NPKC isoforms differential activation of S6K1 in adult cardiac myocytes

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Activation of p70/85 S6 kinase (S6K1) contributes to cardiac hypertrophy by augmenting ribosomal biogenesis and activating cell-size determinants. We have established previously in pressure overloaded myocardium, a PKC-mediated c-Raf/MEK/ERK signaling pathway contributes to S6K1 activation. Stimulation of isolated adult feline cardiac myocytes cultured on laminin-coated plates with 100 uM phenylephrine (PE), 200 nM endothelin-1 (Et-1), 200 nM TPA, or 100 nM insulin resulted in T389 and S421/T424 phosphorylation/activation of S6K1. The cPKC inhibitor, Go6976, did not affect S6K1 activation. Blockade of the c-Raf/MEK/ERK pathway with a dominant negative c-Raf adenoviral construct, blocked Et-1 and TPA activation for S6K1 and ERK. Infection with a dominant negative PKC epsilon (DN PKCe) adenoviral construct significantly blocked ET-1, PE and TPA-stimulated mTOR S2448 and S6K1 S421/T424 and T389 phosphorylation, but not wortmannin sensitive insulin mediated S421/T424 phosphorylation. However, specifically blocking PKC delta (PKCd) using a dominant negative adenoviral construct or rottlerin, blocked both mTOR S2448 and S6K1 T389 phosphorylation, across all agents including insulin. These data indicate that: (i) Et-1, and TPA stimulated S421/T424 phosphorylation requires PKCe but not PKCd, (ii) during Et-1 and TPA treatment both PKCe and PKCd are necessary for mTOR and S6K1 activation but during insulin stimulation, PKCd is critical for mTOR activation and S6K1 phosphorylation at T389. Together, these data delineate specific PKC isoform contributions in mTOR and S6K1 phosphorylation/activation. Funding support: NHLBI PPG HL-48788 and VA Merit Award.