Protein Kinase C Autophosphorylation Research Articles

Regulation of PKC Autophosphorylation by Calponin in Contractile Vascular Smooth Muscle Tissue

Protein kinase C (PKC) is a key enzyme involved in agonist-induced smooth muscle contraction. In some cases, regulatory phosphorylation of PKC is required for full activation of the enzyme. However, this issue has largely been ignored with respect to PKC-dependent regulation of contractile vascular smooth muscle (VSM) contractility. The first event in PKC regulation is a transphosphorylation by PDK at a conserved threonine in the activation loop of PKC, followed by the subsequent autophosphorylation at the turn motif and hydrophobic motif sites. In the present study, we determined whether phosphorylation of PKC is a regulated process in VSM and also investigated a potential role of calponin in the regulation of PKC. We found that calponin increases the level of in vitro PKCα phosphorylation at the PDK and hydrophobic sites, but not the turn motif site. In vascular tissues, phosphorylation of the PKC hydrophobic site, but not turn motif site, as well as phosphorylation of PDK at S241 increased in response to phenylephrine. Calponin knockdown inhibits autophosphorylation of cellular PKC in response to phenylephrine, confirming results with recombinant PKC. Thus these results show that autophosphorylation of PKC is regulated in dVSM and calponin is necessary for autophosphorylation of PKC in VSM.

Nuclear Factor-κB (NF-κB) Mediates a Protective Response in Cancer Cells Treated with Inhibitors of Fatty Acid Synthase

The efficacy of drugs used to treat cancer can be significantly attenuated by adaptive responses of neoplastic cells to drug-induced stress. To determine how cancer cells respond to inhibition of the enzyme fatty acid synthase (FAS), we focused on NF-κB-mediated pathways, which can be activated by various cellular stresses. Treating lung cancer cells with C93, a pharmacological inhibitor of FAS, results in changes indicative of a rapid initiation of NF-κB signaling, including translocation of RelA/p65 NF-κB to the nucleus, activation of a transfected NF-κB-luciferase reporter, and increased expression of NF-κB-dependent transcripts, IL-6, IL-8, and COX-2. Verifying that these responses to C93 are specifically related to inhibition of FAS, we confirmed that levels of these same transcripts increase in response to siRNA targeting FAS. Inhibiting this NF-κB response (either by transfecting a mutant IκBα or treating with bortezomib) resulted in increased cell killing by C93, indicating that the NF-κB response is protective in this setting. Because inhibiting FAS leads to accumulation of intermediate metabolites of fatty acid biosynthesis, we then questioned whether protein kinase C (PKC) is involved in this response to metabolic stress. Immunofluorescence microscopy revealed that C93 treatment results in cellular translocation of PKCα and PKCβ isoforms and increased PKCα-dependent phosphorylation of the IκBα subunit of NF-κB. Furthermore, inhibiting PKC activity with RO-31-8220 or PKCα isoform-specific siRNA attenuates C93-induced IκBα phosphorylation and NF-κB activation and also potentiates C93-induced cell killing. These results suggest a link between PKC and NF-κB in protecting cancer cells from metabolic stress induced by inhibiting FAS.

Sustained Receptor Stimulation Leads to Sequestration of Recycling Endosomes in a Classical Protein Kinase C- and Phospholipase D-dependent Manner

