha, beta, and gamma. Here we report on a differential effect of suramin on PKCmu and various PKC isoforms representing the cPKC, nPKC, and aPKC group of the PKC family. In the absence of any cofactors suramin activates all PKC isoforms in the order of aPKCzeta >> PKCmu > cPKC, nPKCdelta. As judged by the Vmax/KM ratios (0.5 for PKCmu and 2.2 for PKCzeta) the substrate syntide 2 is phosphorylated by suramin-activated PKCzeta around four times more effectively than by suramin-activated PKCmu. Suramin-activated PKCmu behaves like that activated by phosphatidylserine and the phorbol ester TPA regarding autophosphorylation and differential inhibition by the PKC inhibitors Gö 6976 and Gö 6983. In the presence of activating cofactors, such as phosphatidylserine and TPA or cholesterol sulfate (for PKCzeta), the activity of the aPKCzeta is further stimulated, PKCmu is not significantly affected, and the cPKCs and the nPKCdelta are strongly inhibited by suramin. The differential action of suramin on PKC isoenzymes might play a role in some of its biological effects, as for instance inhibition of proliferation and tumor development. Moreover, due to this property suramin will possibly be a valuable tool for discriminating the activities of PKC isoenzymes in vitro and in vivo.

Regulation of protein kinase Cmu by basic peptides and heparin. Putative role of an acidic domain in the activation of the kinase.

Protein kinase Cmu is a novel member of the protein kinase C (PKC) family that differs from the other isoenzymes in structural and enzymatic properties. No substrate proteins of PKCmu have been identified as yet. Moreover, the regulation of PKCmu activity remains obscure, since a structural region corresponding to the pseudosubstrate domains of other PKC isoenzymes has not been found for PKCmu. Here we show that aldolase is phosphorylated by PKCmu in vitro. Phosphorylation of aldolase and of two substrate peptides by PKCmu is inhibited by various proteins and peptides, including typical PKC substrates such as histone H1, myelin basic protein, and p53. This inhibitory activity seems to depend on clusters of basic amino acids in the protein/peptide structures. Moreover, in contrast to other PKC isoenzymes PKCmu is activated by heparin and dextran sulfate. Maximal activation by heparin is about twice and that by dextran sulfate four times as effective as maximal activation by phosphatidylserine plus 12-O-tetradecanoylphorbol-13-acetate, the conventional activators of c- and nPKC isoforms. We postulate that PKCmu contains an acidic domain, which is involved in the formation and stabilization of an active state and which, in the inactive enzyme, is blocked by an intramolecular interaction with a basic domain. This intramolecular block is thought to be released by heparin and possibly also by 12-O-tetradecanoylphorbol-13-acetate/phosphatidylserine, whereas basic peptides and proteins inhibit PKCmu activity by binding to the acidic domain of the active enzyme.

Phosphorylation of protein kinase Cdelta (PKCdelta) at threonine 505 is not a prerequisite for enzymatic activity. Expression of rat PKCdelta and an alanine 505 mutant in bacteria in a functional form.

A structural feature shared by many protein kinases is the requirement for phosphorylation of threonine or tyrosine in the so-called activation loop for full enzyme activity. Previous studies by several groups have indicated that the isotypes alpha, betaI, and betaII of protein kinase C (PKC) are synthesized as inactive precursors and require phosphorylation by a putative "PKC kinase" for permissive activation. Expression of PKCalpha in bacteria resulted in a nonfunctional enzyme, apparently due to lack of this kinase. The phosphorylation sites for the PKC kinase in the activation loop of PKCalpha and PKCbetaII could be identified as Thr497 and Thr500, respectively. We report here that PKCdelta, contrary to PKCalpha, can be expressed in bacteria in a functional form. The activity of the recombinant enzyme regarding substrate phosphorylation, autophosphorylation, and dependence on activation by 12-O-tetradecanoylphorbol-13-acetate as well as the Km values for two substrates are comparable to those of recombinant PKCdelta expressed in baculovirus-infected insect cells. By site-directed mutagenesis we were able to show that Thr505, corresponding to Thr497 and Thr500 of PKCalpha and PKCbetaII, respectively, is not essential for obtaining a catalytically competent conformation of PKCdelta. The mutant Ala505 can be activated and does not differ from the wild type regarding activity and several other features. Ser504 can not take over the role of Thr505 and is not prerequisite for the kinase to become activated, as proven by the unaffected enzyme activity of respective mutants (Ala504 and Ala504/Ala505). These results indicate that phosphorylation of Thr505 is not required for the formation of functional PKCdelta and that at least this PKC isoenzyme differs from the isotypes alpha, betaI, and betaII regarding the permissive activation by a PKC kinase.

Conventional PKC-alpha, novel PKC-epsilon and PKC-theta, but not atypical PKC-lambda are MARCKS kinases in intact NIH 3T3 fibroblasts.

