Protein Kinase C Molecule Research Articles

Smooth muscle hyperplasia in protein kinase C-fused blue naevi: Report of 12 cases.

PKC-fused blue naevi are a recently described group of melanocytic tumours that have distinctive morphological features, including a pigmented epithelioid melanocytoma-like junctional component or a dermal biphasic architecture associating with naevocytoid nests surrounded by dendritic and spindled pigmented melanocytes (so-called 'combined common and blue naevus'). There have been reports of smooth muscle hyperplasia in a hamartoma-like pattern in cases of combined blue naevi without genetic exploration. Herein, we describe 12 cases of protein kinase C (PKC)-fused blue tumours associated with a co-existing smooth muscle hyperplasia, identified from a total of 98 PKC-fused melanocytic tumours. Archived slides of PKC-fused blue naevi with haematoxylin, eosin and phloxin staining, immunohistochemistry and molecular confirmation of a PKC-fusion by fluorescence in-situ hybridisation (FISH) or RNAseq were re-evaluated for identification of notable smooth muscle hyperplasia. Fifty-one of these slides had already been studied in a previous publication from our group. The hyperplasia ranged from hypertrophic arrector pili muscles to extensive horizontal bundles of disorganised fibres constantly associated and limited within a biphasic dermal melanocytic component. At least one arrector pili muscle was always visible within the tumour, with occasionally direct extension of the hyperplastic fibres from the main muscle body. These muscle fibres were devoid of a PKC-fusion signal by FISH. PKC molecules are involved in the regulation of smooth muscle function, offering an explanatory framework. These data suggest incorporating smooth muscle hyperplasia as a diagnostic morphological feature of PKC-fused blue melanocytic tumours.

OSU-2S, a Novel PKC Activator, Mediates PKC Dependent Cell Death, Differentiation and Cell Cycle Arrest in Acute Myeloid Leukemia