Considerable insight has been garnered on initial mechanisms of endocytosis of plasma membrane proteins and their subsequent trafficking through the endosomal compartment. It is also well established that ligand stimulation of many plasma membrane receptors leads to their internalization. However, stimulus-induced regulation of endosomal trafficking has not received much attention. In previous studies, we showed that sustained stimulation of protein kinase C (PKC) with phorbol esters led to sequestration of recycling endosomes in a juxtanuclear region. In this study, we investigated whether G-protein-coupled receptors that activate PKC exerted effects on endosomal trafficking. Stimulation of cells with serotonin (5-hydroxytryptamine (5-HT)) led to sequestration of the 5-HT receptor (5-HT2AR) into a Rab11-positive juxtanuclear compartment. This sequestration coincided with translocation of PKC as shown by confocal microscopy. Mechanistically the observed sequestration of 5-HT2AR was shown to require continuous PKC activity because it was inhibited by pretreatment with classical PKC inhibitor Gö6976 and could be reversed by posttreatment with this inhibitor. In addition, classical PKC autophosphorylation was necessary for receptor sequestration. Moreover inhibition of phospholipase D (PLD) activity and inhibition of PLD1 and PLD2 using dominant negative constructs also prevented this process. Functionally this sequestration did not affect receptor desensitization or resensitization as measured by intracellular calcium increase. However, the PKC- and PLD-dependent sequestration of receptors resulted in co-sequestration of other plasma membrane proteins and receptors as shown for epidermal growth factor receptor and protease activated receptor-1. This led to heterologous desensitization of those receptors and diverted their cellular fate by protecting them from agonist-induced degradation. Taken together, these results demonstrate a novel role for sustained receptor stimulation in regulation of intracellular trafficking, and this process requires sustained stimulation of PKC and PLD.

Mechanism of Inhibition of Sequestration of Protein Kinase C α/βII by Ceramide: ROLES OF CERAMIDE-ACTIVATED PROTEIN PHOSPHATASES AND PHOSPHORYLATION/DEPHOSPHORYLATION OF PROTEIN KINASE C α/βII ON THREONINE 638/641

Sustained activation of protein kinase C (PKC) isoenzymes alpha and betaII leads to their translocation to a perinuclear region and to the formation of the pericentrion, a PKC-dependent subset of recycling endosomes. In MCF-7 human breast cancer cells, the action of the PKC activator 4beta-phorbol-12-myristate-13-acetate (PMA) evokes ceramide formation, which in turn prevents PKCalpha/betaII translocation to the pericentrion. In this study we investigated the mechanisms by which ceramide negatively regulates this translocation of PKCalpha/betaII. Upon PMA treatment, HEK-293 cells displayed dual phosphorylation of PKCalpha/betaII at carboxyl-terminal sites (Thr-638/641 and Ser-657/660), whereas in MCF-7 cells PKCalpha/betaII were phosphorylated at Ser-657/660 but not Thr-638/641. Inhibition of ceramide synthesis by fumonisin B1 overcame the defect in PKC phosphorylation and restored translocation of PKCalpha/betaII to the pericentrion. To determine the involvement of ceramide-activated protein phosphatases in PKC regulation, we employed small interference RNA to silence individual Ser/Thr protein phosphatases. Knockdown of isoforms alpha or beta of the catalytic subunits of protein phosphatase 1 not only increased phosphorylation of PKCalpha/betaII at Thr-638/641 but also restored PKCbetaII translocation to the pericentrion. Mutagenesis approaches in HEK-293 cells revealed that mutation of either Thr-641 or Ser-660 to Ala in PKCbetaII abolished sequestration of PKC, implying the indispensable roles of phosphorylation of PKCalpha/betaII at those sites for their translocation to the pericentrion. Reciprocally, a point mutation of Thr-641 to Glu, which mimics phosphorylation, in PKCbetaII overcame the inhibitory effects of ceramide on PKC translocation in PMA-stimulated MCF-7 cells. Therefore, the results demonstrate a novel role for carboxyl-terminal phosphorylation of PKCalpha/betaII in the translocation of PKC to the pericentrion, and they disclose specific regulation of PKC autophosphorylation by ceramide through the activation of specific isoforms of protein phosphatase 1.

