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Phosphorylated Positive Transcription Elongation Factor b (P-TEFb) Is Tagged for Inhibition through Association with 7SK snRNA

2004; Elsevier BV; Volume: 279; Issue: 6 Linguagem: Inglês

10.1074/jbc.m310044200

ISSN

1083-351X

Autores

Ruichuan Chen, Zhiyuan Yang, Qiang Zhou,

Tópico(s)

RNA modifications and cancer

Resumo

The positive transcription elongation factor b (P-TEFb), comprising CDK9 and cyclin T, stimulates transcription of cellular and viral genes by phosphorylating RNA polymerase II. A major portion of nuclear P-TEFb is sequestered and inactivated by the coordinated actions of the 7SK snRNA and the HEXIM1 protein, whose induced dissociation from P-TEFb is crucial for stress-induced transcription and pathogenesis of cardiac hypertrophy. The 7SK·P-TEFb interaction, which can occur independently of HEXIM1 and does not by itself inhibit P-TEFb, recruits HEXIM1 for P-TEFb inactivation. To study the control of this interaction, we established an in vitro system that reconstituted the specific interaction of P-TEFb with 7SK but not other snRNAs. Using this system, together with an in vivo binding assay, we show that the phosphorylation of CDK9, on possibly the conserved Thr-186 in the T-loop, was crucial for the 7SK·P-TEFb interaction. This phosphorylation was not caused by CDK9 autophosphorylation or the general CDK-activating kinase CAK, but rather by a novel HeLa nuclear kinase. Furthermore, the stress-induced disruption of the 7SK·P-TEFb interaction was not caused by any prohibitive changes in 7SK but by the dephosphorylation of P-TEFb, leading to the loss of the key phosphorylation important for 7SK binding. Thus, the phosphorylated P-TEFb is tagged for inhibition through association with 7SK. We discuss the implications of this mechanism in controlling P-TEFb activity during normal and stress-induced transcription. The positive transcription elongation factor b (P-TEFb), comprising CDK9 and cyclin T, stimulates transcription of cellular and viral genes by phosphorylating RNA polymerase II. A major portion of nuclear P-TEFb is sequestered and inactivated by the coordinated actions of the 7SK snRNA and the HEXIM1 protein, whose induced dissociation from P-TEFb is crucial for stress-induced transcription and pathogenesis of cardiac hypertrophy. The 7SK·P-TEFb interaction, which can occur independently of HEXIM1 and does not by itself inhibit P-TEFb, recruits HEXIM1 for P-TEFb inactivation. To study the control of this interaction, we established an in vitro system that reconstituted the specific interaction of P-TEFb with 7SK but not other snRNAs. Using this system, together with an in vivo binding assay, we show that the phosphorylation of CDK9, on possibly the conserved Thr-186 in the T-loop, was crucial for the 7SK·P-TEFb interaction. This phosphorylation was not caused by CDK9 autophosphorylation or the general CDK-activating kinase CAK, but rather by a novel HeLa nuclear kinase. Furthermore, the stress-induced disruption of the 7SK·P-TEFb interaction was not caused by any prohibitive changes in 7SK but by the dephosphorylation of P-TEFb, leading to the loss of the key phosphorylation important for 7SK binding. Thus, the phosphorylated P-TEFb is tagged for inhibition through association with 7SK. We discuss the implications of this mechanism in controlling P-TEFb activity during normal and stress-induced transcription. 