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

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 ∼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. 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. The positive transcription elongation factor b (P-TEFb) 2The abbreviations used are: P-TEFb, positive transcription elongation factor b; shRNA, short hairpin RNA; CTD, carboxyl-terminal domain; Cdk, cyclin-dependent kinase; PPM, protein phosphatase, magnesium-dependent; FCP, transcription factor IIF-associating CTD phosphatase; SCP, small CTD phosphatase; HA, hemagglutinin; PH, pleckstrin homology. 2The abbreviations used are: P-TEFb, positive transcription elongation factor b; shRNA, short hairpin RNA; CTD, carboxyl-terminal domain; Cdk, cyclin-dependent kinase; PPM, protein phosphatase, magnesium-dependent; FCP, transcription factor IIF-associating CTD phosphatase; SCP, small CTD phosphatase; HA, hemagglutinin; PH, pleckstrin homology. is a cellular kinase complex that regulates elongation of most mammalian protein-coding genes transcribed by RNA polymerase II (1Peterlin B.M. Price D.H. Mol. Cell. 2006; 23: 297-305Abstract Full Text Full Text PDF PubMed Scopus (844) Google Scholar, 2Zhou Q. Yik J.H. Microbiol. Mol. Biol. Rev. 2006; 70: 646-659Crossref PubMed Scopus (209) Google Scholar). P-TEFb enhances the processivity of RNA polymerase II through the phosphorylation of the carboxyl-terminal domain (CTD) of the polymerase as well as antagonizing the actions of negative factors such as negative elongation factor and 5,6-dichloro-1-β-d-ribofuranosylbenzimidazole sensitivity-inducing factor. P-TEFb is comprised of cyclin-dependent kinase 9 (Cdk9), the kinase core, and its cyclin partner, either Cyclin T1, Cyclin T2, or Cyclin K. Two isoforms of Cdk9 exist, a major 42-kDa protein and a minor 55-kDa protein that contains 117 residues at the amino terminus that are absent in the 42-kDa protein (3Shore S.M. Byers S.A. Maury W. Price D.H. Gene (Amst.). 2003; 307: 175-182Crossref PubMed Scopus (81) Google Scholar). In most cells examined, Cyclin T1 is the predominant regulatory subunit for Cdk9. 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. As a key transcription factor, the activity of P-TEFb is carefully regulated within the cell. The first level of control is mediated by the regulated expression of its components, namely Cdk9 and its Cyclin partners. In primary human CD4+ T lymphocytes and monocytes, Cdk9 and Cyclin T2 expression are relatively high, whereas that of Cyclin T1 is low. Upon T cell activation or monocyte differentiation, Cyclin T1 protein expression is strongly up-regulated by post-transcriptional mechanisms, whereas expression of Cdk9 and Cyclin T2 remains relatively constant (4Liou L.Y. Herrmann C.H. Rice A.P. J. Virol. 2002; 76: 10579-10587Crossref PubMed Scopus (67) Google Scholar, 5Rice A.P. Herrmann C.H. Curr. HIV Res. 2003; 1: 395-404Crossref PubMed Scopus (54) Google Scholar, 6Ghose R. Liou L.Y. Herrmann C.H. Rice A.P. J. Virol. 2001; 75: 11336-11343Crossref PubMed Scopus (89) Google Scholar, 7Marshall R.M. Salerno D. Garriga J. Grana X. J. Immunol. 2005; 175: 6402-6411Crossref PubMed Scopus (44) Google Scholar, 8Liou L.Y. Haaland R.E. Herrmann C.H. Rice A.P. J. Leukoc. Biol. 2006; 79: 388-396Crossref PubMed Scopus (27) Google Scholar, 9Sung T.L. Rice A.P. Retrovirology. 2006; 3: 66Crossref PubMed Scopus (63) Google Scholar). Low levels of Cyclin T1 in resting CD4+ T cells and freshly isolated blood monocytes may function as one of the rate-limiting factors not only for transcription of many cellular genes but also for transcription of the human immunodeficiency virus type 1 provirus (10Yu W. Wang Y. Shaw C.A. Qin X.F. Rice A.P. Retrovirology. 