Tamar Liron

Opposing cardioprotective actions and parallel hypertrophic effects of δPKC and ɛPKC

Conflicting roles for protein kinase C (PKC) isozymes in cardiac disease have been reported. Here, δPKC-selective activator and inhibitor peptides were designed rationally, based on molecular modeling and structural homology analyses. Together with previously identified activator and inhibitor peptides of ɛPKC, δPKC peptides were used to identify cardiac functions of these isozymes. In isolated cardiomyocytes, perfused hearts, and transgenic mice, δPKC and ɛPKC had opposing actions on protection from ischemia-induced damage. Specifically, activation of ɛPKC caused cardioprotection whereas activation of δPKC increased damage induced by ischemia in vitro and in vivo. In contrast, δPKC and ɛPKC caused identical nonpathological cardiac hypertrophy; activation of either isozyme caused nonpathological hypertrophy of the heart. These results demonstrate that two related PKC isozymes have both parallel and opposing effects in the heart, indicating the danger in the use of therapeutics with nonselective isozyme inhibitors and activators. Moreover, reduction in cardiac damage caused by ischemia by perfusion of selective regulator peptides of PKC through the coronary arteries constitutes a major step toward developing a therapeutic agent for acute cardiac ischemia.

Evidence of zeta protein kinase C involvement in polymorphonuclear neutrophil integrin-dependent adhesion and chemotaxis.

Classical chemoattractants and chemokines trigger integrin-dependent adhesion of blood leukocytes to vascular endothelium and also direct subsequent extravasation and migration into tissues. In studies of human polymorphonuclear neutrophil responses to formyl peptides and to interleukin 8, we show evidence of involvement of the atypical ζ protein kinase C in the signaling pathway leading to chemoattractant-triggered actin assembly, integrin-dependent adhesion, and chemotaxis. Selective inhibitors of classical and novel protein kinase C isozymes do not prevent chemoattractant-induced neutrophil adhesion and chemotaxis. In contrast, chelerythrine chloride and synthetic myristoylated peptides with sequences based on the endogenous ζ protein kinase C pseudosubstrate region block agonist-induced adhesion to fibrinogen, chemotaxis and F-actin accumulation. Biochemical analysis shows that chemoattractants trigger rapid translocation of ζ protein kinase C to the plasma membrane accompanied by rapid but transient increase of the kinase activity. Moreover, pretreatment with C3 transferase, a specific inhibitor of Rho small GTPases, blocks ζ but not α protein kinase C plasma membrane translocation. Synthetic peptides from ζ protein kinase C also inhibit phorbol ester-induced integrin-dependent adhesion but not NADPH-oxidase activation, and C3 transferase pretreatment blocks phorbol ester-triggered translocation of ζ but not α protein kinase C. These data suggest the involvement of ζ protein kinase C in chemoattractant-induced leukocyte integrin-dependent adhesion and chemotaxis. Moreover, they highlight a potential link between atypical protein kinase C isozymes and Rho signaling pathways leading to integrin-activation.

Cardiotrophic Effects of Protein Kinase C ε Analysis by In Vivo Modulation of PKCε Translocation

Abstract —Protein kinase C (PKC) is a key mediator of many diverse physiological and pathological responses. Although little is known about the specific in vivo roles of the various cardiac PKC isozymes, activation-induced translocation of PKC is believed to be the primary determinant of isozyme-specific functions. Recently, we have identified a catalytically inactive peptide translocation inhibitor (eV1) and translocation activator (ψeRACK [receptors for activated Ckinase]) specifically targeting PKCe. Using cardiomyocyte-specific transgenic expression of these peptides, we combined loss- and gain-of-function approaches to elucidate the in vivo consequences of myocardial PKCe signaling. As expected for a PKCe RACK binding peptide, confocal microscopy showed that eV1 decorated cross-striated elements and intercalated disks of cardiac myocytes. Inhibition of cardiomyocyte PKCe by eV1 at lower expression levels upregulated α–skeletal actin gene expression, increased cardiomyocyte cell size, and modestly impaired left ventricular fractional shortening. At high expression levels, eV1 caused a lethal dilated cardiomyopathy. In contrast, enhancement of PKCe translocation with ψeRACK resulted in selectively increased β myosin heavy chain gene expression and normally functioning concentric ventricular remodeling with decreased cardiomyocyte size. These results identify for the first time a role for PKCe signaling in normal postnatal maturational myocardial development and suggest the potential for PKCe activators to stimulate “physiological” cardiomyocyte growth.

Involvement of protein kinase Cepsilon (PKCepsilon) in thyroid cell death. A truncated chimeric PKCepsilon cloned from a thyroid cancer cell line protects thyroid cells from apoptosis.

The protein kinase C (PKC) family has been implicated in the regulation of apoptosis. However, the contribution of individual PKC isozymes to this process is not well understood. We reported amplification of the chromosome 2p21 locus in 28% of thyroid neoplasms, and in the WRO thyroid carcinoma cell line. By positional cloning we identified a rearrangement and amplification of the PKCε gene, that maps to 2p21, in WRO cells. This resulted in the overexpression of a chimeric/truncated PKCε (Tr-PKCε) mRNA, coding for N-terminal amino acids 1–116 of the isozyme fused to an unrelated sequence. Expression of the Tr-PKCε protein in PCCL3 cells inhibited activation-induced translocation of endogenous PKCε, but its kinase activity was unaffected, consistent with a dominant negative effect of the mutant protein on activation-induced translocation of wild-type PKCε and/or displacement of the isozyme to an aberrant subcellular location. Cell lines expressing Tr-PKCε grew to a higher saturation density than controls. Moreover, cells expressing Tr-PKCε were resistant to apoptosis, which was associated with higher Bcl-2 levels, a marked impairment in p53 stabilization, and dampened expression of Bax. These findings point to a role for PKCε in apoptosis-signaling pathways in thyroid cells, and indicate that a naturally occurring PKCε mutant that functions as a dominant negative can block cell death triggered by a variety of stimuli.