7SK is an abundant (2 × 105 copies/cell) and evolutionarily conserved small nuclear RNA (snRNA) 1The abbreviations used are: snRNAsmall nuclear RNAPolpolymeraseGSTglutathione S-transferaseNEnuclear extractPP1type I protein phosphatasePAPPpotato acid phosphataseDRB5,6-dichloro-1-β-d-ribofuranosylbenzimidazolP-TEFbpositive transcription elongation factor bCTDC-terminal domainCycT1cyclin T1HIVhuman immunodeficiency virusHMBAhexamethylene bisacetamide. of ∼330 nucleotides (1Humphries P. Russell S.E.H. McWilliam P. McQuaid S. Pearson C. Humphries M.M. Biochem. J. 1987; 24: 281-284Crossref Scopus (9) Google Scholar, 2Murphy S. Altruda F. Ullu E. Tripodi M. Silengo L. Melli M. J. Mol. Biol. 1984; 177: 575-590Crossref PubMed Scopus (34) Google Scholar, 3Reddy R. Henning D. Subrahmanyam C.S. Busch H. J. Biol. Chem. 1984; 259: 12265-12270Abstract Full Text PDF PubMed Google Scholar, 4Ullu E. Melli M. Nucleic Acids Res. 1982; 10: 2209-2223Crossref PubMed Scopus (23) Google Scholar). It is transcribed by RNA polymerase (Pol) III from one or more genes belonging to a family of interspersed repeats in the mammalian genome (2Murphy S. Altruda F. Ullu E. Tripodi M. Silengo L. Melli M. J. Mol. Biol. 1984; 177: 575-590Crossref PubMed Scopus (34) Google Scholar, 5Zieve G. Benecke B.J. Penman S. Biochemistry. 1977; 16: 4520-4525Crossref PubMed Scopus (76) Google Scholar). The high conservation and abundance of 7SK suggest an important physiological function of this RNA. In searching for nuclear factors that can bind to and regulate the activity of human positive transcription elongation factor b (P-TEFb), we and others (6Nguyen V.T. Kiss T. Michels A.A. Bensaude O. Nature. 2001; 414: 322-325Crossref PubMed Scopus (557) Google Scholar, 7Yang Z. Zhu Q. Luo K. Zhou Q. Nature. 2001; 414: 317-322Crossref PubMed Scopus (559) Google Scholar) have identified 7SK as a specific P-TEFb-associated factor. Consisting of CDK9 and cyclin T1 (CycT1) (8Peng J. Zhu Y. Milton J.T. Price D.H. Genes Dev. 1998; 12: 755-762Crossref PubMed Scopus (452) Google Scholar, 9Wei P. Garber M.E. Fang S.M. Fischer W.H. Jones K.A. Cell. 1998; 92: 451-462Abstract Full Text Full Text PDF PubMed Scopus (1055) Google Scholar), P-TEFb strongly enhances the processivity of RNA Pol II by phosphorylating the C-terminal domain (CTD) of Pol II and antagonizing the actions of negative elongation factors (10Shim E.Y. Walker A.K. Shi Y. Blackwell T.K. Genes Dev. 2002; 16: 2135-2146Crossref PubMed Scopus (165) Google Scholar, 11Renner D.B. Yamaguchi Y. Wada T. Handa H. Price D.H. J. Biol. Chem. 2001; 276: 42601-42609Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar, 12Wada T. Takagi T. Yamaguchi Y. Watanabe D. Handa H. EMBO J. 1998; 17: 7395-7403Crossref PubMed Scopus (282) Google Scholar, 13Yamaguchi Y. Takagi T. Wada T. Yano K. Furuya A. Sugimoto S. Hasegawa J. Handa H. Cell. 1999; 97: 41-51Abstract Full Text Full Text PDF PubMed Scopus (622) Google Scholar). Studies employing RNA interference in Caenorhabditis elegans and a pharmacological inhibitor of CDK9 in mammalian cells implicate P-TEFb as essential for expression of most protein-coding genes (10Shim E.Y. Walker A.K. Shi Y. Blackwell T.K. Genes Dev. 2002; 16: 2135-2146Crossref PubMed Scopus (165) Google Scholar, 14Chao S.