Proteomics analysis identifies positive transcription elongation factor b as a novel GATA-4-binding partner involved in hypertrophic responses in cardiomyocytes

Proteomics analysis identifies positive transcription elongation factor b as a novel GATA-4-binding partner involved in hypertrophic responses in cardiomyocytes

Yoichi Sunagawa1, Tatsuya Morimoto1, Teruhisa Kawamura1, Tomohide Takaya1, Hiromichi Wada1, Akira Shimatsu1, Koji Hasegawa1, Masatoshi Fujita2, Toru Kita2.

  1. Kyoto Medical Center, National Hospital Organization, Kyoto, Japan.
  2. Graduate School of Medicine, Kyoto University, Kyoto, Japan.

American Heart Association Scientific Sessions 2007 (Orlando, USA), 2007/11/07 (Poster).

Abstract

Introduction: A zinc finger protein GATA-4 is one of the 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 the inhibition of hypertrophic responses in cardiomyocytes.

Hypothesis: We assessed the hypothesis that proteomics analysis may identify novel GATA-4-binding partners required for myocardial cell hypertrophy.

Methods: GATA-4 was stably expressed as FLAG-HA-epitope fusion in HeLa cells by retroviral transduction. GATA-4 complex was purified from nuclear extracts of these cells by sequential immunoprecipitation with an anti-FLAG antibody followed by an anti-HA antibody (Tandem Affinity Purification).

Results: By mass spectrometric analyses, we identified 73 GATA-4 binding proteins, including histone modifying factors, methyltransferase, HDAC and unknown proteins. One was cyclin-dependent kinase-9 (Cdk9), a component of Positive Transcription Elongation Factor b (P-TEFb). Immunoblotting analyses demonstrated that GATA-4 formed a complex with p300, Cdk9 and cyclin T1, another component of P-TEFb. A dominant-negative form of p300 disrupted the interaction of GATA-4 with Cdk9 or cyclin T1. Conversely, a dominant-negative form of Cdk9 and a Cdk9 kinase inhibitor, 5,6-dichloro-1-h-ribofuranosyl-benzimidazole (DRB), inhibited p300-induced activation of GATA-4-dependent transcription as well as the acetylation of GATA-4. Stimulation of cardiomyocytes with phenylephrine (PE) increased the binding of GATA-4/p300 with Cdk9 or cyclin T1. DN-Cdk9 and DRB not only disrupted the complex of GATA-4/p300 with Cdk9 or cyclin T1, but also repressed PE-induced hypertrophic responses such as myofibrillar organization, increase in cell size and promoter activation of the endothelin-1 and atrial natriuretic factors in cardiomyocytes.

Conclusion: These findings demonstrate that Cdk9 and cyclin T1 form a functional protein complex with GATA-4/p300 and that this large complex is a prerequisite for hypertrophic responses in cardiac myocytes.