Abstract 12566: Receptor for Activated Protein Kinase C1 Competes With GATA4 for p300 and Represses Hypertrophic Responses in Cardiomyocyte

Abstract

Introduction: The zinc finger protein GATA4 is one factor involved in transcriptional regulation during myocardial cell hypertrophy, and forms a complex with an intrinsic histone acetyltransferase (HAT), p300. HAT activity of p300 is required for acetylation and the transcriptional activity of GATA4, as well as for cardiomyocyte hypertrophy and the development of heart failure in vivo . We recently identified Receptor for Activated Protein Kinase C1 (RACK1), a multi-functional scaffold protein, as a novel GATA4-binding partner, and revealed that RACK1 regulates the p300/GATA4-dependent transcriptional pathway and hypertrophic responses in cardiomyocytes. However, the molecular mechanism by which RACK1 suppresses p300/GATA4-dependent gene transcription remains unknown. Hypothesis: We hypothesized that RACK1 reduces the phenylephrine (PE)-induced increase of the interaction between GATA4 and p300 in cardiacmyocytes. Methods and Results: To examine the effect of RACK on the binding activity of p300 to GATA4, expression plasmids encoding these proteins were used to co-transfect HEK293 cells. Nuclear extracts from these cells were subjected to immunoprecipitation, followed by Western blotting. Overexpression of RACK1 inhibited p300-induced GATA4 acetylation and the association with p300/GATA4. Based on chromatin immunoprecipitation and reporter assays, RACK1 inhibited not only p300-induced DNA binding activity of GATA4 on atrial natriuretic factors (ANF) and endothelin-1 (ET-1) promoters but also p300/GATA4-induced ANF and ET-1 promoter activities. In cultured neonatal rat ventricular cardiomyocytes, overexpression of RACK1 significantly inhibited PE-induced GATA4 acetylation, activation of ANF and ET-1 promoters, and myocardial cell hypertrophy. Also, PE decreased binding between GATA4 and RACK1. Finally, PE stimulation increased tyrosine phosphorylation of RACK1. This phosphorylation was inhibited by a tyrosine kinase inhibitor, Dasatinib. Conclusions: The findings demonstrated that RACK1 disrupted the p300/GATA4 complex and inhibited hypertrophic responses in cardiomyocytes. Tyrosine phosphorylation of RACK1 may trigger the dissociation of RACK1 from p300/GATA4.