2006; 3: 32Crossref PubMed Scopus (23) Google Scholar, 11Lassen K.G. Ramyar K.X. Bailey J.R. Zhou Y. Siliciano R.F. PLoS Pathog. 2006; 2: e68Crossref PubMed Scopus (168) Google Scholar). 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. In cells examined to date, the HEXIM1 protein is expressed at an approximate 10-fold higher level than the HEXIM2 protein (12Byers S.A. Price J.P. Cooper J.J. Li Q. Price D.H. J. Biol. Chem. 2005; 280: 16360-16367Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar). In HeLa cells, roughly 50% of cellular P-TEFb complexes are associated with 7SK/HEXIM and are catalytically repressed (13Yang Z. Zhu Q. Luo K. Zhou Q. Nature. 2001; 414: 317-322Crossref PubMed Scopus (553) Google Scholar, 14Nguyen V.T. Kiss T. Michels A.A. Bensaude O. Nature. 2001; 414: 322-325Crossref PubMed Scopus (553) Google Scholar). Previous studies have shown that 7SK RNA functions as a scaffold for the formation of the P-TEFb·7SK·HEXIM1 complex. However, the association of 7SK is not sufficient to inactivate the kinase complex as HEXIM1 has been identified as the key suppressor for P-TEFb function both in vitro and in vivo (15Michels A.A. Nguyen V.T. Fraldi A. Labas V. Edwards M. Bonnet F. Lania L. Bensaude O. Mol. Cell. Biol. 2003; 23: 4859-4869Crossref PubMed Scopus (203) Google Scholar, 16Yik J.H. Chen R. Nishimura R. Jennings J.L. Link A.J. Zhou Q. Mol. Cell. 2003; 12: 971-982Abstract Full Text Full Text PDF PubMed Scopus (378) Google Scholar). Although the binding of 7SK to P-TEFb does not require HEXIM1, the binding of HEXIM1 to P-TEFb is strictly 7SK-dependent (16Yik J.H. Chen R. Nishimura R. Jennings J.L. Link A.J. Zhou Q. Mol. Cell. 2003; 12: 971-982Abstract Full Text Full Text PDF PubMed Scopus (378) Google Scholar). It is currently believed that the existence of two pools of P-TEFb in the nucleus, an active 7SK/HEXIM-free and a repressed 7SK/HEXIM-bound pool, allows the cell to maintain P-TEFb activity at a regulated yet dynamic equilibrium (2Zhou Q. Yik J.H. Microbiol. Mol. Biol. Rev. 2006; 70: 646-659Crossref PubMed Scopus (209) Google Scholar). Recent work has also identified LARP7 (17Markert A. Grimm M. Martinez J. Wiesner J. Meyerhans A. Meyuhas O. Sickmann A. Fischer U. EMBO Rep. 2008; 9: 569-575Crossref PubMed Scopus (126) Google Scholar) and PIP7S (18He N. Jahchan N.S. Hong E. Li Q. Bayfield M.A. Maraia R.J. Luo K. Zhou Q. Mol. Cell. 2008; 29: 588-599Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar) as proteins associated in the P-TEFb·7SK small nuclear ribonucleoprotein complex. Given the importance of P-TEFb regulation in RNA polymerase II transcription, we were interested in identifying cellular factors that regulate P-TEFb activation in addition to 7SK and the HEXIM1/2 proteins. A critical regulatory residue in Cdk9 is Thr-186 in a region of the protein termed the T-loop. Cyclin-dependent kinases contain a conserved threonine in their T-loop, and phosphorylation of this residue induces a conformational change that allows Cdk substrates to access the catalytic core of the enzyme (19Pavletich N.P. J. Mol. Biol. 1999; 287: 821-828Crossref PubMed Scopus (565) Google Scholar, 20Morgan D.O. Nature. 1995; 374: 131-134Crossref PubMed Scopus (2930) Google Scholar). In the case of Cdk9, the association of 7SK with P-TEFb has been shown to be dependent on Thr-186 phosphorylation (21Chen R. Yang Z. Zhou Q. J. Biol. Chem. 2004; 279: 4153-4160Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar). A recent study reported that under conditions of stress, either UV irradiation or treatment with high concentrations of hexamethylene bisacetamide, the phosphatases PP2B and PP1α act cooperatively to disrupt P-TEFb from the 7SK small nuclear ribonucleoprotein through the dephosphorylation of Thr-186 (22Chen R. Liu M. Li H. Xue Y. Ramey