-H. Price D.H. J. Biol. Chem. 2001; 276: 31793-31799Abstract Full Text Full Text PDF PubMed Scopus (544) Google Scholar). small nuclear RNA polymerase glutathione S-transferase nuclear extract type I protein phosphatase potato acid phosphatase 5,6-dichloro-1-β-d-ribofuranosylbenzimidazol positive transcription elongation factor b C-terminal domain cyclin T1 human immunodeficiency virus hexamethylene bisacetamide. P-TEFb also functions as a specific host cellular cofactor for the HIV-1 Tat protein. Stimulation of Pol II elongation is essential for HIV transcription, during which P-TEFb is recruited to the nascent mRNA by Tat (15Jeang K.T. Xiao H. Rich E.A. J. Biol. Chem. 1999; 274: 28837-28840Abstract Full Text Full Text PDF PubMed Scopus (379) Google Scholar, 16Price D.H. Mol. Cell Biol. 2000; 20: 2629-2634Crossref PubMed Scopus (571) Google Scholar). Tat binds to CycT1 and recruits P-TEFb through formation of a stable ternary complex containing P-TEFb, Tat, and the HIV TAR RNA structure located at the 5′-end of the nascent viral transcript. Once recruited, P-TEFb phosphorylates the CTD and stimulates the production of the full-length HIV-1 transcripts. It is important to point out that not all forms of P-TEFb in the cell can display transcriptional activity. In fact, at least half of P-TEFb in HeLa cells are sequestered in the 7SK snRNP, where their kinase and transcriptional activities are suppressed (6Nguyen V.T. Kiss T. Michels A.A. Bensaude O. Nature. 2001; 414: 322-325Crossref PubMed Scopus (557) Google Scholar, 7Yang Z. Zhu Q. Luo K. Zhou Q. Nature. 2001; 414: 317-322Crossref PubMed Scopus (559) Google Scholar). Besides P-TEFb and 7SK, the snRNP has recently been shown to also contain HEXIM1 (17Yik J.H.N. Chen R. Nishimura R. Jennings J.L. Link A.J. Zhou Q. Mol. Cell. 2003; 12: 971-983Abstract Full Text Full Text PDF PubMed Scopus (382) Google Scholar, renamed as MAQ1 in 18Michels 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 (205) Google Scholar). HEXIM1 has previously been identified as a nuclear protein whose expression is rapidly induced in cells treated with hexamethylene bisacetamide (HMBA) (19Ouchida R. Kusuhara M. Shimizu N. Hisada T. Makino Y. Morimoto C. Handa H. Ohsuzu F. Tanaka H. Genes Cells. 2003; 8: 95-107Crossref PubMed Scopus (65) Google Scholar), a potent inducer of cell differentiation (20Marks P.A. Richon V.M. Rifkind R.A. Int. J. Hematol. 1996; 63: 1-17Crossref PubMed Scopus (40) Google Scholar). While the binding of 7SK to P-TEFb can occur in the absence of HEXIM1, the binding of HEXIM1 to P-TEFb is strictly 7SK-dependent (17Yik J.H.N. Chen R. Nishimura R. Jennings J.L. Link A.J. Zhou Q. Mol. Cell. 2003; 12: 971-983Abstract Full Text Full Text PDF PubMed Scopus (382) Google Scholar). Furthermore, the 7SK binding alone is not sufficient to inhibit P-TEFb. P-TEFb is inhibited by HEXIM1 in a process that specifically requires 7SK for bridging the HEXIM1·P-TEFb interaction. This allows HEXIM1 to inhibit transcription both in vivo and in vitro (17Yik J.H.N. Chen R. Nishimura R. Jennings J.L. Link A.J. Zhou Q. Mol. Cell. 2003; 12: 971-983Abstract Full Text Full Text PDF PubMed Scopus (382) Google Scholar). The amount of the HEXIM1/7SK-bound P-TEFb does not remain unchanged. In fact, treatment of cells with several stress-inducing agents rapidly releases 7SK and HEXIM1 from P-TEFb (6Nguyen V.T. Kiss T. Michels A.A. Bensaude O. Nature. 2001; 414: 322-325Crossref PubMed Scopus (557) Google Scholar, 7Yang Z. Zhu Q. Luo K. Zhou Q. Nature. 2001; 414: 317-322Crossref PubMed Scopus (559) Google Scholar, 17Yik J.H.N. Chen R. Nishimura R. Jennings J.L. Link A.J. Zhou Q. Mol. Cell. 2003; 12: 971-983Abstract Full Text Full Text PDF PubMed Scopus (382) Google Scholar, 18Michels 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 (205) Google Scholar). These agents have been shown to stimulate CTD phosphorylation and HIV transcription (21Valerie K. Delers A. Bruck C. Thiriart C. Rosenberg H. Debouck C. Rosenberg M. Nature. 