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.

PHLPP is a Ser/Thr phosphatase that has been shown to directly dephosphorylate several members of the AGC family of kinases, which are important regulators of cell growth, proliferation, and survival. We have previously demonstrated that removal of PHLPP1 increases Akt activity in neonatal rat ventricular myocytes (NRVMs) (Miyamoto et al 2010) as well as the heart (Moc et al 2015) leading to protection from pathological hypertrophy and damage, however the effects of PHLPP2 removal on cardiomyocyte hypertrophic growth are unknown. In NRVMs, we found that removal of PHLPP2 induced hypertrophic growth basally without altering Akt activity and immunoprecipitation experiments revealed a novel interaction with an AGC kinase, G-protein coupled receptor kinase 5 (GRK5). GRK5 acts as an HDAC kinase downstream of phenylephrine (PE) to regulate hypertrophy in NRVMs. Thus, our objective was to determine whether PHLPP2 regulates PE-induced cardiomyocyte hypertrophy through its interaction with GRK5. We found that knockdown of PHLPP2, not PHLPP1, potentiated PE-induced hypertrophy (siPH2+PE (109 ± 3.5μm) vs. siCon+PE (88 ± 1.9μm)), and increased mRNA expression of fetal genes (i.e. ANF and BNP). Conversely, overexpression of PHLPP2 attenuated PE-induced hypertrophic growth (PH2+PE (66.6 ±7.7μm) vs. GFP+PE (95.5 ± 2.7μm)) as well as fetal gene re-expression. This inhibition of hypertrophic growth was dependent on phosphatase activity since a PHLPP2 mutant lacking the phosphatase domain (αPP2C) reversed the effect of PHLPP2 overexpression. To determine whether removal of PHLPP2 alters GRK5 translocation, cells were fractionated, and we found that PHLPP2 removal significantly increased basal and PE-induced nuclear accumulation of GRK5 that was blocked by overexpression of PHLPP2. Finally, knockdown studies with siGRK5 revealed that GRK5 was indeed required for the hypertrophic response elicited by PHLPP2 removal. Overall, our data revealed for the first time that PHLPP2 plays a role in regulating PE-induced cardiac hypertrophy via a GRK5 dependent pathway. Understanding the biological function and signaling pathways altered by PHLPP2 in cardiomyocytes may help delineate therapeutic targets for cardiac hypertrophy.