W.N. He N. Ai N. Luo H. Zhu Y. Zhou N. Zhou Q. Genes Dev. 2008; 22: 1356-1368Crossref PubMed Scopus (141) Google Scholar). However, kinases and phosphatases that regulate Cdk9 T-loop phosphorylation under non-stress conditions have not been identified. Phosphatases that dephosphorylate Thr-186 in Cdk9 are likely to be protein-serine/threonine phosphatases. Cellular protein-serine/threonine phosphatases are classified into three families (23Gallego M. Virshup D.M. Curr. Opin. Cell Biol. 2005; 17: 197-202Crossref PubMed Scopus (134) Google Scholar): the PPMs (protein phosphatases, magnesium-dependent), which are mostly monomeric enzymes requiring the presence of Mg2+ or Mn2+ for catalytic activities; PPPs (phosphoprotein phosphatases) such as PP1A and PP2A, which are mostly oligomeric holoenzymes with related catalytic subunits bound by distinct regulatory subunits; and the less well characterized FCP/SCP (transcription factor IIF-associating CTD phosphatase/small CTD phosphatase) family represented by FCP1 and SCPs that dephosphorylate the CTD of RNA polymerase II. In the present study, we utilized a newly developed antiserum that recognizes phosphorylated Thr-186 in the Cdk9 T-loop. Using a phosphatase expression library, we identified two cellular phosphatases, PPM1A and the related PPM1B, that when overexpressed can dephosphorylate the Cdk9 T-loop in vivo. We found that PPM1A associates with Cdk9 in vivo and can directly dephosphorylate the Cdk9 T-loop in vitro. of PPM1A with resulted in an increase in Cdk9 T-loop phosphorylation. data also that 7SK may Thr-186 from dephosphorylated by PPM1B, PPM1A is to this function and dephosphorylate Cdk9 in the presence of 7SK data indicate that PPM1A and PPM1B are negative regulators of P-TEFb function. and cells and human cells were from and in with was from Cell and were from was from from from from and protein were from the expression for Cdk9, the of Cdk9 42-kDa or its were by from or Yang X. Herrmann C.H. Rice A.P. J. Virol. PubMed Google Scholar) and into a that of at the amino terminus of the expression for PPM1A and PPM1B, namely and and negative PPM1A have been X. X. Y. J. M. Wang J. Y. Chen A. Cell. 2006; Full Text Full Text PDF PubMed Scopus Google Scholar). were into HeLa cells at for or at for depletion HeLa cell were with of the or human X. X. Y. J. M. Wang J. Y. Chen A. Cell. 2006; Full Text Full Text PDF PubMed Scopus Google Scholar) cell were and cell were by cells with of of and complex and nuclear were nuclear and to the of cell were for with protein were with of at for by protein of or of were three with and into for or Cell or were on and were as for Y. Rice A.P. 2006; PubMed Scopus Google Scholar). were with the and cells were on in to The cells were and for as C.H. M.A. J. Cell 2001; PubMed Google Scholar) Cyclin T1 and for primary and to and were with and was as C.H. M.A. J. Cell 2001; PubMed Google Scholar). were and from were for In Cdk9 and phosphatases were purified from cells by were from by with In vitro phosphatase were as X. X. Y. J. M. Wang J. Y. Chen A. Cell. 2006; Full Text Full Text PDF PubMed Scopus Google Scholar) by with protein in a phosphatase at for were by the addition of and at for of Cdk9 at Thr-186 was examined in RNA and complex was purified from of HeLa cell nuclear by the of Cdk9. 