Abstract
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.
Highlights
The Cyclin T1/positive transcription elongation factor b (P-TEFb) complex has been studied extensively as this P-TEFb complex is targeted by the human immunodeficiency virus type 1 Tat protein to activate RNA polymerase II transcription of the integrated provirus, and this is essential for viral replication
The identification of PPM1A as a regulator of Cdk9 is not unexpected as PPM1A has been shown previously to dephosphorylate the T-loops of Cdk2 and Cdk6, two Cdk family members involved in cell cycle regulation [26, 27]
PPM1A inhibits mitogen signaling through the c-Jun NH2-terminal kinase (JNK) and p38 pathways through the dephosphorylation of factors that regulate these pathways [25]
Summary
The Cyclin T1/P-TEFb complex has been studied extensively as this P-TEFb complex is targeted by the human immunodeficiency virus type 1 Tat protein to activate RNA polymerase II transcription of the integrated provirus, and this is essential for viral replication. Low levels of Cyclin T1 in resting CD4ϩ T cells and freshly isolated blood monocytes may function as one of the rate-limiting factors for transcription of many cellular genes and for transcription of the human immunodeficiency virus type 1 provirus [10, 11]. In cells where the overall amount of P-TEFb has reached its steady state, another level of control is used At this level, P-TEFb is negatively regulated by its reversible association with a small nuclear RNA known as 7SK and the HEXIM proteins HEXIM1 and HEXIM2. Kinases and phosphatases that regulate Cdk T-loop phosphorylation under non-stress conditions have not been identified
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