Acute myeloid leukemia (AML) is a near incurable disease with 5-year survival rates of 10% in older patients. Poor tolerance to chemotherapy, chemo resistance and high rate of relapse warrants less toxic and more effective regimens in AML. AML leads to a block in normal myeloid differentiation, leading to an accumulation of undifferentiated blasts. Thus, therapies directed towards promoting these cells to differentiate re-establishes the ability of AML cells to undergo cell death, reducing the leukemic population. Such differentiation therapy with all-trans retinoic acid (ATRA) has been successful in acute promyelocytic leukemia (APL), but not in other AML subtypes resistant to ATRA, thus presenting a promising and unexploited avenue for targeted therapies. There is accumulating evidence that Protein Kinase C (PKC) activation induces myeloid differentiation. However, since most PKC activators are either tumor promoters or have inconclusive activity, their potential for use in AML has yet to be realized. OSU-2S is a non-immunosuppressive derivative of FTY720 (a sphingosine analogue with anti-tumorigenic properties) and has previously been reported to directly activate PKC to mediate cytotoxicity, showing promising in vitro and in vivo activity against a number of leukemias and lymphomas, and other malignancies such as hepatocellular carcinoma. This impelled us to evaluate the activity of OSU-2S in causing cell death and differentiation in AML. We found that OSU-2S resulted in increased activation of PKC in HL-60 cells. In addition to effecting significant dose-dependent cytotoxicity against AML cell lines HL-60, MV4-11 and MOLM-13 [mean decrease in viability with OSU-2S in HL-60: 85±7% (p=0.08), MV4-11: 74±20% (p=0.02), MOLM-13: 60±25% (p=0.04)], and in primary samples from leukemic patients, including high risk FLT3-ITD mutated AMLs (65±8% decrease in viability with OSU-2S, p<0.0001, n=13, dose trend p<0.0001), OSU-2S caused a significant upregulation of CD11b, a cell-surface marker associated with myeloid maturation, in HL-60 and primary AML samples [(25±5% and 14±7% increase in CD11b+ cells with OSU-2S vs vehicle control in HL-60 (p =0.0126) and primary AML (p=0.043) cells respectively]. In addition, OSU-2S treated HL-60 and primary AML cells exhibited morphological features indicative of differentiation, as evidenced by lower nuclear to cytoplasmic ratio, and highly vacuolated and granulated cytoplasm. The observed differentiation activity prompted us to test if OSU-2S affected the leukemia-initiating cell population. Colony forming assays showed OSU-2S significantly reduced the number of colony forming units-leukemic (CFU-L) in primary AML blasts (52±13% decrease in CFU-L in response to OSU-2S vs vehicle control; p=0.018, n=4). OSU-2S mediated cytotoxicity was significantly abrogated in the presence of PKC inhibitor Bisindolylmaleimide (BIS) in HL-60 cell line [39±8% decrease in OSU-2S induced cytotoxicity in the presence of BIS vs control (p=0.008), n=3] as well as in primary AML cells [41±16% decrease in OSU-2S induced cytotoxicity (p=0.02), n=6], indicating that OSU-2S mediates its cytotoxic effects in AML via PKC activation.To further gain insight into the mechanism of cell death and differentiation effected by OSU-2S, we performed RNA-Sequencing analysis of total RNA isolated from MV4-11 cells treated with OSU-2S. Ingenuity Pathway Analysis on these transcripts showed significant changes in gene expression affecting cell cycle regulation and proliferation (p=10-13). Cell cycle analysis of MV4-11, HL-60 and U-937 cells by PI staining followed by flow cytometry confirmed a decrease in cells in G2/M phase of the cell cycle associated with increased cells in S but not Go/G1 phase (69.63% decrease in G2/M phase cells with OSU-2S vs vehicle control, p=0.04). Current ongoing studies in cell line-derived xenografted mouse models of AML indicate favorable therapeutic effect of OSU-2S in providing survival advantage in AML. Tumor burden monitoring by bioluminescent imaging shows reduction in tumor burden following OSU-2S treatment. Our results show promising pre-clinical efficacy of OSU-2S against AML, and warrants further studies to establish clinical efficacy as a potential therapeutic agent. Ongoing studies are also directed towards studying the involvement and role of PKC and other target molecules in the cell death and differentiation effected by OSU-2S. DisclosuresWalker:Gilead: Research Funding. Mims:Novartis: Honoraria. Chen:The Ohio State University: Patents & Royalties: OSU-2S. Byrd:Pharmacyclics: Research Funding; Acerta Pharma: Research Funding; Janssen: Research Funding; Genentech: Research Funding; The Ohio State University: Patents & Royalties: OSU-2S. Vasu:Boehringer-Ingelheim: Research Funding; Stemline Therapeutics: Research Funding.

The molecular model of PKC life cycle explained through Systems Biology Approach

Protein kinase C (PKC) is a family of kinases which is poised to transduce signals emanating from the cell surface. Membranes of most cells provide the regulatory platform for supporting the life cycle and function of PKC enzymes. Maturation, activation and termination are three key regulatory steps of PKC life cycle. These regulatory steps precisely control the level of signaling competent PKC enzyme in the cells and any dysfunction in this regulation results in pathophysiological disease state such as cancer, where PKC levels are observed to be altered. Here, we describe a molecular model of PKC life cycle based on computational approach. Our model explains the fate of PKC during denovo synthesis and lipid mediated activation cycle. Through our systems biology approach we show that life cycle of PKC is controlled by multiple phosphorylation and dephosphorylation events. PKC processing events can be divided into two types 1: Maturation processing of newly synthesized enzyme; 2. Activation of enzyme. Newly synthesized PKC enzyme is constitutively processed through three ordered phosphorylations and is stored in cytosol as a signaling competent inactive molecule. First step in this maturation sequence of PKC is PDK1 mediated phosphorylation at activation loop (Thr500 in PKCβII). Once phosphorylated at activation loop PKC molecule binds with mTORC2 complex and undergoes constitutive auto‐phosphorylations at turn and hydrophobic motifs (Thr641 and Ser660 in PKCβII). Upon stimulation by extra‐cellular stimuli the generation of diacylglycerol (DAG) and Ca+2 at membrane engages the PKC molecule through binding at C2 and C1 domains respectively. This activates the enzyme. Our model explains all these steps in PKC lifecycle through a quantitative approach. Our model also shows that once active PKC molecule is prone to de‐phosphorylation (PHLPP‐mediated) and degradation. This model also explains the stabilizing role of HSP‐60 in rescuing the de‐phosphorylated molecule and assisting its re‐phosphorylation, thus re‐building the pool of signaling competent enzyme after activation cycle. This model shows that for small to moderate activation stimuli the pool of PKC molecule is effectively unchanged however, larger stimuli can down‐regulate the enzyme through degradation pathways. Our results also show that blocking the PHLPP de‐phosphorylation pathway could recover the PKC pool in a dose dependent manner even after a large amplitude stimulus has been applied. Our results also points toward a possible role of HSP‐60 overexpression in recovering the PKC signaling competent pool after large amplitude stimuli has been applied. The results presented in this communication unravel some interesting aspects of PKC life cycle through a computational approach. Enhancing the understanding of PKC life cycle could be instrumental in translating new drugs for this important transducing molecule for diseases like breast cancer.Support or Funding InformationBioSystOmics

