Phosphatase PPM1A regulates phosphorylation of Thr-186 in the Cdk9 T-loop.
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 approximately 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.
Wang
Yan
Y
Department of Molecular Virology and Microbiology, Baylor College of Medicine, Houston, Texas 77030, USA.
Dow
Eugene C
EC
Liang
Yao-Yun
YY
Ramakrishnan
Rajesh
R
Liu
Hongbing
H
Sung
Tzu-Ling
TL
Lin
Xia
X
Rice
Andrew P
AP
D020134:Catalytic Domain / Q000502:physiology; D042863:Cyclin-Dependent Kinase 9 / Q000235:genetics / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D010749:Phosphoprotein Phosphatases / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation / Q000502:physiology; D042925:Positive Transcriptional Elongation Factor B / Q000235:genetics / Q000378:metabolism*; D000071636:Protein Phosphatase 2C; D017433:Protein Structure, Secondary / Q000502:physiology; D012342:RNA, Small Nuclear / Q000235:genetics / Q000378:metabolism
D012342:RNA, Small Nuclear; D042925:Positive Transcriptional Elongation Factor B; C467969:CDK9 protein, human; D042863:Cyclin-Dependent Kinase 9; C000607456:PPM1A protein, human; C000607457:PPM1B protein, human; D010749:Phosphoprotein Phosphatases; D000071636:Protein Phosphatase 2C