Protein kinase C (PKC) isozymes translocate to unique subcellular sites following activation. We previously suggested that translocation of activated isozymes is required for their function and that in addition to binding to lipids, translocation involves binding of the activated isozymes to specific anchoring proteins (receptors for activated protein kinase C. Using cultured cardiomyocytes we identified inhibitors, the V1 fragment of ⑀PKC (⑀V1), and an 8-amino acid peptide derived from it that selectively inhibited the translocation of ⑀PKC. Inhibition of ⑀PKC translocation but not inhibition of ␦ or ␤PKC translocation specifically blocked phorbol ester-or norepinephrine-mediated regulation of contraction. These isozyme-selective translocation inhibitors provide novel tools to determine the function of individual PKC isozymes in intact cells. Activation of protein kinase C (PKC)1 isozymes is associated with translocation of the enzymes form the cell soluble to the cell particulate fraction (1). These isozymes are activated by binding to lipid-derived second messengers and negatively charged phospholipids present in the cell particulate fraction (2, 3). In addition to binding to lipids, specific anchoring proteins participate in binding the activated PKC isozymes to this fraction (4 -9). We collectively termed these proteins RACKs, for receptors for activated protein kinase C (7, 10).In cultured neonatal cardiomyocytes, immunofluorescence studies demonstrated isozyme-specific subcellular localization following activation with either 4-␤ phorbol 12-myristate-13-acetate (PMA) or with norepinephrine (NE) via an ␣ 1 -adrenergic receptor (11,12). Similar isozyme-specific localization was found in other cells following PKC activation (e.g. (13)). This isozyme-specific localization suggests that unique sequences in each isozyme (14) contain at least part of the recognition site for the anchoring molecules, the isozyme-specific RACKs.Here, we focus on the ⑀PKC unique region, ⑀V1, which is the largest variable region in this isozyme. Some homology between ⑀V1 and the C2 region of the classical PKCs, ␣, ␤, and ␥, was noted (15). Because C2 contains at least part of the RACKbinding site of classical PKCs (16,17), an ⑀PKC specific RACKbinding site may reside within ⑀V1. In that case, an ⑀V1 fragment should bind to the ⑀PKC-specific RACK when introduced into cells and thus inhibit PMA-or hormone-induced ⑀PKC translocation and binding to that RACK. Translocation of other PKC isozymes should not be affected by ⑀V1. The following study confirms these predictions and demonstrates the use of translocation inhibitors to determine the role of specific isozymes in regulating cardiac contraction. EXPERIMENTAL PROCEDURESPeptides and Reagents-Peptides ⑀V1-2 (EAVSLKPT; ⑀PKC (14 -21)), scrambled ⑀V1-2 (LSETKPAV), ⑀V1-3 (LAVFHDA; ⑀PKC (81-87)), V1-2 (EAVGLQPT; PKC (18 -25)), and ␤C2-4 (SLNPEWNET; ␤PKC (218 -226)) were synthesized at the Beckman Center Protein and Nucleic Acid Facility at Stanford. All the peptides used in this study were ...

The protein kinase C (PKC) family consists of 11 isoenzymes. Following activation, each isoenzyme translocates and binds to a specific receptor for activated C kinase (RACK) Although PKC activation enhances insulin release, the specific function of each isoenzyme is unknown. Here we show that following stimulation with glucose, ␣PKC and ⑀PKC translocate to the cell's periphery, while ␦PKC and PKC translocate to perinuclear sites. ␤C2-4, a peptide derived from the RACK1-binding site in the C2 domain of ␤PKC, inhibits translocation of ␣PKC and reduces insulin response to glucose. Likewise, ⑀V1-2, an ⑀PKC-derived peptide containing the site for its specific RACK, inhibits translocation of ⑀PKC and reduces insulin response to glucose. Inhibition of islet-glucose metabolism with mannoheptulose blocks translocation of both ␣PKC and ⑀PKC and diminishes insulin response to glucose while calcium-free buffer inhibits translocation of ␣PKC but not ⑀PKC and lowers insulin response by 50%. These findings illustrate the unique ability of specific translocation inhibitors to elucidate the isoenzyme-specific functions of PKC in complex signal transduction pathways.(Protein kinase C is a family of 11 lipid-dependent serine/ threonine kinases involved in a wide spectrum of signal transduction (7,8). Upon activation, PKC 1 isoenzymes translocate to new cellular sites, including the plasma membrane (9, 10), cytoskeletal elements (11,12), and the nucleus (13,14), as well as other subcellular compartments (15). Many cells are known to contain several isoenzymes (16,17), each localizing to a different cellular site upon stimulation (18). The multiplicity of isoforms of a single enzyme renders the analysis of enzymefunction relationship difficult. Recent work revealed that activated PKC isoenzymes bind anchoring proteins termed RACKs (1-3), believed to be positioned in close proximity to the isoenzyme's substrate. It was further shown that the functional specificity of the PKC isoenzyme is determined, in part, by the differential localization of the isoenzyme-specific RACKs (19). The RACK for ␤PKC, RACK1, has been cloned, and at least part of its binding site on ␤PKC has been mapped to a short sequence within the C2 domain (1). ␤C2-4, a nonopeptide derived from this region, inhibits phorbol ester-induced translocation of the C2-containing isoenzymes but not the translocation of C2-less isoenzymes such as ␦-and ⑀PKC when tested in intact cells (1). A short peptide derived from the V1 region of ⑀PKC, ⑀V1-2, was similarly shown to inhibit the translocation of ⑀PKC, but not ␣-, ␤-, and ␦PKC (20). Furthermore, these isozyme-specific inhibitors blocked the specific function of individual isoenzymes; for example, ⑀V1-2, but not ␤C2-4, inhibited phorbol 12-myristate 13-acetate-induced regulation of the contraction rate in intact cardiomyocytes. Here we use these novel PKC isozyme-specific inhibitors to determine that PKC activation is part of the signals involved in the regulation of glucose-induced insulin secretion and to identify the specif...