Regulation of mesenchymal stem cell chondrogenesis by glucose through protein kinase C/transforming growth factor signaling

Effective induction of human mesenchymal stem cell (hMSC) differentiation for regenerative medicine applications remains a great challenge. While much research has studied hMSC activity during differentiation, it is unclear whether pre-differentiation culture can modulate differentiation capacity. We investigate the effect of glucose concentration in pre-differentiation/expansion culture on modulating chondrogenic capacity of hMSCs, and explore the underlying molecular mechanism. The extent of chondrogenesis of hMSCs previously cultured with different concentrations of glucose was evaluated. Transforming growth factor-beta (TGF-β) signaling molecules and protein kinase C (PKC) were analyzed to identify the role of these molecules in the regulation of glucose on chondrogenesis. In addition, hMSCs in high-glucose expansion culture were treated with the PKC inhibitor to modulate the activity of PKC and TGF-β signaling molecules. High-glucose maintained hMSCs were less chondrogenic than low-glucose maintained cells upon receiving differentiation signals. Interestingly, we found that high-glucose culture increased the phosphorylation of PKC and expression of type II TGF-β receptor (TGFβRII) in pre-differentiation hMSCs. However, low-glucose maintained hMSCs became more responsive to chondrogenic induction with increased PKC activation and TGFβRII expression than high-glucose maintained hMSCs during differentiation. Inhibiting the PKC activity of high-glucose maintained hMSCs during expansion culture upregulated the TGFβRII expression of chondrogenic cell pellets, and enhanced chondrogenesis. Our findings demonstrate the effect of glucose concentration on regulating the chondrogenic capability of pre-differentiation hMSCs, and provide insight into the mechanism of how glucose concentration regulates PKC and TGF-β signaling molecules to prime pre-differentiation hMSCs for subsequent chondrogenesis.

Identification of Acidic Amino Acid Residues in the Protein Kinase Cα V5 Domain That Contribute to Its Insensitivity to Diacylglycerol

The protein kinase C (PKC) isoforms are maintained in an inactive and closed conformation by intramolecular interactions. Upon activation these are disrupted by activators, binding proteins and cellular membrane. We have seen that autophosphorylation of two sites in the C-terminal V5 domain is crucial to keep PKC alpha insensitive to the activator diacylglycerol, which presumably is caused by a masking of the diacylglycerol-binding C1a domain. Here we demonstrate that the diacylglycerol sensitivity of the PKC beta isoforms also is suppressed by autophosphorylation of the V5 sites. To analyze conformational differences, a fusion protein ECFP-PKC alpha-EYFP was expressed in cells and the FRET signal was analyzed. The analogous mutant with autophosphorylation sites exchanged for alanine gave rise to a substantially lower FRET signal than wild-type PKC alpha indicating a conformational difference elicited by the mutations. Expression of the isolated PKC alpha V5 domain led to increased diacylglycerol sensitivity of PKC alpha. We identified acidic residues in the V5 domain that, when mutated to alanines or lysines, rendered PKC alpha sensitive to diacylglycerol. Furthermore, mutation to glutamate of four lysines in a lysine-rich cluster in the C2 domain gave a similar effect. Simultaneous reversal of the charges of the acidic residues in the V5 and the lysines in the C2 domain gave rise to a PKC alpha that was insensitive to diacylglycerol. We propose that these structures participate in an intramolecular interaction that maintains PKC alpha in a closed conformation. The disruption of this interaction leads to an unmasking of the C1a domain and thereby increased diacylglycerol sensitivity of PKC alpha.

