Abstract 16872: Receptor for Activated Protein Kinase C1 Forms a Functional Complex With p300/GATA4 and Suppress Hypertrophic Responses in Cardiomyocytes
Introduction: A zinc finger protein GATA4 is one of the factors involved in transcriptional regulation during cardiac hypertrophy and forms a functional complex with an intrinsic histone acetyltransferase (HAT), p300. HAT activity of p300 is required for acetylation and the transcriptional activation of GATA4 as well as for cardiac hypertrophy and the development of heart failure in vivo. By tandem affinity purification and mass spectrometric analyses, we identified a Receptor for Activated Protein Kinase C1 (RACK1), a multi-functional scaffold protein, as a novel GATA4-binding partner. However, the precise functional relationships among p300, GATA4, and RACK1 remain unknown.
Hypothesis: We assessed the hypothesis that RACK1 regulates p300/GATA4-dependent transcriptional pathway and hypertrophic responses in cardimyocytes.
Methods and Results: Using glutathione S-transferase pull-down assays, we showed that RACK1 directly bound to GATA4. A series of assays using multiple mutants of RACK1 and GATA4 revealed that the WD4/5 domain of RACK1 bound to both N-terminal domain and C-terminal zinc finger domain of GATA4. Immunoprecipitation-Western blot (IP-WB) analysis demonstrated that RACK1 interacted with GATA4 in HEK293T cells. Overexpression of RACK1 inhibited the increases in p300-dependent acetylation of GATA4 and p300/GATA4-dependent activations of atrial natriuretic factor (ANF) and endothelin-1 (ET-1) promoters. Moreover, overexpression of RACK1 significantly inhibited phenylephrine (PE)-induced hypertrophic responses such as myofibrillar organization, an increase in cell size and the activation of the ANF and ET-1 promoters in cultured cardiomyocytes from neonatal rats. Finally, IP-WB showed that stimulation of cardiomyocytes with PE reduced the binding activity between RACK1 and GATA4.
Conclusions: RACK1 forms a functional complex with p300/GATA4 and plays a repressive role in myocardial cell hypertrophy as well as p300/GATA4-dependent transcription.
- © 2013 by American Heart Association, Inc.