1988; 333: 78-81Crossref PubMed Scopus (187) Google Scholar, 22Casse C. Giannoni F. Nguyen V.T. Dubois M.-F. Bensaude O. J. Biol. Chem. 1999; 274: 16097-16106Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). Thus, the induced release of 7SK/HEXIM1 and activation of P-TEFb may contribute directly to the stress-induced HIV and cellular gene expression. The activation of P-TEFb through release of 7SK also contributes to the pathogenesis of cardiac hypertrophy, a disease characterized by the enlargement of cardiac myocytes due to a global increase in RNA and protein contents. P-TEFb activity is limiting for normal cardiac growth. Hypertrophic signals induce the dissociation of 7SK (presumably also HEXIM1) from P-TEFb, activating P-TEFb to stimulate Pol II transcription (23Sano M. Schneider M.D. Cell Cycle. 2003; 2: 99-104Crossref PubMed Scopus (39) Google Scholar, 24Sano M. Abdellatif M. Oh H. Xie M. Bagella L. Giordano A. Michael L.H. DeMayo F.J. Schneider M.D. Nat. Med. 2002; 8: 1310-1317Crossref PubMed Scopus (204) Google Scholar). Given the importance of the 7SK·P-TEFb interaction in recruiting HEXIM1 for P-TEFb inactivation, we would like to understand how this interaction is regulated. This will aid the future elucidation of the signaling pathways that control the formation and disruption of the 7SK snRNP in response to physiological stimuli. Here, we report the establishment of an in vitro system that reconstituted the specific 7SK·P-TEFb interaction. Our data reveal that the phosphorylation of CDK9, on possibly the conserved Thr-186 in the T-loop, was crucial for this interaction. This phosphorylation was not caused by CDK9 autophosphorylation or the general CDK-activating kinase CAK, but rather by a yet-to-be-identified HeLa nuclear kinase. Finally, the stress-induced dissociation of 7SK from P-TEFb was not caused by any changes or modifications in 7SK but rather by the dephosphorylation of P-TEFb, resulting in the loss of the key phosphorylation important for 7SK binding. In Vitro Reconstitution of 7SK·P-TEFb Interaction—The reconstitution was performed in two main steps. During the immunoprecipitation step, 250 μg of nuclear extract (NE) prepared from either F1C2 (an engineered HeLa cell line stably expressing FLAG-tagged CDK9) (7Yang Z. Zhu Q. Luo K. Zhou Q. Nature. 2001; 414: 317-322Crossref PubMed Scopus (559) Google Scholar) or HeLa cells (as a negative control) were incubated at 4 °C for 2 h with 10 μl of anti-FLAG-agarose beads (Sigma). After binding, the beads were washed extensively with buffer D (20 mm HEPES-KOH, pH 7.9, 15% glycerol, 0.2 mm EDTA, 0.2% Nonidet P-40, 1 mm dithiothreitol, and 1 mm phenylmethylsulfonyl fluoride) containing 0.3 m KCl (D0.3M), then buffer D0.8M, and finally D0.1M. The second wash with D0.8M stripped 7SK and HEXIM1 off P-TEFb. After virtually all the liquid was removed, the beads were preblocked at room temperature for 30 min with 15–20 μl of a blocking solution containing 5 mg/ml bovine serum albumin, 3 mg/ml poly(C) (Amersham Biosciences), and 2.5 mg/ml Escherichia coli tRNA. During the reconstitution step, the preblocked beads were incubated at 30 °C for 30 min with 100 μg of HeLa NE in buffer D0.1M plus 0.5 mm ATP and 5 mm MgCl2 or 100 μg of HeLa NE depleted