Degradation of protein kinase Malpha by mu-calpain in a mu-calpain-protein kinase Calpha complex

In previous studies, we isolated and identified a mu-calpain-PKCalpha complex from rabbit skeletal muscle. At the same time we pointed out that an association between mu-calpain and PKCalpha could occur at the level of the plasma membrane of muscle cells, and that PKCalpha could thus be considered as a potential mu-calpain substrate. In the present study, using the mu-calpain-PKCalpha complex as a model, we report that mu-calpain is activated in the combined presence of physiological calcium concentrations (less than 1 microM) and phosphatidylserine. Furthermore our data also show that: (1) there exists a correlation between the appearance of autolyzed mu-calpain forms and PKCalpha hydrolysis which leads to the formation of PKMalpha; (2) in certain experimental conditions, autolyzed mu-calpain forms are able to hydrolyze PKMalpha independently of the presence of diacylglycerol.     

Retinoic acid as a modulator of the activity of protein kinase Calpha

All-trans-retinoic acid (atRA) is a derivative of vitamin A and possesses antitumor activity. We demonstrate that atRA is able to modulate the activity of protein kinase C alpha (PKCalpha), which is related to tumor development. In vitro, it was found that atRA activated PKCalpha in the presence of Ca(2+) and in the absence of phosphatidylserine, although such activity is considerably inhibited in mutations affecting residues D246 and D248 and also residue N189, all of which are known to be essential for the interaction with Ca(2+) and phosphatidylserine in the C2 domain. It was concluded that atRA substitutes phosphatidylserine although with lower specific activities. However, atRA had a biphasic effect on PKCalpha activity in the presence of activating phospholipids, such as phosphatidylserine and phosphatidylinositol 4,5-bisphosphate, yielding activation at low concentrations but inactivation at higher ones. This second inhibitory characteristic was not shown with K209 and K211 mutations (residues located in the Lys-rich cluster in the C2 domain) in PKCalpha. This interesting effect revealed the importance of phospholipid binding at this site to ensure maximum activity for the wild-type PKCalpha. The C1 domain was not related with the atRA effect on PKCalpha. It was concluded that whereas atRA may activate PKCalpha through the Ca(2+)-phosphatidylserine-binding site of the C2 domain, it may also inhibit the activity of this enzyme when displacing the phospholipid from the Lys-rich cluster also located in the C2 domain.     

Competitive binding of protein kinase Calpha to membranes and Rho GTPases

Previously, we have shown that protein kinase C (PKC) forms a direct high-affinity, isozyme-specific and membrane lipid-independent interaction with Rho GTPases [Slater, S. J., Seiz, J. L., Stagliano, B. A., and Stubbs, C. D. (2001) Biochemistry 40, 4437-4445]. Since the cellular activation of PKCalpha involves an initial translocation from cytosolic to membrane compartments, the present study investigates the interdependence between the direct protein-protein interaction of PKCalpha with the Rho GTPase, Cdc42, and the protein-lipid interactions of PKCalpha with membranes. It was hypothesized that the interaction of PKCalpha with membrane-bound Cdc42 would contribute to the overall membrane-binding affinity of the kinase by providing an additional anchor. However, it was found that the incorporation of isoprenylated Cdc42 into membranes resulted in an apparent decrease in the membrane-binding affinity of PKCalpha, whereas the association of PKCbetaI, PKCdelta, PKCepsilon, and PKCzeta was each unaffected. The presence of membrane-bound Cdc42 resulted in a rightward shift in both the PS- and Ca2+-concentration response curves for PKCalpha membrane association and for the ensuing activation, whereas the maximal levels of binding and activation attained at saturating PS and Ca2+ concentrations were in each case unaffected. Overall, these findings suggest that PKCalpha undergoes a isozyme-specific interaction with membrane-bound Cdc42 to form a PKCalpha-Cdc42 complex, which possesses a membrane-binding affinity that is reduced relative to that of the individual components due to competition between Cdc42 and PS/Ca2+ for binding to PKCalpha. Consistent with this, it was found that the interaction of PKCalpha with membrane-bound Cdc42 was accompanied by the physical dissociation of the PKCalpha-Cdc42 complex from membranes. Thus, the study provides a novel mechanism by which the membrane association and activation of PKCalpha and Cdc42 may be regulated by competing protein-protein and protein-lipid interactions.     

