Phosphatase PPM1A Regulates Phosphorylation of Thr-186 in the Cdk9 T-loop

• Published in: The Journal of biological chemistry 2008-11, Vol.283 (48), p.33578-33584
• Main Authors: Wang, Yan, Dow, Eugene C., Liang, Yao-Yun, Ramakrishnan, Rajesh, Liu, Hongbing, Sung, Tzu-Ling, Lin, Xia, Rice, Andrew P.
• Format: Article
• Language: English
• Subjects: Catalytic Domain - physiology; Cyclin-Dependent Kinase 9 - genetics; Cyclin-Dependent Kinase 9 - metabolism; HeLa Cells; Humans; Phosphoprotein Phosphatases - genetics; Phosphoprotein Phosphatases - metabolism; Phosphorylation - physiology; Positive Transcriptional Elongation Factor B - genetics; Positive Transcriptional Elongation Factor B - metabolism; Protein Phosphatase 2C; Protein Structure, Secondary - physiology; RNA, Small Nuclear - genetics; RNA, Small Nuclear - metabolism; Transcription, Chromatin, and Epigenetics
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M807495200

Cdk9 is the catalytic subunit of a general RNA polymerase II elongation factor known as positive transcription elongation factor b (P-TEFb). The kinase function of P-TEFb requires phosphorylation of Thr-186 in the T-loop of Cdk9 to allow substrates to access the catalytic core of the enzyme. To identify human phosphatases that dephosphorylate the T-loop of Cdk9, we used a Thr-186-phosphospecific antiserum to screen a phosphatase expression library. Overexpression of PPM1A and the related PPM1B greatly reduced Cdk9 T-loop phosphorylation in vivo. PPM1A and Cdk9 appear to associate in vivo as the proteins could be co-immunoprecipitated. The short hairpin RNA depletion of PPM1A resulted in an increase in Cdk9 T-loop phosphorylation. In phosphatase reactions in vitro, purified PPM1A could dephosphorylate Thr-186 both with and without the association of 7SK RNA, a small nuclear RNA that is bound to ∼50% of total cellular P-TEFb. PPM1B only efficiently dephosphorylated Cdk9 Thr-186 in vitro when 7SK RNA was depleted from P-TEFb. Taken together, our data indicate that PPM1A and to some extent PPM1B are important negative regulators of P-TEFb function.