Phosphorylation of myristoylated alanine-rich protein kinase C substrate (MARCKS) in intact cells has been employed as an indicator for activation of protein kinase C (PKC). Specific PKC isoenzymes responsible for MARCKS phosphorylation under physiological conditions, however, remained to be identified. In our present study using stably transfected NIH 3T3 cell clones we demonstrate that expression of constitutively active mutants of either conventional cPKC-alpha or novel nPKC-epsilon increased phosphorylation of endogenous MARCKS in the absence of phorbol 12,13-dibutyrate in intact mouse fibroblasts, implicating that each of these PKC isoforms itself is sufficient to induce enhanced MARCKS phosphorylation. Similarly, ectopic expression of a constitutively active mutant of PKC-theta significantly increased MARCKS phosphorylation compared to vector controls, identifying PKC-theta as a MARCKS kinase. The PKC-specific inhibitor GF 109203X (bisindolylmaleimide I) reduced MARCKS phosphorylation in intact cells at a similar dose-response as enzymatic activity of recombinant isoenzymes cPKC-alpha, nPKC-epsilon, and nPKC-theta in vitro. Consistently, phorbol 12,13-dibutyrate-dependent MARCKS phosphorylation was significantly reduced in cell lines expressing dominant negative mutants of either PKC-alpha K368R or (dominant negative) PKC-epsilon K436R. The fact, that the constitutively active PKC-lambda A119E mutant did not alter the MARCKS phosphorylation underscores the assumption that atypical PKC isoforms are not involved in this process. We conclude that under physiological conditions, conventional cPKC-alpha and novel nPKC-epsilon, but not atypical aPKC-lambda are responsible for MARCKS phosphorylation in intact NIH 3T3 fibroblasts.

Suppression of apoptosis in COLO 205 cells by the phorbol ester TPA may be mediated by the PKC isoenzyme alpha.

Apoptosis induced by an antibody to CD95/APO-1/FAS in the colon carcinoma cells COLO 205 and HT-29 is suppressed by the phorbol ester TPA. Inhibition is much more effective in COLO 205 than in HT-29 cells. The TPA effect is abrogated by the protein kinase C (PKC)-specific inhibitor Go6983 indicating a role of PKC in this process. Bryostatin 1, unlike TPA, is unable to suppress apoptosis, but inhibits the TPA-induced suppression of apoptosis. TPA also inhibits indomethacin-induced apoptosis in COLO 205 cells. COLO 205 and HT-29 cells contain the PKC isoenzymes alpha, beta(II) delta, epsilon, eta, mu and zeta. Expression and activity of PKC alpha are at least 5 times higher in COLO 205 than in HT-29 cells. This correlates with the fact that inhibition of CD95-mediated apoptosis by TPA is more prominent in COLO 205 than in HT-29 cells. Thus, these findings suggest that PKC alpha has an important role in the TPA-induced inhibition of apoptosis.

Immunological demonstration of protein kinase C mu in murine tissues and various cell lines. Differential recognition of phosphorylated forms and lack of down-regulation upon 12-O-tetradecanoylphorphol-13-acetate treatment of cells.

13 murine tissues and 12 cell lines were tested for the expression of the novel protein kinase C (PKC) isoenzyme mu. Using two different PKC mu antibodies (sc-639 and P26720), PKC mu was detected in all tissues and cells and thus proved to be an ubiquitous PKC isotype. However, in some tissues, PKC mu was recognized only by the antibody P26720 and not by sc-639. Thymus, lung and peripheral blood mononuclear cells expressed the greatest amount of PKC mu. Recognition of PKC mu by the antibody sc-639 was drastically impaired when treating keratinocytes or mouse skin in vivo with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA), thus mimicking down-regulation of PKC mu. The lack of a decrease in the PKC mu amount and, thus, the lack of down-regulation could be proved using the antibody P26720. This antibody was able to recognize PKC mu in extracts of untreated as well as TPA-treated tissues or cells. Phosphorylation of proteins in a cell-free system (cell or tissue extracts) in the presence and absence of TPA or other PKC activators and various protein kinase inhibitors indicated that phosphorylation of activated PKC mu caused its reduced interaction with the antibody sc-639. Therefore, this antibody might present a well suited tool for the detection of activated PKC mu in vivo. Moreover, our results clearly show that some antibodies, such as sc-639, might be able to selectively detect non-phosphorylated or phosphorylated forms of a protein, and that such properties of an antibody have to be studied carefully before the latter can be used for reliable quantitative determination of this protein. We consider this information important to avoid misinterpretation of data concerning the immunological quantification of proteins such as PKC mu.

Inhibition of protein kinase C mu by various inhibitors. Differentiation from protein kinase c isoenzymes.

