Proteomics analysis identifies novel GATA-4-binding partners involved in hypertrophic responses in cardiac myocytes

Proteomics analysis identifies novel GATA-4-binding partners involved in hypertrophic responses in cardiac myocytes

Tatsuya Morimoto1, Yoichi Sunagawa2, Teruhisa Kawamura2, Tomohide Takaya2, Hiromichi Wada2, Akira Shimatsu2, Masatoshi Fujita3, Toru Kita1, Koji Hasegawa2.

  1. Department of Cardiovascular Medicine, Graduate School of Medicine, Kyoto University, Kyoto, Japan.
  2. Division of Translational Research, Kyoto Medical Center, National Hospital Organization, Kyoto, Japan.
  3. School of Health Sciences, Faculty of Medicine, Kyoto University, Kyoto, Japan.

第71回日本循環器学会学術集会 (神戸), 2007/03/17 (ポスター).

Abstract

A zinc finger protein GATA-4 is one of factors involved in transcriptional regulation during myocardial cell hypertrophy. In response to hypertrophic stimuli, GATA-4 forms a large complex with ERK, NFATc and an intrinsic histone acetyltransferase, p300. Disruption of this complex results in inhibition of hypertrophic responses in cardiac myocytes. To identify novel GATA-4-binding partners required for myocardial cell hypertrophy, we performed proteomics analysis. GATA-4 was stably expressed as FLAG-HA-epitope fusion in HeLa cells by retroviral transcription. A GATA-4 complex was purified from nuclear extracts of these cells by sequential immunoprecipitation with anti-FLAG antibody followed by anti-HA antibody (Tandem Affinity Purification). By mass spectrometric analyses, we identify 73 GATA-4-binding proteins including histone, chromatin modifying factors, methyltransferase, histone de-acetyltransferases, basal transcription factors and unknown proteins. Chromatin modifying factors includes CAF-1 (chromatin assembly factor-1), and a SWI/SNF complex involved in transcriptional activation via ATP-dependent remodeling of nucleosome structure. Basal transcriptional factors include cyclin-dependent kinase-9 (CDK9), a component of Positive Transcription Elongation Factor b (P-TEFb) that causes phosphorylation of RNA polymerase II and transcriptional elongation of target promoters. We confirmed interaction of these proteins with GATA-4 in cardiac myocytes and demonstrated that one of these, CDK9, was required for myocardial cell hypertrophy. Thus, proteomics analysis of functional GATA-4 complex is useful to identify novel transcriptional pathways that mediate hypertrophic responses in cardiac myocytes.