Abstract
PH domain leucine-rich repeat protein phosphatase (PHLPP) is a serine/threonine phosphatase that has been shown to regulate cell growth and survival through dephosphorylation of several members of the AGC family of kinases. G-protein-coupled receptor kinase 5 (GRK5) is an AGC kinase that regulates phenylephrine (PE)-induced cardiac hypertrophy through its noncanonical function of directly targeting proteins to the nucleus to regulate transcription. Here we investigated the possibility that the PHLPP2 isoform can regulate GRK5-induced cardiomyocyte hypertrophy in neonatal rat ventricular myocytes (NRVMs). We show that removal of PHLPP2 by siRNA induces hypertrophic growth of NRVMs as measured by cell size changes at baseline, potentiated PE-induced cell size changes, and re-expression of fetal genes atrial natriuretic factor and brain natriuretic peptide. Endogenous GRK5 and PHLPP2 were found to interact in NRVMs, and PE-induced nuclear accumulation of GRK5 was enhanced upon down-regulation of PHLPP2. Conversely, overexpression of PHLPP2 blocked PE-induced hypertrophic growth, re-expression of fetal genes, and nuclear accumulation of GRK5, which depended on its phosphatase activity. Finally, using siRNA against GRK5, we found that GRK5 was necessary for the hypertrophic response induced by PHLPP2 knockdown. Our findings demonstrate for the first time a novel regulation of GRK5 by the phosphatase PHLPP2, which modulates hypertrophic growth. Understanding the signaling pathways affected by PHLPP2 has potential for new therapeutic targets in the treatment of cardiac hypertrophy and failure.
Highlights
PH domain leucine-rich repeat protein phosphatase (PHLPP) is a serine/threonine phosphatase that has been shown to regulate cell growth and survival through dephosphorylation of several members of the AGC family of kinases
neonatal rat ventricular myocytes (NRVMs) were transfected with siRNA for PHLPP1, PHLPP2, or control and treated with PE for 48 h to induce hypertrophic growth
PHLPP1 and PHLPP2 are a family of serine/threonine protein phosphatases that target multiple AGC kinases including Akt and PKC [2, 16, 27]
Summary
PH domain leucine-rich repeat protein phosphatase (PHLPP) is a serine/threonine phosphatase that has been shown to regulate cell growth and survival through dephosphorylation of several members of the AGC family of kinases. Overexpression of PHLPP2 blocked PE-induced hypertrophic growth, re-expression of fetal genes, and nuclear accumulation of GRK5, which depended on its phosphatase activity. Activation of G-protein– coupled receptors (GPCRs) by hypertrophic agonists such as PE engages a number of intracellular signaling pathways that are important transducers of the hypertrophic response These pathways include calcineurin-nuclear factor of activated T cells (NFAT) [4], Ca2ϩ/calmodulin-dependent kinase II [5, 6], mitogen-activated protein kinases [7, 8], PKC [9], GRK5 [10], and the Akt-mechanistic target of rapamycin pathway [11, 12] among many others. Our data revealed for the first time that the phosphatase PHLPP2 plays a novel role in regulating PE-induced cardiac hypertrophy via a GRK5-dependent pathway
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