Dual mechanism of autoregulation of protein kinase C in myocardial ischemia

Recently, a dual activation mechanism of protein kinase C (PKC) in ischemia has been reported, consisting of early translocation and late expressional regulation. Moreover, autophosphorylation of the enzyme has been shown in vitro during its activation. This study aimed to show modes of late activation of PKC in myocardial ischemia in intact hearts. Isolated perfused hearts of male Wistar rats were used. A: To examine if the early translocation of PKC influences the late transcriptional activation, hearts were treated with the PKC-inhibitor Bisindolylmaleimid (BIS, 0.25 microM) before the onset of ischemia and then subjected to ischemia (30 min). PKC-isoform mRNA was quantified by RT-PCR. In these experiments, ischemia leads to a selective increase of mRNA specific for the isoforms PKC-delta and PKC-epsilon (163% and 168% of control, p<0.05). This ischemia-induced upregulation could be completely blocked by BIS given before the onset of ischemia. B: To test the capacity of PKC to undergo phosphorylation during ischemia, hearts were perfused with [32P]-phosphorus and then subjected to ischemia. Ischemia (30 min) induced a significant 3-fold increase of PKC phosphorylation. Stimulation of heart with the PKC-activator tetradecanoylphorbol-13-acetate (TPA) lead to a comparable phosphorylation, suggesting that ischemia leads to autophosphorylation of PKC. Ischemia activates two distinct forms of autoregulation of PKC. The expressional upregulation of PKC-delta and PKC-epsilon is dependent on early activation of the enzyme. At the same time, processes of enzyme phosphorylation occur. Both the mechanisms may contribute to enzyme activation processes beyond the classical enzyme translocation.

A Ras activation pathway dependent on Syk phosphorylation of protein kinase C.

Protein kinase C (PKC) and Syk protein tyrosine kinase play critical roles in immune cell activation including that through the high-affinity IgE receptor, FcepsilonRI. Mechanisms by which PKC activation leads to the activation of Ras, a family of GTPases essential for immune cell activation, have been elusive. We present evidence that Tyr-662 and Tyr-658 of PKCbetaI and PKCalpha, respectively, are phosphorylated by Syk in the membrane compartment of FcepsilonRI-stimulated mast cells. These phosphorylations require prior PKC autophosphorylation of the adjacent serine residues (Ser-661 and Ser-657, respectively) and generate a binding site for the SH2 domain of the adaptor protein Grb-2. By recruiting the Grb-2/Sos complex to the plasma membrane, these conventional PKC isoforms contribute to the full activation of the Ras/extracellular signal-regulated kinase signaling pathway in FcepsilonRI-stimulated mast cells.

Alterations in protein kinase C isoenzyme expression and autophosphorylation during the progression of pressure overload-induced left ventricular hypertrophy.

Cardiomyocytes express several isoenzymes of protein kinase C (PKC), which as a group have been implicated in the induction of left ventricular hypertrophy (LVH) and its transition to heart failure. Individual PKC isoenzymes also require transphosphorylation and autophosphorylation for enzymatic activity. To determine whether PKC isoenzyme expression and autophosphorylation are altered during LVH progression in vivo, suprarenal abdominal aortic coarctation was performed in Sprague-Dawley rats. Quantitative Western blotting was performed on LV tissue 1, 8 and 24 weeks after aortic banding, using antibodies specific for total PKCalpha, PKCdelta and PKCepsilon, and their C-terminal autophosphorylation sites. Aortic banding produced sustained hypertension and gradually developing LVH that progressed to diastolic heart failure over time. PKCepsilon levels and autophosphorylation were not significantly different from sham-operated controls during any stage of LVH progression. PKCalpha expression levels were also unaffected during the induction of LVH, but increased 3.2 +/- 0.8 fold during the transition to heart failure. In addition, there was a high degree of correlation between PKCalpha levels and the degree of LVH in 24 week banded animals. However, autophosphorylated PKCalpha was not increased at any time point. In contrast, PKCdelta autophosphorylation was increased prior to the development of LVH, and also during the transition to heart failure. The increased PKCdelta autophosphorylation in 1 week banded rats was not accompanied by an increase in total PKCdelta, whereas total PKCdelta levels were markedly increased (6.0 +/- 1.7 fold) in 24 week banded animals. Furthermore, both phosphorylated and total PKCdelta levels were highly correlated with the degree of LVH in 24 week banded rats. In summary, we provide indirect evidence to indicate that PKCdelta may be involved in the induction of pressure overload LVH, whereas both PKCdelta and PKCalpha may be involved in the transition to heart failure.