Mechanisms of regulation of phospholipase D1 by protein kinase Calpha

It has been suggested that protein-protein interaction is important for protein kinase C (PKC) alpha to activate phospholipase D1 (PLD1). To determine the one or more sites on PKCalpha that are involved in binding to PLD1, fragments containing the regulatory domain, catalytic domain, and C1-C3 domain of PKCalpha were constructed and shown to be functional, but they all failed to bind and activate PLD1 in vivo and in vitro. A C-terminal 23-amino acid (aa) deletion mutant of PKCalpha was also found to be inactive. To define the binding/activation site(s) in the C terminus of PKCalpha, 1- to 11-aa deletion mutants were made in this terminus. Deletion of up to 9 aa did not alter the ability of PKCalpha to bind and activate PLDl, whereas a 10-aa deletion was inactive. The residue at position 10 was Phe(663). Mutations of this residue (F663D and F663A) caused loss of binding, activation, and phosphorylation of PLD1, indicating that Phe(663) is essential for these activities. Time course experiments showed that the activation of PLD1 by PMA was much faster than its phosphorylation, and its activity decreased as phosphorylation increased with time. Staurosporine, a PKC inhibitor, completely inhibited PLD1 phosphorylation in response to 4beta-phorbol 12-myristate 13-acetate PMA and blocked the later decrease in PLD activity. The same results were found with the D481E mutant of PKCalpha, which is unable to phosphorylate PLD1. These results indicate that neither the regulatory nor catalytic domains of PKCalpha alone can bind to or activate PLD1 and that a residue in the C terminus of PKCalpha (Phe(663)) is required for these effects. The initial activation of PLD1 by PMA is highly correlated with the binding of PKCalpha. Although PKCalpha can phosphorylate PLD1, this is a relatively slow process and is associated with inactivation of the enzyme.     

Interaction of fascin and protein kinase Calpha: a novel intersection in cell adhesion and motility

Coordination of protrusive and contractile cell-matrix contacts is important for cell adhesion and migration, but the mechanisms involved are not well understood. We report an unexpected direct association between fascin, an actin-bundling component of filopodia, microspikes and lamellipodial ribs, and protein kinase Calpha (PKCalpha), a regulator of focal adhesions. The association is detectable by protein-protein binding in vitro, by coimmunoprecipitation from cell extracts, and in live cells as fluorescence resonance energy transfer detected by fluorescence imaging lifetime microscopy. The interaction is physiologically regulated by the extracellular matrix context of cells, depends on activation of PKCalpha and is mediated by the C1B domain of PKCalpha. Strikingly, a fascin mutant, fascin S39D, associates constitutively with PKCalpha. Through use of a newly developed set of membrane-permeable peptides that separately inhibit either fascin/PKCalpha or fascin/actin binding, we have uncovered that specific blockade of the fascin/PKCalpha interaction increases cell migration on fibronectin in conjunction with increased fascin protrusions and remodeling of focal adhesions. These results identify the fascin-PKCalpha interaction as an important novel intersection in the regulation and networking of cell-matrix contacts.     

Identification of interaction sites of protein kinase Calpha on phospholipase D1

Phospholipase D (PLD) is regulated by many factors, including protein kinase C (PKC) and small G-proteins of the Rho and ADP-ribosylation factor families. Previous studies revealed that the activation of PLD1 by phorbol ester is associated with the binding of PKCalpha to a site in the N-terminus of PLD1. The purpose of the present study was to determine this site more precisely. Immunoprecipitation with a series of four PLD1 deletion mutants confirmed that PKCalpha strongly interacted with the amino acid sequence 1-318 at the N-terminus of PLD1 and weakly with the sequence 841-1036 at the C-terminus. Further immunoprecipitation studies with deletion mutants of the 1-318 and 1-215 PLD1 fragments revealed that there were binding sites in the 1-49 N-terminal sequence and also in the 216-318 sequence containing the PH domain. Studies of N-terminal deletion mutants of full-length PLD1 confirmed the presence of a binding site in the 1-49 sequence and a further site in the 1-318 sequence. Both deletion mutants showed impaired activation by PKCalpha in vivo, but unchanged activation by active V(14)RhoA. These findings identify the 1-49 sequence is a major binding/activation site for PKCalpha on PLD1, but also indicate involvement of the PH domain.     

