Abstract P218: PKCε Regulates PI3K/mTOR Complex-2-Dependent AKT Activation Independent of c-Raf/MEK/ERK Pathway

Abstract

Mammalian target of rapamycin (mTOR) has been shown to influence hypertrophic growth of the myocardium through two unique complexes, mTOR complexed with Raptor (mTORC1) and mTOR complexed to Rictor (mTORC2) with downstream activation of S6K1 and AKT respectively. Previously, we characterized the role of novel PKC isoforms (epsilon and delta) on mTORC1-dependent activation of S6K1 downstream of both PI3K-dependent and independent pathways in adult cardiomyocytes. PKC epsilon (PKCε) has been implicated in AKT activation during ischemia-reperfusion injury and has been co-immunoprecipitated in transgenic PKCε overexpressing murine myocardium. In the present study, we explored the role of PKCε on PI3K-dependent AKT S473 phosphorylation. As such, insulin stimulated pAKT S473 was significantly reduced by pretreatment with wortmannin, a selective PI3K inhibitor. However, insulin stimulated activation of AKT was not reduced by pretreatment with either Gö6976 blockade of the classical PKC isoforms (PKC alpha/beta) or U0126 blockade of MEK. Thus, insulin stimulated activation of AKT is mediated primarily through a PI3K-dependent pathway independent of the classical PKC isoforms. To begin to evaluate the role of the novel PKCs, we utilized adenoviral expression of dominant negative PKCε (dnPKCε). After 48 hours of expression of dnPKCε, insulin stimulated pAKT S473 was significantly reduced as compared to B-gal infection controls. Next we identified for the first time in isolated cardiomyocytes a direct link between PKCε and AKT through immunoprecipitation of a unique signaling complex comprised of PKCε bound to mTORC2 and pAKT (S473) only during insulin stimulation. These data establish for the first time in adult isolated cardiomyocytes: i) the necessity of PKCε during insulin stimulated PI3K-dependent AKT activation and ii) a signaling complex containing mTOR, Rictor, PKCε, and pAKT that forms during insulin stimulation. Given the importance of AKT in physiologic growth and survival, these studies indicate that PKCε plays a pivotal role during AKT activation and for the first time displays a direct link between PKCε, pAKT and mTORC2 in isolated cardiomyocytes.