Membrane Translocation of Novel Protein Kinase Cs Is Regulated by Phosphorylation of the C2 Domain

Ca(2+)-independent or novel protein kinase Cs (nPKCs) contain an N-terminal C2 domain of unknown function. Removal of the C2 domain of the Aplysia nPKC Apl II allows activation of the enzyme at lower concentrations of phosphatidylserine, suggesting an inhibitory role for the C2 domain in enzyme activation. However, the mechanism for C2 domain-mediated inhibition is not known. Mapping of the autophosphorylation sites for protein kinase C (PKC) Apl II reveals four phosphopeptides in the regulatory domain of PKC Apl II, two of which are in the C2 domain at serine 2 and serine 36. Unlike most PKC autophosphorylation sites, these serines could be phosphorylated in trans. Interestingly, phosphorylation of serine 36 increased binding of the C2 domain to phosphatidylserine membranes in vitro. In cells, PKC Apl II phosphorylation at serine 36 was increased by PKC activators, and PKC phosphorylated at this position translocated more efficiently to membranes. Moreover, mutation of serine 36 to alanine significantly reduced membrane translocation of PKC Apl II. We suggest that translocation of nPKCs is regulated by phosphorylation of the C2 domain.

Regulation of Receptor-mediated Protein Kinase C Membrane Trafficking by Autophosphorylation

Signal transduction via protein kinase C (PKC) is closely regulated by its subcellular localization. In response to activation of cell-surface receptors, PKC is directed to the plasma membrane by two membrane-targeting domains, namely the C1 and C2 regions. This is followed by the return of the enzyme to the cytoplasm, a process shown recently to require PKC autophosphorylation (Feng, X., and Hannun, Y. A. (1998) J. Biol. Chem. 273, 26870-26874). In the present study, we examined mechanisms of translocation and reverse translocation and the role of autophosphorylation in these processes. By visualizing the trafficking of wild-type as well as mutant PKCbetaII in live cells, we demonstrated that in response to cell-surface receptor activation, the function of the C1 region is required but not sufficient for recruitment of the enzyme to the plasma membrane. The C2 region is also critical in anchoring the enzyme to the plasma membrane. Furthermore, the inability of a kinase-deficient PKC to undergo reverse translocation was restored by the addition of intracellular calcium chelators, suggesting a role for the C2 region in the persistent phase of translocation. On the other hand, the inability of a C2 deletion mutant (C1 region intact) to translocate in response to agonist was reversed in mutants lacking kinase activity or by mutation of the Ser(660) autophosphorylation site to alanine, suggesting that autophosphorylation of this site is required for opposing the action of the C2 region. Therefore, the membrane-targeting function of the C1 region is facilitated by the C2 region and appears to be opposed by autophosphorylation. Taken together, these findings provide novel evidence of the functional regulation of reversible PKC membrane localization by autophosphorylation, and they show that the dynamic translocation of PKC in response to agonists is tightly regulated in a collaborative fashion by the C1 and C2 regions in balance with the effects of autophosphorylation.

A synthetic peptide derived from the non-structural protein 3 of hepatitis C virus serves as a specific substrate for PKC.