We have previously identified a phorbol ester-induced PKCϵ (protein kinase Cϵ) interaction with the (∼18 kDa) COIV [CO (cytochrome c oxidase) subunit IV] in NCMs (neonatal cardiac myocytes). Since PKCϵ has been implicated as a key mediator of cardiac PC (preconditioning), we examined whether hypoxic PC could induce PKCϵ–COIV interactions. Similar to our recent study with phorbol esters [Ogbi, Chew, Pohl, Stuchlik, Ogbi and Johnson (2004) Biochem. J. 382, 923–932], we observed a time-dependent increase in the in vitro phosphorylation of an approx. 18 kDa protein in particulate cell fractions isolated from NCMs subjected to 1–60 min of hypoxia. Introduction of a PKCϵ-selective translocation inhibitor into cells attenuated this in vitro phosphorylation. Furthermore, when mitochondria isolated from NCMs exposed to 30 min of hypoxia were subjected to immunoprecipitation analyses using PKCϵ-selective antisera, we observed an 11.1-fold increase in PKCϵ–COIV co-precipitation. In addition, we observed up to 4-fold increases in CO activity after brief NCM hypoxia exposures that were also attenuated by introducing a PKCϵ-selective translocation inhibitor into the cells. Finally, in Western-blot analyses, we observed a >2-fold PC-induced protection of COIV levels after 9 h index hypoxia. Our studies suggest that a PKCϵ–COIV interaction and an enhancement of CO activity occur in NCM hypoxic PC. We therefore propose novel mechanisms of PKCϵ-mediated PC involving enhanced energetics, decreased mitochondrial reactive oxygen species production and the preservation of COIV levels.

We have developed an improved, less disruptive procedure for the transient permeabilization of neonatal cardiac myocytes using saponin. The method allows delivery of peptides to a high percentage of cells in culture without effects on long-term cell viability. Permeation was confirmed microscopically by cellular uptake of a fluorescently labeled peptide and biochemically by uptake of 125I-labeled calmodulin and a 20-kD protein kinase C epsilon fragment into the cells. The intracellular molar concentration of the introduced peptide was approximately 10% of that applied outside. We found no significant effects of permeabilization on spontaneous, phorbol ester-modulated, or norepinephrine-modulated contraction rates. Similarly, the expression of c-fos mRNA (measured 30 minutes after permeabilization) and the incorporation of [-14C]phenylalanine following agonist stimulation (measured 3 days after permeabilization) were not altered by saponin permeabilization. Finally, permeabilization of cells in the presence of a protein kinase C pseudosubstrate peptide, but not a control peptide, inhibited phorbol ester-induced [14C]phenylalanine incorporation into proteins by 80%. Our results demonstrate a methodology for the introduction of peptides into neonatal cardiac myocytes that allows study of their actions without substantial compromises in cell integrity.

Rationale: MicroRNAs (miRs) are small, non-coding RNAs that function to post-transcriptionally regulate gene expression. First transcribed as long primary miR transcripts (pri-miRs), they are enzymatically processed in the nucleus by Drosha into hairpin intermediate miRs (pre-miRs) and further processed in the cytoplasm by Dicer into mature miRs where they regulate cellular processes following activation by a variety of signals such as those stimulated by β-adrenergic receptors (βARs). Initially discovered to desensitize βAR signaling, β-arrestins are now appreciated to transduce multiple effector pathways independent of G protein-mediated second messenger accumulation, a concept known as biased signaling. We previously showed that the β-arrestin-biased βAR agonist carvedilol activates cellular pathways in the heart. Objective: Here, we tested whether carvedilol could activate β-arrestin-mediated miR maturation, thereby providing a novel potential mechanism for its cardioprotective effects. Methods and Results: In human cells and mouse hearts, carvedilol upregulates a subset of mature and pre-miRs but not their pri-miRs in β1AR-, G protein-coupled receptor kinase 5/6- and β-arrestin1-dependent manner. Mechanistically, β-arrestin1 regulates miR processing by forming a nuclear complex with hnRNPA1 and Drosha on pri-miRs. Conclusions: Our findings indicate a novel function for β1AR-mediated β-arrestin1 signaling activated by carvedilol in miR biogenesis, which may be linked, in part, to its mechanism for cell survival.