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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.

Pubmed ID: 18829461

Authors

  • Wang Y
  • Dow EC
  • Liang YY
  • Ramakrishnan R
  • Liu H
  • Sung TL
  • Lin X
  • Rice AP

Journal

The Journal of biological chemistry

Publication Data

November 28, 2008

Associated Grants

  • Agency: NIAID NIH HHS, Id: AI35381
  • Agency: NIDDK NIH HHS, Id: DK073932

Mesh Terms

  • Catalytic Domain
  • Cyclin-Dependent Kinase 9
  • HeLa Cells
  • Humans
  • Phosphoprotein Phosphatases
  • Phosphorylation
  • Positive Transcriptional Elongation Factor B
  • Protein Structure, Secondary
  • RNA, Small Nuclear