A synthetic peptide corresponding to the amino acid sequence Arg1487-Arg-Gly-Arg-Thr-Gly-Arg-Gly-Arg-Arg-Gly-Ile-Tyr-Arg1500 of the hepatitis C virus (HCV) polyprotein was found to be a selective substrate for protein kinase C (PKC). In the presence of Ca2+, TPA and phospholipid, PKC phosphorylates the peptide [termed HCV(1487-1500)] with a Km of 11 microM and Vmax of 24 micromol x min(-1) x mg(-1). HCV(1487-1500) acts as a competitive inhibitor of PKC towards other peptide or protein substrates and inhibits the kinase activity with an IC50 corresponding to the Km values measured for the substrates. N- or C-terminally deleted analogs of HCV(1487-1500) did not show inhibitory effects and were only marginally or not phosphorylatable. We designed an additional peptide in which the tyrosine residue was replaced by phenylalanine ([Phe1499]HCV(1487-1500)). This peptide was neither phosphorylated by other serine/threonine kinases tested nor by whole cell extracts prepared from PKC-depleted cells. [Phe1499]HCV(1487-1500) was used to monitor the TPA-induced translocation of PKC activity to the particulate fraction in JB6 cells. The use of SDS/PAGE to separate the peptide from ATP and Pi allowed to monitor simultaneously PKC autophosphorylation and phosphorylation of the peptide. The data presented here show that[Phe1499]HCV(1487-1500) can serve as a convenient tool for investigations of PKC activity also in the presence of other kinases in tissues or in crude cell extracts.

Phage display identifies thioredoxin and superoxide dismutase as novel protein kinase C-interacting proteins: thioredoxin inhibits protein kinase C-mediated phosphorylation of histone

Using phage display we identify the redox proteins thioredoxin and superoxide dismutase (SOD) as novel protein kinase C (PKC)-interacting proteins. Overlay assays demonstrated that PKC bound to immobilized thioredoxin, providing supporting evidence for the phage display results. Kinase assays demonstrated that SOD and thioredoxin were not direct substrates for PKC but that both proteins blocked autophosphorylation of PKC. Moreover, thioredoxin inhibited PKC-mediated phosphorylation of histone (IC50of approx. 20 ng/ml).

Phage display identifies thioredoxin and superoxide dismutase as novel protein kinase C-interacting proteins: thioredoxin inhibits protein kinase C-mediated phosphorylation of histone

Using phage display we identify the redox proteins thioredoxin and superoxide dismutase (SOD) as novel protein kinase C (PKC)-interacting proteins. Overlay assays demonstrated that PKC bound to immobilized thioredoxin, providing supporting evidence for the phage display results. Kinase assays demonstrated that SOD and thioredoxin were not direct substrates for PKC but that both proteins blocked autophosphorylation of PKC. Moreover, thioredoxin inhibited PKC-mediated phosphorylation of histone (IC(50) of approx. 20 ng/ml).

Contributions to Maxima in Protein Kinase C Activation

In many lipid systems, the activity of protein kinase C (PKC) exhibits a peak followed by a decline as the mol % of one component is increased. In these systems, an increase in one lipid component is always at the expense of another or accompanied by a change in total lipid concentration. Here we report that in saturated phosphatidylserine (PS)/phosphatidylcholine (PC)/diacylglycerol (DAG) mixtures, increasing PS or DAG at the expense of PC revealed an optimal mol % PS, dependent on mol % DAG, with higher mol % PS diminishing activity. The decrease at high mol % PS is probably not attributable simply to more gel-phase lipid due to the higher melting temperature of saturated PS versus PC because a similar peak in activity occurred in unsaturated lipid systems. Increasing the total lipid concentration at suboptimal mol % PS provided the same activity as higher mol % PS at lower total lipid concentration. However, at optimal mol % PS, activity increased and then decreased as a function of total lipid concentration. PKC autophosphorylation also exhibited an optimum as a function of mol % PS, and increasing the PKC concentration increased the mol % PS at which activity decreased, both for autophosphorylation and for heterologous phosphorylation. Formation of two-dimensional crystals of PKC on lipid monolayers also exhibited a peak as a function of mol % PS, and the unit cell size of the crystals formed shifts from 50 x 50 A at low mol % PS to 75 x 75 A at higher PS. Collectively, these data suggest the existence of optimal lipid compositions for PKC activation, with increased quantity of these domains serving to dilute out enzyme-substrate aggregates and/or enzyme-enzyme aggregates on the lipid surface.