of the endogenous ATP by treatment with hexokinase and glucose (25Zamore P.D. Tuschl T. Sharp P.A. Bartel D.P. Cell. 2000; 101: 25-33Abstract Full Text Full Text PDF PubMed Google Scholar). After the incubation, the beads were washed extensively with buffer D0.4M and then D0.1M. The reconstituted 7SK snRNP was finally eluted off the beads with 0.4 mg/ml of FLAG peptide (Sigma) prepared in D0.1M. RNA used in the specified reconstitution reactions was transcribed in vitro from a 7SK DNA template by T7 RNA polymerase (Stratagene) or extracted from HeLa NE or the D0.8M-stripped 7SK fraction with phenol and chloroform. After precipitation with ethanol, the RNA pellet was dissolved in D0.1M. Phosphatase Treatment of Immobilized P-TEFb—After washing away nonspecific factors with D0.3M, the immobilized 7SK(+) P-TEFb was washed with 90 mm sodium citrate and incubated with 0.3 units of PAPP (potato acid phosphatase, Sigma) in 50 μl. After incubation at 30 °C for 45 min, the complex was washed with D0.3M and then eluted by Flag peptide. For treatment of the immobilized 7SK/HEXIM1-free P-TEFb with PP1 (type 1 protein phosphatase, NEB), the D0.8M-stripped P-TEFb was washed with the PP1 solution (50 mm Tris-HCl pH 7.0, 0.1 mm EDTA, 5 mm dithiothreitol, 0.01% Nonidet P-40, and 2 mm MnCl2) and then incubated at 30 °C with 12.5 units of PP1 in 20 μl for 45 min. After washing with D0.3M, the dephosphorylated P-TEFb was used in the reconstitution reactions. Affinity Purification of CDK9, CycT1, and Their Associated Factors for Northern and Western Analyses—FLAG-tagged wild-type and mutant CDK9 and CycT1 and their associated factors were isolated by anti-FLAG immunoprecipitation from NE of transfected HeLa cells. After incubation at 4 °C for 2 h, the immunoprecipitates were washed with D0.3M. The FLAG peptide-eluted materials were analyzed by Western blotting with anti-FLAG (M2, Sigma), anti-CDK9, and anti-CycT1 antibodies (Santa Cruz Biotechnology), and Northern blotting using the full-length 7SK antisense RNA as a probe. In Vitro Kinase Reactions—P-TEFb containing wild-type or mutant CDK9-FLAG were immunoprecipitated from NE of transfected HeLa cells, washed with D0.8M to remove the associated 7SK and HEXIM1, and eluted with FLAG peptide prepared in D0.1M. The kinase reactions contained P-TEFb, 1 μg of immobilized GST-CTD, 5 mm MgCl2, 50 μm cold ATP, and 1 μl of [γ-P32]ATP (3000 Ci/mmol) and were incubated at 30 °C for 25 min in 20-μl volume. The phosphorylated GST-CTD was analyzed by SDS-PAGE followed by autoradiography. Treatment of Cells with Drugs—The HeLa-based F1C2 cells (3 × 106) expressing CDK9-FLAG (7Yang Z. Zhu Q. Luo K. Zhou Q. Nature. 2001; 414: 317-322Crossref PubMed Scopus (559) Google Scholar) were seeded in 15-cm dishes 1 day before the treatment. 5,6-Dichloro-1-β-d-ribofuranosylbenzimidazol (DRB) (100 μm), H7 (100 μm), staurosporine (0.5 μm), and actinomycin D (1.2 μg/ml) were added to the culture medium for 1 h. NE was prepared from the treated cells and subjected to further analysis. Phosphorylation-dependent Interaction of 7SK with P-TEFb— To investigate the mechanism controlling the 7SK·P-TEFb interaction, we asked whether protein phosphorylation might play a role in this process. Previously, it has been shown that treatment of HeLa cells with the kinase inhibitor DRB causes a rapid dissociation of 7SK from P-TEFb (6Nguyen V.T. Kiss T. Michels A.A. Bensaude O. Nature. 