Mechanism of membrane redistribution of protein kinase C by its ATP-competitive inhibitors

ATP-competitive inhibitors of PKC (protein kinase C) such as the bisindolylmaleimide GF 109203X, which interact with the ATP-binding site in the PKC molecule, have also been shown to affect several redistribution events of PKC. However, the reason why these inhibitors affect the redistribution is still controversial. In the present study, using immunoblot analysis and GFP (green fluorescent protein)-tagged PKC, we showed that, at commonly used concentrations, these ATP-competitive inhibitors alone induced redistribution of DAG (diacylglycerol)-sensitive PKCalpha, PKCbetaII, PKCdelta and PKCepsilon, but not atypical PKCzeta, to the endomembrane or the plasma membrane. Studies with deletion and point mutants showed that the DAG-sensitive C1 domain of PKC was required for membrane redistribution by these inhibitors. Furthermore, membrane redistribution was prevented by the aminosteroid PLC (phospholipase C) inhibitor U-73122, although an ATP-competitive inhibitor had no significant effect on acute DAG generation. Immunoblot analysis showed that an ATP-competitive inhibitor enhanced cell-permeable DAG analogue- or phorbol-ester-induced translocation of endogenous PKC. Furthermore, these inhibitors also enhanced [3H]phorbol 12,13-dibutyrate binding to the cytosolic fractions from PKCalpha-GFP-overexpressing cells. These results clearly demonstrate that ATP-competitive inhibitors cause redistribution of DAG-sensitive PKCs to membranes containing endogenous DAG by altering the DAG sensitivity of PKC and support the idea that the inhibitors destabilize the closed conformation of PKC and make the C1 domain accessible to DAG. Most importantly, our findings provide novel insights for the interpretation of studies using ATP-competitive inhibitors, and, especially, suggest caution about the interpretation of the relationship between the redistribution and kinase activity of PKC.

New bivalent PKC ligands linked by a carbon spacer: enhancement in binding affinity.

Protein kinase C (PKC) is known to play an important role in many signal transduction pathways involved in hormone release, mitogenesis, and tumor promotion. In continuation of our efforts to find highly potent activators of PKC for possible use as Alzheimer's disease therapeutics, we designed and synthesized molecules containing two binding moieties (amides of benzolactams or esters of naphthylpyrrolidones) connected by a flexible spacer chain, which could theoretically bind to both the C1a and C1b activator binding domains of the catalytic region or to the C1 domains of two adjacent PKC molecules. The dimers 2a-g of benzolactam showed a 200-fold increase in affinity to PKCalpha and -delta as the spacer length increased from 4 to 20 carbon atoms. Replacement of the oligomethylene chain with an oligoethylene glycol unit (compounds 2h, 2i) showed a 4000- to 7000-fold decrease in affinity to PKCalpha. The dimers of naphthylpyrrolidones 4a-g did not show any marked improvement in binding affinities to PKC in comparison to the monomers synthesized earlier. The dimer of benzolactam 2e did not show much selectivity for PKCalpha, -betaIota, -delta, -epsilon, and -gamma. The high binding affinity of compounds 2d-g to PKCs gives us the impetus to design additional molecules that would retain this enhanced activity and would also show selectivity for the PKC isoforms.

Evidence for a role of protein kinase C in FGF signal transduction in the developing chick limb bud

In developing limbs, numerous signaling molecules have been identified but less is known about the mechanisms by which such signals direct patterning. We have explored signal transduction pathways in the chicken limb bud. A cDNA encoding RACK1, a protein that binds and stabilizes activated protein kinase C (PKC), was isolated in a screen for genes induced by retinoic acid (RA) in the chick wing bud. Fibroblast growth factor (FGF) also induced RACK1 and such induction of RACK1 expression was accompanied by a significant augmentation in the number of active PKC molecules and an elevation of PKC enzymatic activity. This suggests that PKCs mediate signal transduction in the limb bud. Application of chelerythrine, a potent PKC inhibitor, to the presumptive wing region resulted in buds that did not express sonic hedgehog (Shh) and developed into wings that were severely truncated. This observation suggests that the expression of Shh depends on PKCs. Providing ectopic SHH protein, RA or ZPA grafts overcome the effects of blocking PKC with chelerythrine and resulted in a rescue of the wing morphology. Taken together, these findings suggest that the responsiveness of Shh to FGF is mediated, at least in part, by PKCs.