Protected-site phosphorylation of protein kinase C in hippocampal long-term potentiation.

One important aspect of synaptic plasticity is that transient stimulation of neuronal cell surface receptors can lead to long-lasting biochemical and physiological effects in neurons. In long-term potentiation (LTP), generation of autonomously active protein kinase C (PKC) is one biochemical effect persisting beyond the NMDA receptor activation that triggers plasticity. We previously observed that the expression of early LTP is associated with a phosphatase-reversible alteration in PKC immunoreactivity, suggesting that autophosphorylation of PKC might be elevated in LTP. In the present studies we tested the hypothesis that PKC phosphorylation is persistently increased in the early maintenance of LTP. We generated an antiserum that selectively recognizes the alpha and betaII isoforms of PKC autophosphorylated in the C-terminal domain. Using western blotting with this antiserum we observed an NMDA receptor-mediated increase in phosphorylation of PKC 1 h after LTP was induced. How is the increased phosphorylation maintained in the cell in the face of ongoing phosphatase activity? We observed that dephosphorylation of PKC in vitro requires the presence of cofactors normally serving to activate PKC, i.e., Ca2+, phosphatidylserine, and diacylglycerol. Based on these observations and computer modeling of the three-dimensional structure of the PKC catalytic core, we propose a "protected site" model of PKC autophosphorylation, whereby the conformation of PKC regulates accessibility of the phosphates to phosphatase. Although we have proposed the protected site model based on our studies of PKC phosphorylation in LTP, phosphorylation of protected sites might be a general biochemical mechanism for the generation of stable, long-lasting physiologic changes.

The in vitro phosphorylation of p53 by calcium-dependent protein kinase C--characterization of a protein-kinase-C-binding site on p53.

We show that, in vitro, Ca2+-dependent protein kinase C (PKC) phosphorylates recombinant murine p53 protein on several residues contained within a conserved basic region of 25 amino acids, located in the C-terminal part of the protein. Accordingly, synthetic p53-(357-381)-peptide is phosphorylated by PKC at multiple Ser and Thr residues, including Ser360, Thr365, Ser370 and Thr377. We also establish that p53-(357-381)-peptide at micromolar concentrations has the ability to stimulate sequence-specific DNA binding by p53. That stimulation is lost upon phosphorylation by PKC. To further characterise the mechanisms that regulate PKC-dependent phosphorylation of p53-(357-381)-peptide, the phosphorylation of recombinant p53 and p53-(357-381)-peptide by PKC were compared. The results suggest that phosphorylation of full-length p53 on the C-terminal PKC sites is highly dependent on the accessibility of the phosphorylation sites and that a domain on p53 distinct from p53-(357-381)-peptide is involved in binding PKC. Accordingly, we have identified a conserved 27-amino-acid peptide, p53-(320-346)-peptide, within the C-terminal region of p53 and adjacent to residues 357-381 that interacts with PKC in vitro. The interaction between p53-(320-346)-peptide and PKC inhibits PKC autophosphorylation and the phosphorylation of substrates, including p53-(357-381)-peptide, neurogranin and histone H1. Conventional Ca2+-dependent PKC alpha, beta and gamma and the catalytic fragment of PKC (PKM) were nearly equally susceptible to inhibition by p53-(320-346)-peptide. The Ca2+-independent PKC delta was much less sensitive to inhibition. The significance of these findings for understanding the in vivo phosphorylation of p53 by PKC are discussed.

