Regulation of mTOR and S6K1 activation by the nPKC isoforms, PKCepsilon and PKCdelta, in adult cardiac muscle cells.
Activation of both mTOR and its downstream target, S6K1 (p70 S6 kinase) have been implicated to affect cardiac hypertrophy. Our earlier work, in a feline model of 1-48 h pressure overload, demonstrated that mTOR/S6K1 activation occurred primarily through a PKC/c-Raf pathway. To further delineate the role of specific PKC isoforms on mTOR/S6K1 activation, we utilized primary cultures of adult feline cardiomyocytes in vitro and stimulated with endothelin-1 (ET-1), phenylephrine (PE), TPA, or insulin. All agonist treatments resulted in S2248 phosphorylation of mTOR and T389 and S421/T424 phosphorylation of S6K1, however only ET-1 and TPA-stimulated mTOR/S6K1 activation was abolished with infection of a dominant negative adenoviral c-Raf (DN-Raf) construct. Expression of DN-PKC(epsilon) blocked ET-1-stimulated mTOR S2448 and S6K1 S421/T424 and T389 phosphorylation but had no effect on insulin-stimulated S6K1 phosphorylation. Expression of DN-PKC(delta) or pretreatment of cardiomyocytes with rottlerin, a PKC(delta) specific inhibitor, blocked both ET-1 and insulin stimulated mTOR S2448 and S6K1 T389 phosphorylation. However, treatment with Gö6976, a specific classical PKC (cPKC) inhibitor did not affect mTOR/S6K1 activation. These data indicate that: (i) PKC(epsilon) is required for ET-1-stimulated T421/S424 phosphorylation of S6K1, (ii) both PKC(epsilon) and PKC(delta) are required for ET-1-stimulated mTOR S2448 and S6K1 T389 phosphorylation, (iii) PKC(delta) is also required for insulin-stimulated mTOR S2448 and S6K1 T389 phosphorylation. Together, these data delineate both distinct and combinatorial roles of specific PKC isoforms on mTOR and S6K1 activation in adult cardiac myocytes following hypertrophic stimulation.
Moschella
Phillip C
PC
Cardiology Division of the Department of Medicine, Gazes Cardiac Research Institute, Medical University of South Carolina, 114 Doughty Street, Charleston, SC 29425, USA.
Rao
Vijay U
VU
McDermott
Paul J
PJ
Kuppuswamy
Dhandapani
D
D000730:Androstadienes / Q000494:pharmacology; D000818:Animals; D002415:Cats; D002478:Cells, Cultured; D004789:Enzyme Activation / Q000187:drug effects; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D005799:Genes, Dominant; D007527:Isoenzymes / Q000378:metabolism; D008954:Models, Biological; D032383:Myocytes, Cardiac / Q000187:drug effects / Q000201:enzymology*; D010766:Phosphorylation / Q000187:drug effects; D011493:Protein Kinase C / Q000378:metabolism*; D011494:Protein Kinases / Q000378:metabolism*; D019908:Proto-Oncogene Proteins c-raf / Q000378:metabolism; D019893:Ribosomal Protein S6 Kinases / Q000378:metabolism*; D058570:TOR Serine-Threonine Kinases; D000077191:Wortmannin
D000730:Androstadienes; D007527:Isoenzymes; D011494:Protein Kinases; C115197:calcium-independent protein kinase C; D019908:Proto-Oncogene Proteins c-raf; D019893:Ribosomal Protein S6 Kinases; D058570:TOR Serine-Threonine Kinases; D011493:Protein Kinase C; D048049:Extracellular Signal-Regulated MAP Kinases; D000077191:Wortmannin