Direct Interaction of All-trans-retinoic Acid with Protein Kinase C (PKC): IMPLICATIONS FOR PKC SIGNALING AND CANCER THERAPY

Protein kinase C (PKC) regulates fundamental cellular functions including proliferation, differentiation, tumorigenesis, and apoptosis. All-trans-retinoic acid (atRA) modulates PKC activity, but the mechanism of this regulation is unknown. Amino acid alignments and crystal structure analysis of retinoic acid (RA)-binding proteins revealed a putative atRA-binding motif in PKC, suggesting existence of an atRA binding site on the PKC molecule. This was supported by photolabeling studies showing concentration- and UV-dependent photoincorporation of [(3)H]atRA into PKCalpha, which was effectively protected by 4-OH-atRA, 9-cis-RA, and atRA glucuronide, but not by retinol. Photoaffinity labeling demonstrated strong competition between atRA and phosphatidylserine (PS) for binding to PKCalpha, a slight competition with phorbol-12-myristate-13-acetate, and none with diacylglycerol, fatty acids, or Ca(2+). At pharmacological concentrations (10 micrometer), atRA decreased PKCalpha activity through the competition with PS but not phorbol-12-myristate-13-acetate, diacylglycerol, or Ca(2+). These results let us hypothesize that in vivo, pharmacological concentrations of atRA may hamper binding of PS to PKCalpha and prevent PKCalpha activation. Thus, this study provides the first evidence for direct binding of atRA to PKC isozymes and suggests the existence of a general mechanism for regulation of PKC activity during exposure to retinoids, as in retinoid-based cancer therapy.

Conformation of the C1 phorbol-ester-binding domain participates in the activating conformational change of protein kinase C

The fluorescent phorbol ester 12-N-methylanthraniloylphorbol 13-acetate [sapintoxin D (SAPD)] was used as both the activator and the probe for the activating conformational change of the C1 domain of recombinant protein kinase C (PKC)alpha. Fluorescence emission spectra and steady-state anisotropy measurements of SAPD in fully active membrane-associated PKC show that there is a relatively hydrophobic environment and restricted motional freedom characterizing the phorbol-ester-binding site. SAPD also interacts with the membrane lipids so that it was necessary to resort to time-resolved anisotropy measurements to resolve the signals corresponding to PKC-bound SAPD from that associated with buffer and lipid. In the presence of membrane lipids (unilamellar vesicles of phosphatidylcholine and phosphatidylserine, 4:1 molar ratio) and Ca(2+), at a concentration sufficient to activate the enzyme fully, a long correlation time characteristic of highly restricted motion was observed for PKC-associated SAPD. The fraction of SAPD molecules displaying this restricted motion, in comparison with the total SAPD including that in lipids and in buffer, increased with increasing concentrations of Ca(2+) and paralleled the appearance of enzyme activity, whereas the rotational correlation time remained constant. This could be rationalized as an increase in the number of active PKC conformers in the total population of PKC molecules. It therefore seems that there is a distinct conformation of the C1 activator-binding domain associated with the active form of PKC. The addition of SAPD and dioleoyl-sn-glycerol together produced an activity higher than that achievable by either activator alone both at concentrations that alone induced maximal activity for the respective activator; this higher activity was associated with a further restriction in SAPD motion. Increasing the cholesterol concentration, the phosphatidylethanolamine concentration, the sn-2 unsaturation in phosphatidylcholine and the vesicle curvature each also elevated SAPD-induced PKC activity and again increased the PKC-associated SAPD rotational correlation time. In summary, the rotational correlation time of PKC-bound SAPD, extractable from a single time-resolved fluorescence anisotropy measurement, provides a novel probe for the involvement of interactions between the C1 domain and phorbol ester in the modulation of PKC activity.

