Abstract 9686: Novel GATA4 Binding Proteins, RbAp48/46 Regulate Post-Translational Modification During Hypertrophic Responses in Cardiomyocytes

Abstract

Introduction: A zinc finger protein GATA4 associates with not only ERK1/2 but also an intrinsic histone acetyltransferase p300 and regulates myocardial transcriptional activities in response to hypertrophic stimuli. We recently reported that Retinoblastoma protein (Rb)-associated protein 48 and 46 (RbAp48/46) are novel components of the p300/GATA4 complex and form a repressor complex with HDACs in cardiomyocytes. However, precise functional regulation by RbAp48/46 during hypertrophic responses is still unknown. Hypothesis: We assessed the hypothesis that RbAp48/46 suppressed cardiac hypertrophic responses by regulating post-translational modification of GATA4 in cardiomyocytes. Methods and Results: Overexpression of RbAp48/46 inhibited phenylephrine (PE)-induced acetylation of GATA4, activation of atrial natriuretic factors (ANF) and endotheline-1 (ET-1) promoters, and increase in cell size in cultured cardiomyocytes. Conversely, knockdown of RbAp48/46 by shRNA augmented such responses. In the absence of HDAC1/2, RbAp48/46 failed to suppress cardiac hypertrophy. GST pull down assay revealed that RbAp48/46 interacted with a portion near the S105 phosphorylation site within N-terminal domain of GATA4. Immunoprecipitation and Western Blot demonstrated that a constituted active form of MEK1 induced S105 phosphorylation of GATA4, the dissociation between RbAp48/46 and GATA4, the increase in p300-dependant acetylation of GATA4, and p300/GATA4-dependant synergistic activations of ANF and ET-1 promoters. Such inductions did not occur in phosphorylation-mutated GATA4. Stimulation of cardiomyocytes with PE increased S105 phosphorylation of GATA4 and the dissociation of GATA4 from RbAp48/46 and HDAC1/2, and decreased recruitments of RbAp48/46 onto GATA element of the ANF promoter. These changes are suppressed by PD98059, a MEK1 inhibitor. Conclusion: These findings demonstrate that RbAp48 and RbAp46 form repressor complex with GATA4 as well as HDAC1/2, release p300 from GATA4, and inhibit hypertrophic responses in cardiomyocytes. The phosphorylation of GATA4 may be a trigger of the disassociation between RbAp/HDAC complex and GATA4, and of subsequent formation of p300/GATA4 complex and hypertrophic gene activation.