Human T-cell lymphotropic virus type 1 Tax1 activation of NF-kappa B: involvement of the protein kinase C pathway

Human T-cell lymphotropic virus type 1 Tax1 induces the activation and nuclear localization of the cellular transcription factor, NF-kappa B. Treatment of cells with calphostin C, a protein kinase C (PKC) inhibitor, blocked induction of NF-kappa B DNA binding activity in human T-cell lymphotropic virus type 1-transformed C81 cells and Tax1-stimulated murine pre-B cells, suggesting that PKC was an important intermediate in the NF-kappa B induction pathway. We further demonstrate that Tax1 associates with, and activates, PKC. PKC was coimmunoprecipitated with anti- Tax1 sera from Tax1-expressing MT4 extracts and Jurkat extracts in the presence of exogenous Tax1 protein. In addition, the glutathione-S-transferase-Tax1 protein bound specifically to the alpha, delta, and eta PKC isoenzymes synthesized in rabbit reticulocyte lysates. The addition of Tax1 to in vitro kinase reaction mixtures leads to the phosphorylation of Tax1 and an 18-fold increase in the autophosphorylation of PKC. Transfection of Jurkat cells with wild-type Tax1 stimulated membrane translocation of PKC. In contrast, Tax1 mutant M22, which fails to stimulate NF-kappa B-dependent transcription, failed to stimulate membrane translocation of PKC. Tax1 did not directly increase PKC phosphorylation of I kappa B alpha. Our results are consistent with a model in which Tax1 interacts with PKC and stimulates membrane translocation and triggering of the PKC pathway. Subsequent steps in the PKC cascade likely stimulate phosphorylation of I kappa B alpha.

Conformation-dependent phosphorylation of Na,K-ATPase by protein kinase A and protein kinase C.

Phosphorylation of sodium and potassium ion-activated adenosine triphosphatase (Na,K-ATPase) by protein kinase A (PKA) and protein kinase C (PKC) was investigated in vitro, where substrate conformation, kinase activity, and consequent effects on Na,K-ATPase activity could be controlled. With Na, K-ATPase from rat kidney, optimal stoichiometries were close to 1 mol 32P/mol Na,K-ATPase for both kinases. Addition of Na+, K+, P(i), or ouabain is known to stabilize the Na,K-ATPase in different states and was found to affect phosphorylation by the two kinases in a reciprocal way. This indicates that exposure of the phosphorylation sites varies with conformation and suggests a structural basis for the variable responses to kinase activation in intact cells. Further evidence for the importance of Na,K-ATPase conformation in its interaction with kinase came from the autophosphorylation of PKC, which varied in proportion to both the concentration and conformation of rat Na,K-ATPase. With pig and dog Na,K-ATPase, little phosphorylation by PKC was detected, and yet the PKC phosphorylated itself when the Na,K-ATPase was in the optimal conformation. The location of the PKA phosphorylation site was confirmed to be Ser-938 by sequence analysis of a tryptic peptide. Effects of PKA on Na,K-ATPase activity could not be measured because of inhibition by the Triton X-100 needed to obtain phosphorylation. Phosphorylation by PKC, even in optimal conditions, failed to result in inhibition of Na,K-ATPase activity. This suggests that any physiological role of phosphorylation either entails a subtle modulation of enzyme properties, or requires additional regulatory proteins.

Synergy Between Phorbol Esters and Retinoic Acid in Inducing Protein Kinase C Activation

All-trans retinoic acid (RA) activates brain protein kinase C (PKC) in a unique fashion. Co-factors such as Ca2+ or PtdSer are not required for histone phosphorylation. Binding experiments have provided evidence that RA does not act as a phorbol-ester-like activator. However, phorbol esters synergistically enhance this activation in a dose-dependent manner and increase the reaction rate up to five-fold when combined with 10μM RA. Phospholipid-interacting drugs such as phenothiazines and 1-N-(6 aminohexyl) 5-chloro-1-naphtalene-sulfonamide (W7), which compete with PtdSer and inhibit phorbol ester / PtdSer-mediated activation, have potentiating effects on the RA-mediated reaction. RA elicites Ca2+-dependent PKC autophosphorylation. The activation resulting from the combined treatment with PtdSer and RA is more than additive in the presence of Ca2+, indicating that PtdSer-and RA-binding sites are distinct. RA shares several characteristics of activation with sodium deoxycholate and arachidonic acid. These present results suggest that the direct activation of PKC may have physiological and/or pharmacological relevance in the signaling triggered by retinoids.