Both activators and inhibitors of protein kinase C promote the inhibition of phenylephrine-induced [Ca2+] i oscillations in single intact rat hepatocytes

In single isolated rat hepatocytes Ca 2+-mobilising hormones induce oscillations in cytosolic free Ca 2+ ([Ca 2+] i) in which the frequency of spiking depends on agonist dose, but the time course of individual spikes depends on the hormone species, rather than agonist concentration. We have previously presented data using sphingosine and staurosporine as evidence of a negative feedback role for protein kinase C (PKC) in the elongation of the falling phase of [Ca 2+] i spikes. We show here that the principal effect of three specific PKC inhibitors, namely the bis-indolylmaleimide GF 109203X, the tetracyclic aromatic alkaloid chelerythrine, and a myristoylated PKC pseudosubstrate peptide, that act at different sites on the PKC molecule, is a reduction in, or a complete suppression of, the phenylephrine-induced [Ca 2+] i oscillation frequency. These results resemble the effects of activators of PKC and modulators of diacylglycerol (DAG) metabolism. Furthermore, following phorbol ester-induced inhibition of the hepatocyte [Ca 2+] i oscillator, the addition of all three of these PKC inhibitors further reduces the [Ca 2+] i oscillation frequency, with high concentrations of chelerythrine being the only agent that overcomes this inhibition by phorbol ester. These paradoxical results point to the need for caution in interpreting the effects of protocols involving PKC activators and inhibitors in assessing the feedback control from PKC on cellular [Ca 2+] i oscillations.

Direct Activation of Protein Kinase C by 1α,25-Dihydroxyvitamin D3

The key metabolite of vitamin D3, 1 alpha,25-dihydroxyvitamin D3 (1,25-D3), induces rapid cellular responses that constitute a so-called "non-genomic" response. This effect is distinguished from its "classic" genomic role in calcium homeostasis involving the nuclear 1,25-D3 receptor. Evidence is presented that protein kinase C (PKC) is directly activated by 1,25-D3 at physiological concentrations (EC50 = 16 +/- 1 nM). The effect was demonstrable with single PKC-alpha, -gamma, and -epsilon isoform preparations, assayed in a system containing only purified enzyme, substrate, co-factors, and lipid vesicles, from which it is inferred that a direct interaction with the enzyme is involved. The finding that calcium-independent isoform PKC-epsilon was also activated by 1,25-D3 shows that the calcium binding C2 domain is not required. The level of 1,25-D3-induced activation, paired with either diacylglycerol or 4 beta-12-O-tetradecanoylphorbol-13-acetate, was greater than that achievable by any individual activator alone, each at a saturating concentration, a result that implies two distinct activator sites on the PKC molecule. Phosphatidylethanolamine present in the lipid vesicles potentiated 4 beta-12-O-tetradecanoylphorbol-13-acetate- and diacylglycerol-induced PKC activities, whereas 1,25-D3-induced activity decreased, consistent with 1,25-D3-activated PKC possessing a distinct conformation. The results suggest that PKC is a "membrane-bound receptor" for 1,25-D3 and that it could be important in the control of non-genomic cellular responses to the hormone.

Characterization of protein kinase C activities in postsynaptic density fractions prepared from cerebral cortex, hippocampus, and cerebellum

Protein kinase C (PKC) activities, especially, substrates and PKC isozymes, associated with postsynaptic density (PSD) fractions isolated from rat cerebral cortex, hippocampus, or cerebellum were investigated. The 17k M r major substrate for PKC was associated with PSD fractions prepared from cerebral cortex and hippocampus, and several substrates including 18k M r, protein were associated with PSD fraction isolated from cerebellum. The content of 17k M r, substrate was extremely low in the PSD fraction prepared from cerebellum. PKCs-β and λ were associated with PSD fractions and PKC-α was virtually absent in the fraction prepared from the three different regions of the brain. All of PKCs-α, β, and λ were associated with synaptosome fractions. The 36k M r bands immunoreactive with anti-PKC-β antibody, probably degradation products of native PKC-β, were detected in both the PSD and synaptosome fractions from the three regions, and the ratio of the degradation fragments to native PKC molecule was higher in PSD fractions than in synaptosome fractions. The results suggest postsynaptic roles of PKCs-β and λ and involvement of proteolytic activation of PKC-β in the postsynaptic signal processing.