2001; 414: 322-325Crossref PubMed Scopus (557) Google Scholar). To determine whether other kinase inhibitors such as staurosporine and H7 (1-(5-isoquinolinesulfonyl)-2-methylpiperazine) would produce a similar effect, a HeLa-based cell line (F1C2) stably expressing CDK9-FLAG (7Yang Z. Zhu Q. Luo K. Zhou Q. Nature. 2001; 414: 317-322Crossref PubMed Scopus (559) Google Scholar) was treated with DRB, staurosporine, H7, or the control solvent Me2SO (DMSO). CDK9-FLAG and its associated CycT1 and 7SK were immunoprecipitated from the NE of the treated cells and analyzed by Western and Northern blotting. All three kinase inhibitors significantly weakened the binding of 7SK to P-TEFb without affecting the CycT1-CDK9 interaction or the steady state levels of CDK9, CycT1, and 7SK in NE (Fig. 1A), suggesting that a protein phosphorylation event may be crucial in promoting the binding of 7SK to P-TEFb. To determine whether a direct phosphorylation of a component of the 7SK·P-TEFb complex is required for this binding, we incubated the immobilized 7SK-bound P-TEFb with PAPP. While the CycT1-CDK9 interaction remained unaffected, 7SK was dissociated from P-TEFb following the incubation with PAPP and the subsequent washes (Fig. 1B, left panel). This effect was blocked by phosphatase inhibitors (data not shown). The PAPP preparation was free of any RNases and did not degrade 7SK when incubated with the peptide-eluted 7SK·P-TEFb complex in solution (Fig. 1B, right panel). Together, these in vivo and in vitro data implicate the phosphorylation of a component of the 7SK-bound P-TEFb as important for the binding of 7SK to P-TEFb. Requirement of Both CDK9 and CycT1 Subunits but Not CycT1 Phosphorylation for 7SK·P-TEFb Binding—CDK9 and CycT1 are both phosphoproteins, and CycT1 is phosphorylated by CDK9 in the C-terminal half (26Fong Y.W. Zhou Q. Mol. Cell Biol. 2000; 20: 5897-5907Crossref PubMed Scopus (78) Google Scholar, 27Garber M.E. Mayall T.P. Suess E.M. Meisenhelder J. Thompson N.E. Jones K.A. Mol. Cell Biol. 2000; 20: 6958-6969Crossref PubMed Scopus (142) Google Scholar) (Fig. 2A). To determine whether the phosphorylation of CycT1 is important for the 7SK·P-TEFb binding, a C-terminally truncated CycT1-(1–333)-FLAG lacking all the phosphorylation sites (26Fong Y.W. Zhou Q. Mol. Cell Biol. 2000; 20: 5897-5907Crossref PubMed Scopus (78) Google Scholar, 27Garber M.E. Mayall T.P. Suess E.M. Meisenhelder J. Thompson N.E. Jones K.A. Mol. Cell Biol. 2000; 20: 6958-6969Crossref PubMed Scopus (142) Google Scholar) was analyzed for its binding to CDK9 and 7SK in anti-FLAG immunoprecipitates derived from NE of transfected HeLa cells. To test the contribution of CDK9 to the 7SK·P-TEFb binding, two additional CycT1 mutants 1–254 and 2M were also analyzed (Fig. 2A). Wild-type CycT1 and its two C-terminal deletion mutants 1–333 and 1–254 all contained an intact cyclin-box, and as expected, were able to bind to CDK9 (Fig. 2B). Although 1–333 was expressed to a higher level than wild-type CycT1 and 1–254 based on per microgram of DNA transfected, a similar level of CDK9 was found to co-precipitate with all three CycT1 molecules, indicating the formation of a similar amount of the CycT1/CDK9 heterodimers (Fig. 2B). Under these conditions, 7SK bound strongly to the P-TEFb containing either wild-type CycT1 or 1–333 but not 1–254. Thus, a region between amino acids 255 and 333, which is C-terminal to the Tat/TAR recognition motif (TRM) (28Garber M.E. Wei P. KewalRamani V.N. Mayall T.P. Herrmann C.H. Rice A.P. Littman D.R. Jones K.A. Genes Dev. 