Regulation of protein kinase B/Akt activity and Ser473 phosphorylation by protein kinase Calpha in endothelial cells

Protein kinase Balpha (PKBalpha/Akt-1) is a key mediator of multiple signaling pathways involved in angiogenesis, cell proliferation and apoptosis among others. The unphosphorylated form of Akt-1 is virtually inactive and its full activation requires two phosphatidylinositol-3,4,5-triphosphate-dependent phosphorylation events, Thr308 by 3-phosphoinositide-dependent kinase-1 (PDK1) and Ser473 by an undefined kinase that has been termed PDK2. Recent studies have suggested that the Ser473 kinase is a plasma membrane raft-associated kinase. In this study we show that protein kinase Calpha (PKCalpha) translocates to the membrane rafts in response to insulin growth factor-1 (IGF-1) stimulation. Overexpression of PKCalpha increases Ser473 phosphorylation and Akt-1 activity, while inhibition of its activity or expression decreases IGF-1-dependent activation of Akt-1. Furthermore, in vitro, in the presence of phospholipids and calcium, PKCalpha directly phosphorylates Akt-1 at the Ser473 site. We conclude, therefore, that PKCalpha regulates Akt-1 activity via Ser473 phosphorylation and may function as PDK2 in endothelial cells.     

VCAM-1 signals activate endothelial cell protein kinase Calpha via oxidation

Lymphocyte binding to VCAM-1 activates endothelial cell NADPH oxidase, resulting in the generation of 1 muM H(2)O(2). This is required for VCAM-1-dependent lymphocyte migration. In this study, we identified a role for protein kinase Calpha (PKCalpha) in VCAM-1 signal transduction in human and mouse endothelial cells. VCAM-1-dependent spleen cell migration under 2 dynes/cm(2) laminar flow was blocked by pretreatment of endothelial cells with dominant-negative PKCalpha or the PKCalpha inhibitors, R?-32-0432 or G?-6976. Phosphorylation of PKCalpha(Thr638), an autophosphorylation site indicating enzyme activity, was increased by Ab cross-linking of VCAM-1 on endothelial cells or by the exogenous addition of 1 muM H(2)O(2). The anti-VCAM-1-stimulated phosphorylation of PKCalpha(Thr638) was blocked by scavenging of H(2)O(2) and by inhibition of NADPH oxidase. Furthermore, anti-VCAM-1 signaling induced the oxidation of endothelial cell PKCalpha. Oxidized PKCalpha is a transiently active form of PKCalpha that is diacylglycerol independent. This oxidation was blocked by inhibition of NADPH oxidase. In summary, VCAM-1 activation of endothelial cell NADPH oxidase induces transient PKCalpha activation that is necessary for VCAM-1-dependent transendothelial cell migration.     

Agonist-induced association of tropomyosin with protein kinase Calpha in colonic smooth muscle

Smooth muscle contraction regulated by myosin light chain phosphorylation is also regulated at the thin-filament level. Tropomyosin, a thin-filament regulatory protein, regulates contraction by modulating actin-myosin interactions. Present investigation shows that acetylcholine induces PKC-mediated and calcium-dependent phosphorylation of tropomyosin in colonic smooth muscle cells. Our data also shows that acetylcholine induces a significant and sustained increase in PKC-mediated association of tropomyosin with PKCalpha in the particulate fraction of colonic smooth muscle cells. Immunoblotting studies revealed that in colonic smooth muscle cells, there is no significant change in the amount of tropomyosin or actin in particulate fraction in response to acetylcholine, indicating that the increased association of tropomyosin with PKCalpha in the particulate fraction may be due to acetylcholine-induced translocation of PKCalpha to the particulate fraction. To investigate whether the association of PKCalpha with tropomyosin was due to a direct interaction, we performed in vitro direct binding assay. Tropomyosin cDNA amplified from colonic smooth muscle mRNA was expressed as GST-tropomyosin fusion protein. In vitro binding experiments using GST-tropomyosin and recombinant PKCalpha indicated direct interaction of tropomyosin with PKCalpha. PKC-mediated phosphorylation of tropomyosin and direct interaction of PKCalpha with tropomyosin suggest that tropomyosin could be a substrate for PKC. Phosphorylation of tropomyosin may aid in holding the slided tropomyosin away from myosin binding sites on actin, resulting in actomyosin interaction and sustained contraction.     