The protein kinase C family for the regulation of cellular functions

The physiological importance of protein kinase C (PKC) activation is widely appreciated and well documented. It is now clear that there is more than one species of PKC molecule, and several discrete subspecies have been defined. These proteins are derived from both multiple genes and from alternative splicing of a single mRNA transcript, yet possess a primary structure containing conserved structural motifs with a high degree of sequence homology. In mammalian tissues at least seven subspecies can be distinguished, one of which is expressed only in the central nervous tissues. Biochemical and immunocytochemical studies have revealed that these PKC subspecies are differently located in particular cell types, and at limited intracellular locations. The enzyme subspecies purified from tissue show subtle differences in their mode of activation, sensitivity to Ca2+ and catalytic activity. It is worth noting that unsaturated free fatty acids including arachidonic, oleic, and linoleic acids dramatically activate several members of the PKC family in the presence of diacylglycerol at the basal level of Ca2+. It is possible that activation of the enzyme is an integral part of the signal-induced degradation cascade of various membrane phospholipids catalyzed by phospholipases C, A2 and perhaps D as well. Evidence now accumulates that PKC plays pivotal roles in control of a number of membrane functions, such as exocytosis, release reactions, and ion channel conductivity, as well as in cross-talks of various cell-signaling systems. It is also clear that PKC plays roles of crucial importance for regulation of gene expression and cell growth. Biomedical Reviews 1992; 1: 1-6.

Protein kinase C in rat brain myelin.

It has been suggested that phosphorylation of myelin basic protein (MBP) in CNS is catalyzed by protein kinase C (PKC). In order to demonstrate that PKC in the myelin phosphorylates MBP, PKC was partially purified from rat CNS myelin by solubilization with Triton X-100 followed by a DEAE-cellulose column. MBP and histone III-S were phosphorylated in the presence of Ca2+ and phospholipid by rat myelin PKC. High voltage electrophoresis revealed that the phosphoamino acids in MBP by this kinase was serine residue, which is known to be the amino acid phosphorylated by PKC. The activity of PKC extracted from myelin was inhibited by the addition of psychosine to the incubation mixture. To confirm the presence of PKC molecule and to identify the isoform of PKC in the myelin, the solubilized myelin fraction was applied on SDS-PAGE, transferred to a nitrocellulose sheet and stained with anti-PKC monoclonal antibodies. Rat CNS myelin contained the PKC of about 80 kDa (intact PKC), and no proteolytic fragments were observed. PKC isozymes in myelin were type II and III. A developmental study from 14 to 42 postnatal days showed that PKC activity in CNS myelin seemed to parallel the deposition of myelin protein.

The protein kinase C-related PKC-L(eta) gene product is localized in the cell nucleus.

The tumor promoters phorbol esters are thought to induce changes in cell growth and gene expression by direct activation of protein kinase C (PKC). However, the molecular mechanisms by which PKC molecules transduce signals into the cell nucleus are unknown. In this study, we provide evidence for a direct target for phorbol esters in the nucleus. We demonstrate that the new PKC-related family member, PKC-L, recently isolated by us, is expressed specifically in the cell nucleus. Localization of PKC-L in the cell nucleus is shown both by immunofluorescence staining and by subcellular fractionation experiments of several human cell lines, including the human epidermoid carcinoma line A431. Treatment of these cells by phorbol esters does not induce the down-regulation of PKC-L, in contrast to their effect on classical PKC family members. This is the only PKC isoenzyme described so far that resides permanently and specifically in the cell nucleus. PKC-L may function as an important link in tumor promoting, e.g., as a nuclear regulator of gene expression that changes the phosphorylation state of transcriptional components such as the AP-1 complex.

The protein kinase C-related PKC-L(eta) gene product is localized in the cell nucleus.

