The kinase activity of Cdk9 is required for phosphorylation of p300 and acetylation of GATA4 as well as cardiomyocyte hypertrophy

Abstract

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 complex with an intrinsic histone acetyltransferase (HAT), p300, and increases its transcriptional activity by acetylation. By tandem affinity purification and mass spectrometric analyses, we identified cyclin-dependent kinase-9 (Cdk9), a component of positive transcription elongation factor b (P-TEFb), as a novel GATA4-binding partner. Cdk9 also forms a complex with p300 as well as GATA-4. However, precise functional relationship between p300/GATA-4 and P-TEFb is unknown. Intact p300 induced not only the acetylation of GATA-4 but also the interaction of GATA-4 with P-TEFb. Furthermore, p300 induced the hyperphosphorylation of RNA Pol II, suggesting that p300 is involved in regulating the kinase activity of Cdk9. All of these effects were inhibited by a dominant-negative form of (DN-) p300. Meanwhile, DN-Cdk9, which loses its kinase activity by a single amino acid substitution, inhibited p300-induced hyperphophorylation of RNA Pol II. Notably, DN-Cdk9 inhibited phosphorylation of p300 as well as p300-induced acetylation and DNA binding of GATA-4, indicating a requirement of Cdk9 in the HAT activity of p300. Finally, both DN-p300 and DN-Cdk9 inhibited phenylephrine-induced hypertrophic responses in cardiac myocytes. These findings demonstrate that Cdk9 forms a functional complex with the p300/GATA4 and is required for p300/GATA4-transcriptional pathway during cardiomyocyte hypertrophy.