Various inhibitors were tested for their potential to suppress the kinase activity of protein kinase C mu (PKC mu) in vitro and in vivo. Among the staurosporine-derived, rather selective PKC inhibitors the indolocarbazole Gö 6976 previously shown to inhibit preferentially cPKC isotypes proved to be a potent inhibitor of PKC mu with an IC50 of 20 nM, whereas the bisindolylmaleimide Gö 6983 was extremely ineffective in suppressing PKC mu kinase activity with a thousand-fold higher IC50 of 20 microM. Other strong inhibitors of PKC mu were the rather unspecific inhibitors staurosporine and K252a. Contrary to the poor inhibition of PKC mu by Gö 6983, this compound was found to suppress in vitro kinase activity of PKC isoenzymes from all three subgroups very effectively with IC50 values from 7 to 60 nM. Thus, Gö 6983 was able to differentiate between PKC mu and other PKC isoenzymes being useful for selective determination of PKC mu kinase activity in the presence of other PKC isoenzymes.

Loss of protein kinase C delta from human HaCaT keratinocytes upon ras transfection is mediated by TGF alpha.

The spontaneously immortalized human skin keratinocytes HaCaT contain protein kinase C (PKC) alpha, -delta, -epsilon, and -zeta. All PKC isoenzymes except PKC zeta are down-regulated by TPA as well as by bryostatin. However, with PKC delta, bryostatin but not TPA was found to be much less effective at high concentrations than at low ones. PKC delta expression at the protein and mRNA level is significantly suppressed in HaCaT cells I-7 and II-4, which are transfected with mutated c-Ha-ras. The expression of the other isoenzymes remains essentially unchanged in the ras-transfected cells compared to normal ones. PKC delta is lost when growing HaCaT cells in a medium obtained from the cultivation of ras-transfected cells ("ras-conditioned" medium). The factor secreted into the medium by the ras-transfected cells that is responsible for this effect appears to be TGF alpha, since the action of ras-conditioned medium on PKC delta expression can be overcome by the addition of an anti-TGF alpha antibody. Moreover, treatment of HaCaT cells with TGF alpha suppresses selectively the expression of the PKC isoenzyme delta.

Protein kinase C delta-specific phosphorylation of the elongation factor eEF-alpha and an eEF-1 alpha peptide at threonine 431.

Two cytosolic proteins of murine epidermis or porcine spleen with molecular masses of 37 kDa (p37) and 50 kDa (p50) are differentially phosphorylated in vitro by the purified protein kinase C (PKC) isoenzymes alpha, beta, gamma (cPKC) and PKC delta. p37, identified as annexin I, is preferentially phosphorylated by cPKC, whereas p50, identified as elongation factor eEF-1 alpha, is phosphorylated with much greater efficacy by PKC delta than by cPKC. Using the recombinant PKC isoenzymes alpha, beta, gamma, delta, epsilon, eta, and zeta, we could show that purified eEF-1 alpha is indeed a specific substrate of PKC delta. It is not significantly phosphorylated by PKC epsilon, -eta, and -zeta and only slightly by PKC alpha, -beta, and -gamma. PKC delta phosphorylates eEF-1 alpha at Thr-431 (based on the murine amino acid sequence). The peptide RFAVRDMRQTVAVGVIKAVDKK with a sequence corresponding to that of 422-443 from murine eEF-1 alpha and containing Thr-431 is an absolutely specific substrate for the delta-type of PKC. The single basic amino acid close to Thr-431 (Arg-429) is essential for recognition of the peptide as a substrate by PKC delta and for the selectivity of this recognition. Substitution of Arg-429 by alanine abolishes the ability of PKC delta to phosphorylate the peptide, and insertion of additional basic amino acids in the vicinity of Thr-431 causes a complete loss of selectivity.

Lack of an effect of novel inhibitors with high specificity for protein kinase C on the action of the phorbol ester 12-O-tetradecanoylphorbol-13-acetate on mouse skin in vivo.

The inhibitory effects of three novel staurosporine-derived compounds were tested with five different types of protein kinases, including protein kinase C (PKC). IC50 values of two of these compounds were found to be 300 to > 5000 times lower for PKC alpha beta gamma (a mixture of the PKC isoenzymes alpha, beta and gamma) than for any of the other protein kinases. The inhibitory action of the most selective inhibitor was tested also with the Ca(2+)-unresponsive PKC isoenzyme delta and was found to suppress PKC alpha beta gamma and PKC delta differentially. The highly specific PKC inhibitors are active both in cell culture and in vivo. They inhibit the PKC-catalyzed phosphorylation of the specific PKC substrate MARCKS in Swiss-3T3 fibroblasts and the okadaic acid-induced edema of the mouse ear. However, the more complex biological processes triggered by the phorbol ester 12-O-tetradecanoylphorbol-13-acetate in mouse skin, such as inflammation, stimulation of cellular hyperproliferation and tumor promotion, remain largely unaffected upon topical application of these compounds.