The tumor promoters phorbol esters are thought to induce changes in cell growth and gene expression by direct activation of protein kinase C (PKC). However, the molecular mechanisms by which PKC molecules transduce signals into the cell nucleus are unknown. In this study, we provide evidence for a direct target for phorbol esters in the nucleus. We demonstrate that the new PKC-related family member, PKC-L, recently isolated by us, is expressed specifically in the cell nucleus. Localization of PKC-L in the cell nucleus is shown both by immunofluorescence staining and by subcellular fractionation experiments of several human cell lines, including the human epidermoid carcinoma line A431. Treatment of these cells by phorbol esters does not induce the down-regulation of PKC-L, in contrast to their effect on classical PKC family members. This is the only PKC isoenzyme described so far that resides permanently and specifically in the cell nucleus. PKC-L may function as an important link in tumor promoting, e.g., as a nuclear regulator of gene expression that changes the phosphorylation state of transcriptional components such as the AP-1 complex.

Phosphorylation of Type II (β) Protein Kinase C by Casein Kinase II1

Rat brain type II (beta) protein kinase C (PKC) was phosphorylated by rat lung casein kinase II (CK-II). Neither type I (gamma) nor type III (alpha) PKC was significantly phosphorylated by CK-II. CK-II incorporated 0.2-0.3 mol of phosphate into 1 mol of type II PKC. This phosphate was located at the single seryl residue (Ser-11) in the V1-variable region of the regulatory domain of the PKC molecule. A glutamic acid cluster was located at the carboxyl-terminal side of Ser-11, showing the consensus sequence for phosphorylation by CK-II. The velocity of this phosphorylation was enhanced by the addition of Ca2+, diolein, and phosphatidylserine, which are all required for the activation of PKC. Phosphorylation of casein or synthetic oligopeptides by CK-II was not affected by Ca2+, diolein, or phosphatidylserine. Available evidence suggests that CK-II phosphorylates preferentially the activated form of type II PKC. It remains unknown, however, whether this reaction has a physiological significance.

H1 Histone Stimulates Limited Proteolysis of Protein Kinase C Subspecies by Calpain II1

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 acute and chronic response of protein kinase C in cultured neonatal rat heart myocytes to α 1-adrenergic and phorbol ester stimulation

Both α 1-adrenergic agonists (e.g. norepinephrine, NE ∗ ∗ Abbreviations used: DG, diacylglycerol; MEM, minimum essential medium (Eagle's); NE, norepinephrine; PDB, phorbol dibutyrate; PKC, protein kinase C (calcium/lipid-dependent protein kinase); PMA, phorbol myristate acetate. ) and tumor-promoting phorbol esters (e.g. phorbol myristate acetate, PMA) are known to activate protein kinase C (PKC) ( Abdel-Latif, 1986, Niedel and Blackshear, 1986). However, α 1 agonists and PMA produce very different effects on cardiac function (see Simpson, 1985; Benfey, 1987; Meidell et al., 1986 ; Leatherman et al., 1987 ; Yuan et al., 1987 ; for examples). PKC activation in heart cells has been studied only for PMA treated perfused heart ( Yuan et al., 1987 ). Therefore, acute activation and chronic regulation of PKC by NE and PMA were compared in cultured neonatal rat heart myocytes. NE acutely and transiently activated PKC, as measured by translocation of PKC activity to the cell particulate fraction ( Niedel and Blackshear, 1986). Particulate PKC activity peaked at 23% of total after NE for 30 s, as compared with 8% for control ( P < 0.001). By contrast, acute PKC activation by PMA was more pronounced and persistent, with particulate PKC activity 62% of total at 5 min ( P < 0.001). Calcium/lipid-independent kinase activity increased acutely with PMA, but not with NE. Chronic treatment with NE (24 to 48 h) increased total per cell PKC activity and 3H-phorbol dibutyrate (PDB) binding sites, an index of the number of PKC molecules ( Niedel and Blackshear, 1986), by 30 to 60% over control (all P < 0.05 to 0.01). In contrast with NE, chronic treatment with PMA down-regulated PKC, reducing total per cell PKC activity and 3H-PDB binding sites to 3% and 12% of control, respectively ( P < 0.01). Both stimuli increased total protein per cell by 18 to 28% over control ( P < 0.01). This is the first direct demonstration of α 1-adrenergic acute activation and chronic up-regulation of PKC in heart myocytes. Acute and chronic regulation of PKC by α 1-adrenergic stimulation is very different from that by PMA.