Autophosphorylation suppresses whereas kinase inhibition augments the translocation of protein kinase Calpha in response to diacylglycerol

We have seen that protein kinase Calpha (PKCalpha) is transiently translocated to the plasma membrane by carbachol stimulation of neuroblastoma cells. This is induced by the Ca2+ increase, and PKCalpha does not respond to diacylglycerol (DAG). The unresponsiveness is dependent on structures in the catalytic domain of PKCalpha. This study was designed to investigate if and how the kinase activity and autophosphorylation are involved in regulating the translocation. PKCalpha enhanced green fluorescent protein translocation was studied in living neuroblastoma cells by confocal microscopy. Carbachol stimulation induced a transient translocation of PKCalpha to the plasma membrane and a sustained translocation of kinase-dead PKCalpha. In cells treated with the PKC inhibitor GF109203X, wild-type PKCalpha also showed a sustained translocation. The same effects were seen with PKCbetaI, PKCbetaII, and PKCdelta. Only kinase-dead and not wild-type PKCalpha translocated in response to 1,2-dioctanoylglycerol. To examine whether autophosphorylation regulates relocation to the cytosol, the autophosphorylation sites in PKCalpha were mutated to glutamate, to mimic phosphorylation, or alanine, to mimic the non-phosphorylated protein. After stimulation with carbachol, glutamate mutants behaved like wild-type PKCalpha, whereas alanine mutants behaved like kinase-dead PKCalpha. When the alanine mutants were treated with 1,2-dioctanoylglycerol, all cells showed a sustained translocation of the protein. However, neither carbachol nor GF109203X had any major effects on the level of autophosphorylation, and GF109203X potentiated the translocation of the glutamate mutants. We, therefore, hypothesize that 1) autophosphorylation of PKCalpha limits its sensitivity to DAG and 2) that kinase inhibitors augment the DAG sensitivity of PKCalpha, perhaps by destabilizing the closed conformation.     

The origin of C1A-C2 interdomain interactions in protein kinase Calpha

The regulatory domain of protein kinase Calpha (PKCalpha) contains three membrane-targeting modules, two C1 domains (C1A and C1B) that bind diacylglycerol and phorbol ester, and the C2 domain that is responsible for the Ca2+-dependent membrane binding. Accumulating evidence suggests that C1A and C2 domains of PKCalpha are tethered in the resting state and that the tethering is released upon binding to the membrane containing phosphatidylserine. The homology modeling and the docking analysis of C1A and C2 domains of PKCalpha revealed a highly complementary interface that comprises Asp55-Arg252 and Arg42-Glu282 ion pairs and a Phe72-Phe255 aromatic pair. Mutations of these residues in the predicted C1A-C2 interface showed large effects on in vitro membrane binding, enzyme activity, phosphatidylserine selectivity, and cellular membrane translocation of PKCalpha, supporting their involvement in interdomain interactions. In particular, D55A (or D55K) and R252A (or R252E) mutants showed much higher basal membrane affinity and enzyme activity and faster subcellular translocation than wild type, whereas a double charge-reversal mutant (D55K/R252E) behaved analogously to wild type, indicating that a direct electrostatic interaction between the two residues is essential for the C1A-C2 tethering. Collectively, these studies provide new structural insight into PKCalpha C1A-C2 interdomain interactions and the mechanism of lipid-mediated PKCalpha activation.     

Analysis of the subcellular distribution of protein kinase Calpha using PKC-GFP fusion proteins

One important factor for the determination of the specific functions of protein kinase C (PKC) isoforms is their specific subcellular localization. In NIH 3T3 fibroblasts phorbol esters induce translocation of PKCalpha to the plasma membrane and the nucleus. In order to investigate PKCalpha's subcellular distribution and especially its nuclear accumulation in more detail we used fusion proteins consisting of PKCalpha and the green fluorescent protein (GFP). Purified GFP-PKCalpha from baculovirus-infected insect cells undergoes nuclear accumulation without any further stimuli in digitonin-permeabilized cells. Interestingly, permeabilization appears to be a trigger for PKCalpha's nuclear translocation, since the fusion protein also translocates to the nucleus in transiently transfected cells following permeabilization. This suggests that PKCalpha has a high nuclear binding capacity even in the case of large protein amounts. In contrast to endogenous PKCalpha, overexpressed GFP-PKCalpha as well as overexpressed PKCalpha itself translocates mainly to the plasma membrane and only to a smaller extent to the nucleus following stimulation with phorbol ester. Use of fusion proteins of GFP and different mutants of PKCalpha enabled determination of motifs involved PKCalpha's subcellular distribution: A25E and K368R point mutations of PKCalpha showed enhanced affinity for the plasma membrane, whereas sequences within the regulatory domain probably confer PKCalpha's nuclear accumulation.     