7SK small nuclear RNA was from the complex as by first with and with of the purified complex was used for whereas RNA in the was with and on a for the of 7SK and small nuclear RNA was as R.E. Herrmann C.H. Rice A.P. 2003; 17: PubMed Scopus Google Scholar). of Cdk9 identify phosphatases that regulate phosphorylation of the Cdk9 T-loop, we first the of an antiserum a from Cdk9 a phosphorylated HeLa cells were with that either a type Cdk9 42-kDa protein or a Cdk9 protein an for Thr-186 were and expression of the type and Cdk9 proteins was examined in The antiserum that both type and Cdk9 proteins were expressed at The antiserum the the overexpressed type but not the data and of in vitro dephosphorylation and indicate that the antiserum recognizes the phosphorylated Cdk9 T-loop. antiserum be to as the antiserum a at the terminus of Cdk9 and that recognizes total Cdk9 be to as depletion of PPM1A Cdk9 Thr-186 phosphorylation. HeLa cells were with a that human or Cell were at and levels of or total Cdk9 were examined in were as under and protein levels in human PPM1A cells are shown to of 1 in the negative control with Cdk9 and in vitro dephosphorylation HeLa cells were with and Cell were at and phosphatases were with proteins were from by and the association of Cdk9 with phosphatases was in an the Cdk9 was purified by from HeLa of purified and PPM1B shown in A were with under phosphatase The of phosphatase reactions were examined in an and Overexpression of PPM1A or PPM1B Cdk9 Thr-186 that regulate Thr-186 phosphorylation in the Cdk9 T-loop to the protein-serine/threonine phosphatase We utilized a phosphatase expression X. X. Y. J. M. Wang J. Y. Chen A. Cell. 2006; Full Text Full Text PDF PubMed Scopus Google Scholar) that contains the catalytic subunits of protein-serine/threonine phosphatases, phosphatases, transcription factor IIF-associating CTD phosphatase/small CTD phosphatases, one domain phosphatase domain and protein protein phosphatases, and two We used the antiserum to screen for phosphatases that Cdk9 phosphorylation in of cells were with Cdk9 and phosphatase expression were and the levels of Thr-186 phosphorylation were examined in Two phosphatases were identified in this the related PPM1A and PPM1B proteins. PPM1A and PPM1B and are expressed monomeric enzymes and require Mg2+ or Mn2+ for catalytic activity T. Rev. Mol. Biol. PubMed Scopus (126) Google Scholar). both PPM1A and PPM1B have been shown to dephosphorylate the T-loop of and A. Genes Dev. 1999; PubMed Scopus Google Scholar, A. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). To that phosphatases regulate Cdk9 T-loop expression for the two phosphatases and for a negative control were with a Cdk9 protein into were and with the and proteins present in were in Overexpression of PPM1B, and not the overall expression level of the protein present in as with the However, of either PPM1A or PPM1B, but not greatly reduced Cdk9 T-loop phosphorylation. Overexpression of PPM1A and PPM1B also greatly reduced the amount of the HEXIM1 protein that with The in HEXIM1 associated with Cdk9 was as the association of HEXIM1 and 7SK RNA with P-TEFb requires phosphorylation of Thr-186 in the Cdk9 T-loop (21Chen R. Yang Z. Zhou Q. J. Biol. Chem. 2004; 279: 4153-4160Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar). We also in another that of PPM1A reduced the association of 7SK RNA and HEXIM1 with a protein not PPM1A is both in the and nucleus, whereas PPM1B is found in the T. Rev. Mol. Biol. PubMed Scopus (126) Google that PPM1A is to dephosphorylate Thr-186 the whereas the activity of PPM1B for Cdk9 may be to the our from that PPM1A may a than PPM1B in regulation of Cdk9 T-loop phosphorylation and We on PPM1A and an as an to the of on Cdk9 T-loop phosphorylation. A control that of HeLa cells with a phosphorylated Thr-186 the with the the of the antiserum not HeLa cells were with a expression and expression of Cyclin T1, Cdk9, and were examined Cdk9 T-loop phosphorylation was high in cells not the In T-loop phosphorylation was reduced in cells the The of Cyclin T1 with Cdk9 was also reduced in cells the protein to cells as by the in the Cyclin T1 and Cdk9. To the of PPM1A in Cdk9 T-loop HeLa cell were with a that