Hiroki Nakabayashi

Immunochemical identification of protein kinase C isozymes as products of discrete genes

Immunocytochemical studies of rat cerebellum using specific antibodies against type I, II, and III PKC revealed the presence of the type I PKC in the Purkinje cells where transcripts of tau cDNA were localized, the type II PKC in the granule cells where transcripts of beta cDNA were detected, and the type III PKC in both the Purkinje and granule cells. Immunoblot analysis revealed that the expressed PKC in COS cells transfected with either alpha, beta, or tau cDNA of PKC were recognized by specific antibodies against the type III, II, and I PKC isozymes, respectively. These results prove that the type I, II, and III PKC are products of PKC genes, tau, beta, and alpha, respectively. With these specific antibodies we have identified the presence of multiple species of PKC in a variety of cell types.

Catalytic fragment of protein kinase C exhibits altered substrate specificity toward smooth muscle myosin light chain

Smooth muscle myosin light chain (LC) can be phosphorylated by myosin light chain kinase (MLCK) at Ser19 and Thr18 and by protein kinase C (PKC) at Thr9 and Ser1 or Ser2 under the in vitro assay conditions. Conversion of PKC to the spontaneously active protein kinase M (PKM) by proteolysis resulted in a change in the substrate specificity of the kinase. PKM phosphorylated both sets of sites in LC recognized by MLCK and PKC as analyzed by peptide mapping analysis. The PKM‐catalyzed phosphorylation of these sites was not greatly affected by a MLCK inhibitor, ML‐9, nor by the activators of MLCK, Ca2+ and calmodulin.

Increased degradation of protein kinase C without diminution of mRNA level after treatment of WEHI-231 B lymphoma cells with phorbol esters

Immunoblot analysis of WEHI-231 B lymphoma cell homogenates revealed that both type II, a major component, and type III, a minor component, protein kinase C (PKC) were present. Northern blot analysis of PKC mRNA showed a higher level of beta II and beta I mRNA (encoding type II PKC) than of alpha mRNA (encoding type III PKC). Short term (3 min) treatment with phorbol 12-myristate 13-acetate (PMA) caused a rapid loss of PKC in cytosol and a concomitant increase in the particulate fraction. After prolonged (24 hr) exposure, the level of both PKC isozymes were decreased. However, the corresponding mRNA levels remained intact. PMA did not inhibit the anti-IgM-mediated increase in [Ca2+]i in PKC-depleted cells.

Role of protein kinase C in the regulation of rat liver glycogen synthase

Rat liver glycogen synthase was phosphorylated by purified protein kinase C in a Ca2+- and phospholipid-dependent fashion to 1-1.4 mol PO4/subunit. Analysis of the 32P-labeled tryptic peptides derived from the phosphorylated synthase by isoelectric focusing and two-dimensional peptide mapping revealed the presence of a major radioactive peptide. The sites in liver synthase phosphorylated by protein kinase C appears to be different from those phosphorylated by other kinases. Prior phosphorylation of the synthase by protein kinase C has no significant effect on the subsequent phosphorylation by glycogen synthase (casein) kinase-1 or kinase Fa, but prevents the synthase from further phosphorylation by cAMP-dependent protein kinase, Ca2+/calmodulin-dependent protein kinase, phosphorylase kinase, or casein kinase-2. Additive phosphorylation of liver glycogen synthase can be observed by the combination of protein kinase C with the former set of kinases but not with the latter. Phosphorylation of liver synthase by protein kinase C alone did not cause an inactivation nor did the combination of this kinase with glycogen synthase (casein) kinase-1 or kinase Fa produce a synergistic effect on the inactivation of the synthase. Based on these findings we conclude that the phorbol ester-induced inactivation of glycogen synthase previously observed in hepatocytes cannot be accounted for entirely by the activation of protein kinase C.

Phosphorylation of magainin-2 by protein kinase C and inhibition of protein kinase C isozymes by a synthetic analogue of magainin-2-amide

Magainins are a family of antimicrobial peptides present in the skin extracts of Xenopus laevis. Both magainin‐1 and ‐2 do not have any significant effect on the activity of protein kinase C (PKC). Magainin‐2 was found to be readily phosphorylated by PKC to 0.5 mol‐32P/mol of peptide. Neither magainin‐1, which has a sequence of S8AGK and not S8AKK as in the case of magainin‐2, nor the magainin‐2 analogue with substitution of Ala for Ser8 was phosphorylated by the kinase, suggesting that Ser8 is the phosphorylation site of magainin‐2. One synthetic analogue of magainin, designated magainin B, which has a greater tendency for α‐helix formation in non‐aqueous environment than the parent peptide resulting from substitution of Ser8, Gly13, and Gly18 with Ala in magainin‐2‐amide, is a potent inhibitor of PKC. This peptide inhibits all three PKC isozymes with IC50 less than 20 μM. Magainin B also inhibits the binding of [3H]phorbol 12,13‐dibutyrate to the kinase. These results suggest that magainin‐2 may be modified by PKC through phosphorylation and that certain synthetic analogues of magainins may be used as inhibitors of PKC.

Roles of Protein Kinase C Isozymes in Cellular Regulation

The importance of protein kinase C (PKC) in the regulation of cellular function was fully recognized after the discovery of diacylglycerol (DG) as an activator of PKC and that the tumor-promoting phorbol esters elicit their pleiotropic responses through binding to PKC as a phorbol ester receptor (for review see 1, 2). Both DG and phorbol ester stimulate PKC activity by increasing the affinity of the enzyme for Ca2+ and phospholipid so that the enzyme becomes active under the physiological conditions. This hypothetical scheme appears to provide a satisfactory explanation for the activation of PKC inside the cells.

Mechanism of Protein Kinase C-Mediated Signal Transduction

Protein kinase C (PKC) plays a pivotal role in the regulation of numerous cellular functions in response to a variety of external stimuli that cause the increase in [Ca2+], sn-1,2-diacylglycerol (DAG), and cis-unsaturated fatty acids. In vivo, a majority of PKC is believed to be loosely associated with membrane components. Among the various phospholipids tested, the polyphosphoinositides appear to be the best candidate as anchoring sites for PKC., which readily interacts with these phospholipids under the physiological concentrations of Mg2+. PKC anchored to phosphatidylinositol-4, 5-bisphosphate can further interact with Ca2+ and phosphatidylserine to generate a partially active enzyme. In the presence of DAG, the kinase becomes fully active. The activated PKC is susceptible to proteolysis to generate constitutively active PKM, which exhibits a broader substrate specificity than PKC. Phosphorylation of a group of calmodulin (CaM)-binding proteins by PKC provides a link between PKC-mediated responses and other signaling pathways that make use of CaM as activator. Phosphorylation of one of those CaM-binding proteihs, neurogranin (RC3), reduces its affinity for CaM and thus renders CaM available for other enzymes. Activation of PKC in the nucleus is important in controlling gene expression. Phosphorylation of CCAAT/enhancer-binding protein at a serine residue within the basic DNA-binding region attenuates the binding of this protein to specific DNA probes. Control of PKC gene expression has been linked to the regulation of cellular growth and differentiation. Structural features of PKCγ gene promoter region have been characterized and potential regulatory elements identified. This article illustrates several possible mechanisms that confer the functional diversity of PKC.