Identification and characterization of novel mutations in the human gene encoding the catalytic subunit Calpha of protein kinase A (PKA)

The genes PRKACA and PRKACB encode the principal catalytic (C) subunits of protein kinase A (PKA) Cα and Cβ, respectively. Cα is expressed in all eukaryotic tissues examined and studies of Cα knockout mice demonstrate a crucial role for Cα in normal physiology. We have sequenced exon 2 through 10 of PRKACA from the genome of 498 Norwegian donors and extracted information about PRKACA mutations from public databases. We identified four interesting nonsynonymous point mutations, Arg45Gln, Ser109Pro, Gly186Val, and Ser263Cys, in the Cα1 splice variant of the kinase. Cα variants harboring the different amino acid mutations were analyzed for kinase activity and regulatory (R) subunit binding. Whereas mutation of residues 45 and 263 did not alter catalytic activity or R subunit binding, mutation of Ser(109) significantly reduced kinase activity while R subunit binding was unaltered. Mutation of Cα Gly(186) completely abrogated kinase activity and PKA type I but not type II holoenzyme formation. Gly(186) is located in the highly conserved DFG motif of Cα and mutation of this residue to Val was predicted to result in loss of binding of ATP and Mg(2+), which may explain the kinetic inactivity. We hypothesize that individuals born with mutations of Ser(109) or Gly(186) may be faced with abnormal development and possibly severe disease.     

Phospholipase D1 in caveolae: regulation by protein kinase Calpha and caveolin-1

Caveolae are small plasma membrane invaginations that have been implicated in cell signaling, and caveolin is a principal structural component of the caveolar membrane. Previously we have demonstrated that protein kinase Calpha (PKCalpha) directly interacts with phospholipase D1 (PLD1), activating the enzymatic activity of PLD1 in the presence of phorbol 12-myristate 13-acetate (PMA) [Lee, T. G., et al. (1997) Biochim. Biophys. Acta 1347, 199-204]. In this study, using a detergent-free procedure for the purification of a caveolin-enriched membrane fraction (CEM) and immunoblot analysis, we show that PLD1 is enriched in the CEMs of 3Y1 rat fibroblasts. Purified PLD1 directly bound to a glutathione S-transferase-caveolin-1 fusion protein in in vitro binding assays. The association of PLD1 with caveolin-1 could be completely eliminated by preincubation of PLD1 with an oligopeptide corresponding to the scaffolding domain (amino acids 82-101) of caveolin-1, indicating that caveolin-1 interacts with PLD1 through the scaffolding domain. The peptide also inhibited PKCalpha-stimulated PLD1 activity and the interaction between PLD1 and PKCalpha with an IC50 of 0.5 microM. PMA elicits translocation of PKCalpha to the CEMs, inducing PLD activation through the interaction of PKCalpha with PLD1 in the CEMs. Caveolin-1 also coimmunoprecipitated with PLD1 in the absence of PMA, and the amounts of coimmunoprecipitated caveolin-1 decreased in response to treatment with PMA. Taken together, our results suggest a new mechanism for the regulation of the PKCalpha-dependent PLD activity through the molecular interaction between PLD1, PKCalpha, and caveolin-1 in caveolae.     

Evidence that protein kinase Calpha interacts with and regulates the glial glutamate transporter GLT-1

Many of the sodium‐dependent neurotransmitter transporters are rapidly (within minutes) regulated by protein kinase C (PKC), with changes in activity being correlated with changes in transporter trafficking to or from the plasma membrane. Our recent studies suggest that one of the classical subtypes of PKC, PKCα, may selectively mediate redistribution of the neuronal glutamate transporter, excitatory amino acid carrier (EAAC)1, and show that PKCα can be co‐immunoprecipitated with EAAC1. When the glial glutamate transporter GLT‐1a is transfected into C6 glioma cells, this transporter is internalized in response to activation of PKC, but the PKC subtype involved in this regulation is unknown. In the present study, expression of the phorbol ester‐activated subtypes of PKC was examined in C6 glioma transfected with GLT‐1. Of the classical subtypes, only PKCα was detected, and of the non‐classical subtypes, PKCδ and PKCε were detected. In this system, phorbol ester‐dependent internalization of GLT‐1 was blocked by a general inhibitor of PKCs (bisindolylmaleimide II) and by concentrations of Gö6976 that selectively block classical PKCs, but not by an inhibitor of PKCδ (rottlerin). PKCα immunoreactivity was found in GLT‐1 immunoprecipitates obtained from transfected C6 cells and from crude rat brain synaptosomes, a milieu that better mimics in vivo conditions. The amount of PKCα in both types of immunoprecipitate was modestly increased by phorbol ester, and this increase was blocked by a PKC antagonist. These studies suggest that PKCα may be required for the regulated redistribution of GLT‐1.     