Agonists and antagonists of protein kinase C function, derived from its binding proteins.

Physical association between proteins involved in signal transduction is required for their functions. Therefore, identification of the interacting sites in the signaling molecules can lead to the development of means to modulate these interactions. We applied this approach to study signal transduction by protein kinase C (PKC). We have previously identified potential PKC binding sites in two PKC binding proteins (annexin I and RACK1). Peptides derived from these sequences inhibit PKC binding to RACK1 in vitro. Here, we tested the ability of two of these peptides, I (KGDYEKILVALCGGN) and rVI (DIINALCF), to affect PKC-mediated function in vivo. The peptides were microinjected into Xenopus oocytes, and insulin-induced beta PKC translocation and oocyte maturation were examined. The peptides had opposite activities on oocyte; peptide I inhibited whereas peptide rVI stimulated insulin-induced Xenopus oocyte maturation. As expected, beta PKC translocation from the cytosol to the particulate fraction of the Xenopus oocytes was inhibited after microinjection of peptide I and induced after microinjection of peptide rVI. Moreover, peptide rVI caused translocation of beta PKC and oocyte maturation without hormone stimulation. In the absence of PKC activators, peptide rVI but not peptide I, activated PKC in vitro as demonstrated in three assays: increased sensitivity to Arg-C endopeptidase, PKC autophosphorylation, and histone phosphorylation. Therefore, although peptides I and rVI have sequence homology, one mimicked hormone-induced PKC-mediated function whereas the other inhibited this hormone-induced function. The molecular mechanisms underlying these opposing effects of the peptides are discussed.

Activation of protein kinase C by selective binding of arginine-rich polypeptides

Protein substrates for protein kinase C (PKC) have phosphorylation domains that are typically rich in the basic amino acids arginine and lysine. However, arginine-rich proteins may interact with PKC differently than lysine-rich proteins, i.e. lysine-rich histone requires phospholipid and Ca2+ to be phosphorylated, whereas the arginine-rich protein, protamine, can bypass these effector requirements. We have studied the interaction of PKC with protamine, histone, poly-L-arginine, and poly-L-lysine to better understand the role of basic protein domains in PKC activation, effector dependence, and substrate specificity. Using a microtiter binding assay, PKC was found to bind tightly to protamine and poly-L-arginine, but not to histone or poly-L-lysine, in the absence of phospholipid, Ca2+, and MgATP. Furthermore, poly-L-arginine was much more potent than poly-L-lysine at inhibiting protamine phosphorylation; i.e. 1-2 nM poly-L-arginine was sufficient to cause 50% inhibition of protamine phosphorylation, whereas over 300 microM poly-L-lysine was needed to reach 50% inhibition. Autophosphorylation of PKC in the absence of activators was potently stimulated by protamine and poly-L-arginine, but not by histone or poly-L-lysine, suggesting selective stimulation of PKC by arginine-rich polypeptides. Double-reciprocal plots of protamine phosphorylation using either a mixture of isozymes (alpha/beta/gamma) or isolated PKC-beta were parabolic, and analysis of the kinetic data on velocity/[protamine] versus velocity plots indicated positive cooperativity with respect to protamine. These findings are consistent with those from autophosphorylation experiments in that PKC appears to be selectively stimulated by arginine-rich polypeptides. These results suggest that PKC can preferentially bind arginine-rich proteins in the absence of phospholipid and Ca2+. This interaction appears to be distal to the catalytic site and thus binding of arginine-rich proteins may allosterically activate PKC. Selective stimulation of PKC by arginine-rich proteins may be a mechanism by which protamine can bypass activator requirements. Furthermore, control of PKC activity by activator-independent binding of arginine-rich polypeptides suggests that altering access to certain cellular proteins may be a mechanism for PKC regulation in vivo.