Abstract 1377: PHLPP, a Novel Akt Phosphatase, Expressed and is Regulated in Cardiomyocytes
Akt is known to be a strong survival signaling in various cell types including the heart. Although activation of Akt by PI3K, PDK-1 and a putative PDK-2 has been extensively studied, signals terminating Akt activation have only recently been discovered. Studies by the Newton laboratory identified a novel protein phosphatase, PH domain Leucine-rich repeat Protein Phosphatase, PHLPP, that specifically dephosphorylates the ser-473 site, but not the thr-308 site on Akt. PHLPP expression markedly diminished Akt kinase activity and triggered apoptosis, while PHLPP downregulation selectively increased Akt phoshorylation at ser-473 and prevented apoptosis. The possibility that PHLPP is regulated and contributes to the activation of Akt in cardiomyocytes has not yet been explored. Two isoforms of PHLPP, PHLPP-1 and -2 have been identified and PHLPP-1 mRNA is strongly expressed, while PHLPP-2 shows lower expression in adult mouse ventricular myocytes (AMVMs). Western blotting revealed PHLPP-1 expression in both AMVMs and neonatal rat ventricular myocytes (NRVMs). Immunostaining demonstrated that PHLPP-1 was located not only in the cytosol but also in the nucleus implying that PHLPP-1 can regulate Akt signaling in multiple cellular components. Studies were then carried out to determine whether PHLPP expression was regulated when Akt activity was chronically altered. We found that PHLPP-1 protein was upregulated 2–3 fold in NRVMs treated for 48 hrs with leukemia inhibitory factor an efficacious activator of Akt. Conversely treatment with rapamycin, an mTOR inhibitor for 48 hrs, significantly decreased PHLPP expression, consistent with a postulated role of mTOR in regulating Akt activation state. PHLPP was also downregulated in both ES cells and cardiomyocytes from PDK-1 knock out (KO) mice in which Akt activation was severely limited. These data demonstrate that PHLPP expression is regulated to compensate for chronic changes in Akt activity and suggest a potential role for PHLPP in regulating cardiomyocyte survival. Studies are currently in progress using PHLPP-1 adenovirus and PHLPP-1 KO mice to examine the functional importance of PHLPP in regulation of Akt and cardiomyocyte survival in vitro and in response to pathophysiological interventions in vivo.