Identification of two distinct pathways of protein kinase Calpha down-regulation in intestinal epithelial cells

Signal transduction pathways are controlled by desensitization mechanisms, which can affect receptors and/or downstream signal transducers. It has long been recognized that members of the protein kinase C (PKC) family of signal transduction molecules undergo down-regulation in response to activation. Previous reports have indicated that key steps in PKCalpha desensitization include caveolar internalization, priming site dephosphorylation, ubiquitination of the dephosphorylated protein, and degradation by the proteasome. In the current study, comparative analysis of PKCalpha processing induced by the PKC agonists phorbol 12-myristate 13-acetate and bryostatin 1 in IEC-18 rat intestinal epithelial cells demonstrates that: (a) at least two pathways of PKCalpha down-regulation can co-exist within cells, and (b) a single PKC agonist can activate both pathways at the same time. Using a combined biochemical and morphological approach, we identify a novel pathway of PKCalpha desensitization that involves ubiquitination of mature, fully phosphorylated activated enzyme at the plasma membrane and subsequent down-regulation by the proteasome. The phosphatase inhibitors okadaic acid and calyculin A accelerated PKCalpha down-regulation and inhibitors of vesicular trafficking did not prevent degradation of the protein, indicating that neither internalization nor priming site dephosphorylation are requisite intermediate steps in this ubiquitin/proteasome dependent pathway of PKCalpha down-regulation. Instead, caveolar trafficking and dephosphorylation are involved in a second, proteasome-independent mechanism of PKCalpha desensitization in this system. Our findings highlight subcellular distribution and phosphorylation state as critical determinants of PKCalpha desensitization pathways.     

Cell volume regulation in response to hypotonicity is impaired in HeLa cells expressing a protein kinase Calpha mutant lacking kinase activity

The chloride conductance (G(Cl,swell)) that participates in the regulatory volume decrease process triggered by osmotic swelling in HeLa cells was impaired by removal of extracellular Ca(2+), depletion of intracellular Ca(2+) stores with thapsigargin, or by preloading the cells with BAPTA-AM (1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid). Furthermore, overnight exposure to the phorbol ester tetradecanoyl phorbol acetate and acute incubation with inhibitors of the conventional protein kinase C (PKC) isoforms bisindolylmaleimide I and G?6976 inhibited G(Cl,swell). Treatment of HeLa cells with U73122, a phospholipase C inhibitor, also prevented G(Cl,swell). Hypotonicity induced selective PKC alpha accumulation in the membrane/cytoskeleton fraction in fractionation experiments and translocation of a green fluorescent protein-PKC alpha fusion protein to the plasma membrane of transiently transfected HeLa cells. To further explore the role of PKCs in hypotonicity-induced G(Cl,swell), HeLa clones stably expressing either a kinase-dead dominant negative variant of the Ca(2+)-dependent PKC isoform alpha (PKC alpha K386R) or of the atypical PKC isoform zeta (PKCzeta K275W) were generated. G(Cl,swell) was significantly reduced in HeLa cells expressing the dominant negative PKC alpha mutant but remained unaltered in cells expressing dominant negative PKCzeta. These findings strongly implicate PKC alpha as a critical regulatory element that is required for efficient regulatory volume decrease in HeLa cells.     

Cyclic nucleotide-independent protein kinase from rat liver. Purification and characterization of a multifunctional protein kinase

A rat liver cAMP-independent protein kinase that phosphorylates peptide b of ATP-citrate lyase (Ramakrishna, S., Pucci, D. L., and Benjamin, W. B. (1983) J. Biol. Chem. 258, 4950-4956) has been purified to apparent homogeneity. The molecular weight, determined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, sucrose density gradient, and by gel filtration, was found to be 36,000. This protein kinase phosphorylates in vitro ATP-citrate lyase, acetyl-CoA carboxylase, and glycogen synthase and does not phosphorylate phosphorylase, phosphorylase kinase, histone, phosvitin, and casein. It has Fa (activity factor) activity stimulating the ATP X Mg-dependent phosphatase and is therefore named a multifunctional protein kinase. This kinase differs from glycogen synthase kinase-3 with regard to substrate specificity, kinetic parameters, and physicochemical properties.