human PPM1A or a negative control that the human and in PPM1A are and the the human PPM1A protein X. X. Y. J. M. Wang J. Y. Chen A. Cell. 2006; Full Text Full Text PDF PubMed Scopus Google Scholar). of cell were and were to total Cdk9, and Cdk9 The small RNA human PPM1A was to PPM1A levels to the negative control PPM1A small Although the overall level of Cdk9 was by the PPM1A T-loop phosphorylation was by the that a in PPM1A protein expression in an level of Cdk9 T-loop phosphorylation. We from the data in that PPM1A is a cellular phosphatase that can negatively regulate the phosphorylation of Thr-186 in the Cdk9 T-loop. of PPM1A with Cdk9 in and of T-loop in to PPM1A or PPM1B can associate with Cdk9 in vivo. of cells were with expression for a catalytically protein X. X. Y. J. M. Wang J. Y. Chen A. Cell. 2006; Full Text Full Text PDF PubMed Scopus Google or were were and was used to the phosphatases from the The association of the phosphatases with Cdk9 was in Cdk9 could be in with both PPM1A and but not of the purified and were in a and the data indicate that the of and present in are data indicate that PPM1A and Cdk9 can associate in for the that PPM1A is a phosphatase for Cdk9. The negative the to Cdk9, that phosphatase activity is not for the We that the level of the protein that was associated with Cdk9 was higher than that of the type PPM1A that the dephosphorylation of the Cdk9 T-loop by PPM1A may to the of the association the phosphatase and Cdk9. To PPM1A and PPM1B can directly dephosphorylate Thr-186 in the T-loop in Cdk9, we utilized an in vitro phosphatase X. X. Y. J. M. Wang J. Y. Chen A. Cell. 2006; Full Text Full Text PDF PubMed Scopus Google Scholar). Cdk9 was expressed by in HeLa cell and purified by The purified and from the shown in were for under phosphatase conditions with The of dephosphorylation reactions were examined in the antiserum PPM1A was to greatly dephosphorylate the Cdk9 T-loop, whereas PPM1B was to dephosphorylate ∼50% of Cdk9. data that PPM1A and to some extent PPM1B can directly dephosphorylated the Cdk9 T-loop. of Mg2+ for T-loop and of 7SK to the of 7SK on Cdk9 T-loop dephosphorylation in vitro. We the of the phosphatases to dephosphorylate Thr-186 with and without the depletion of 7SK RNA from Cdk9 phosphatases were purified from HeLa cells by and with complexes were from HeLa cells, and complexes were used for in vitro phosphatase of 7SK were by high of The of of that of 7SK RNA was depleted PPM1A was to dephosphorylate Cdk9 both in and and of the T-loop was when Cdk9 was with the catalytically that the dephosphorylation of Cdk9 can be to the phosphatase activity of PPM1B was to dephosphorylate Cdk9 in the but was less active on the and with the shown in where ∼50% of Thr-186 was to dephosphorylation by PPM1B, the associated with 7SK/HEXIM proteins. A of the family is the on Mg2+ or Mn2+ for phosphatase As both PPM1A and PPM1B were to dephosphorylate Cdk9 when reactions were without the addition of Mg2+ and In the data in and indicate that PPM1A can directly dephosphorylate Cdk9 at Thr-186 in both and P-TEFb In PPM1B to only efficiently dephosphorylate Cdk9 Thr-186 when is not associated with 7SK We have shown that of PPM1A greatly phosphorylation of Thr-186 in the Cdk9 T-loop. that PPM1A and Cdk9 associate in vivo. phosphatase reactions in vitro that purified PPM1A can directly dephosphorylate the Cdk9 T-loop both with and without the association of 7SK RNA with the complex. Taken together, that PPM1A the Cdk9 T-loop and is a negative of P-TEFb function. The of PPM1A as a of Cdk9 is not as PPM1A has been shown to dephosphorylate the of and two Cdk family in cell regulation A. Genes Dev. 1999; PubMed Scopus Google Scholar, A. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). PPM1A is a key of cell and and a of its substrates have been identified. PPM1A regulates the cell through the dephosphorylation of and A. Genes Dev. 1999; PubMed Scopus Google Scholar, A. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). PPM1A through the kinase and through the dephosphorylation of factors that regulate T. Rev. Mol. Biol. PubMed Scopus (126) Google Scholar). PPM1A has an important in and by through the dephosphorylation of X. X. Y. J. M. Wang J. Y. Chen A. Cell. 2006; Full Text Full Text PDF PubMed Scopus Google Scholar). P-TEFb is for the expression of many not most protein-coding the of PPM1A as a negative of Cdk9 into that can control in where the of the cell are such as in resting lymphocytes or monocytes, dephosphorylation of Cdk9 by PPM1A may be in a general of In this study, we also found that of PPM1B reduced Cdk9 Thr-186 phosphorylation in vivo. However, our data that PPM1B may be a minor of Cdk9 to the regulatory of PPM1B is a that PPM1B may have activity only for Cdk9, whereas PPM1A is found in both the and and may have activity for Cdk9 in the cell. PPM1B to efficiently dephosphorylate Cdk9 in vitro only the depletion of 7SK RNA, that the association of Cdk9 with 7SK/HEXIM proteins from the phosphatase activity of The association of Cdk9 with 7SK/HEXIM proteins is dynamic and 7SK/HEXIM-free and 7SK/HEXIM-bound (2Zhou Q. Yik J.H. Microbiol. Mol. Biol. Rev. 2006; 70: 646-659Crossref PubMed Scopus (209) Google Scholar). the association of Cdk9 with 7SK/HEXIM is dependent Thr-186 phosphorylation (21Chen R. Yang Z. Zhou Q. J. Biol. Chem. 2004; 279: 4153-4160Abstract Full Text Full Text PDF PubMed Scopus (144) Google the of PPM1B on the 7SK/HEXIM-free Cdk9 may to a dephosphorylation of the Thr-186 as Cdk9 can associate with 7SK/HEXIM the of Although we were to Cdk9 and overexpressed PPM1A in such as the one shown in our not the of the association PPM1A with P-TEFb. It that PPM1A a with Cdk9 as we were to either or overexpressed PPM1A with overexpressed Cdk9 in However, we could Cyclin T1 in with overexpressed PPM1A not that PPM1A associates with Cdk9 that is bound to a Cyclin In our screen of a phosphatase expression library, we also that both PP1A and PP2B reduced Cdk9 T-loop phosphorylation but not to the extent of PPM1A and PPM1B not PP1A and PP2B not the association of HEXIM1 with Cdk9 PPM1A and PPM1B, which this A recent study reported that PP2B can act cooperatively with PP1α to P-TEFb from the 7SK small nuclear ribonucleoprotein through dephosphorylation of Cdk Thr-186 when cells are to by either UV irradiation or hexamethylene treatment (22Chen R. Liu M. Li H. Xue Y. Ramey W.N. He N. Ai N. Luo H. Zhu Y. Zhou N. Zhou Q. Genes Dev. 2008; 22: 1356-1368Crossref PubMed Scopus (141) Google Scholar). Although we not phosphatase reactions with purified PP1A or is that of phosphatases under conditions may to reduced Cdk9 T-loop has been shown to regulate phosphorylation of and threonine residues in the terminus of Cdk9 T. K. Z. J. Retrovirology. 2005; 2: PubMed Scopus Google Scholar). It is that of PP1A or PP2B may in dephosphorylation in Cdk9 residues the T-loop, and this may to of Cdk9 in which Thr-186 is to dephosphorylation by PPM1A or PPM1A and PPM1B are monomeric enzymes and not require a regulatory subunit for either catalytic function or important PPM1A and PPM1B is the of the phosphatases for Cdk9 are It is that are regulated through some or by regulatory partners. the of PPM1A and PPM1B for Cdk9 may be Thr-186 phosphorylation may be regulated by the reversible association of 7SK RNA and HEXIM1/2 and proteins with Cdk9. In this and that regulate the association of Cdk9 with 7SK/HEXIM may to the cell the to Thr-186 phosphorylation levels to We of for and critical on the We and at Cell for the