1998; 12: 3512-3527Crossref PubMed Scopus (385) Google Scholar), contributed to 7SK·P-TEFb binding. However, the C-terminal phosphorylation of CycT1 after residue 334 was clearly dispensable for the binding. Finally, unlike the HIV TAR RNA, which can interact with only the monomeric CycT1 in conjunction with Tat (9Wei P. Garber M.E. Fang S.M. Fischer W.H. Jones K.A. Cell. 1998; 92: 451-462Abstract Full Text Full Text PDF PubMed Scopus (1055) Google Scholar, 28Garber M.E. Wei P. KewalRamani V.N. Mayall T.P. Herrmann C.H. Rice A.P. Littman D.R. Jones K.A. Genes Dev. 1998; 12: 3512-3527Crossref PubMed Scopus (385) Google Scholar), 7SK appeared to require also CDK9 for binding to the CycT1/CDK9 heterodimer. For example, the mutant CycT1(2M), with Lys-93 changed to Leu and Glu-96 to Lys within the cyclin-box, bound to neither CDK9 (29Bieniasz P.D. Grdina T.A. Bogerd H.P. Cullen B.R. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 7791-7796Crossref PubMed Scopus (155) Google Scholar) nor 7SK (Fig. 2B). Requirement of Conserved Thr-186 in CDK9 T-loop for in Vivo 7SK·P-TEFb Binding—Since CDK molecules have built-in structural flexibility that is most often controlled by phosphorylation (30Pavletich N.P. J. Mol. Biol. 1999; 287: 821-828Crossref PubMed Scopus (572) Google Scholar, 31Morgan D.O. Nature. 1995; 374: 131-134Crossref PubMed Scopus (2938) Google Scholar), we next investigated the requirement of CDK9 phosphorylation for the 7SK·P-TEFb binding. A group of Ser/Thr residues, located near the CDK9 C terminus in a region not conserved with other CDKs, are phosphorylated when CDK9 undergoes autophosphorylation (27Garber M.E. Mayall T.P. Suess E.M. Meisenhelder J. Thompson N.E. Jones K.A. Mol. Cell Biol. 2000; 20: 6958-6969Crossref PubMed Scopus (142) Google Scholar). This induces a conformational change in P-TEFb critical for the formation of the P-TEFb·Tat·TAR complex (26Fong Y.W. Zhou Q. Mol. Cell Biol. 2000; 20: 5897-5907Crossref PubMed Scopus (78) Google Scholar, 27Garber M.E. Mayall T.P. Suess E.M. Meisenhelder J. Thompson N.E. Jones K.A. Mol. Cell Biol. 2000; 20: 6958-6969Crossref PubMed Scopus (142) Google Scholar). As a member of the CDK family, the activity and conformation of CDK9 are also affected by phosphorylation on other amino acids, which are conserved among several or all CDK family members (31Morgan D.O. Nature. 1995; 374: 131-134Crossref PubMed Scopus (2938) Google Scholar). For example, phosphorylation of a conserved threonine (Thr-186 in CDK9) located at the tip of a flexible loop (the T-loop) in all CDKs is known to induce a major movement of the loop and allow the access to the catalytic center by the substrate (32Russo A.A. Jeffrey P.D. Pavletich N.P. Nat. Struct. Biol. 1996; 3: 696-700Crossref PubMed Scopus (505) Google Scholar). Furthermore, a conserved Ser residue in the T-loops of CDK7 (Ser-164) and CDK9 (Ser-175) can also be phosphorylated (33Martinez A.M. Afshar M. Martin F. Cavadore J.C. Labbe J.C. Doree M. EMBO J. 1997; 16: 343-354Crossref PubMed Scopus (66) Google Scholar, 34Larochelle S. Chen J. Knights R. Pandur J. Morcillo P. Erdjument-Bromage H. Tempst P. Suter B. Fisher R.P. EMBO J. 2001; 20: 3749-3759Crossref PubMed Scopus (104) Google Scholar). Next, FLAG-tagged CDK9 mutants containing point mutations at these phosphorylation sites were analyzed for their interactions with 7SK and CycT1 in transfected HeLa cells. Unlike the binding of P-TEFb to Tat/TAR, which requires the phosphorylation of several Ser/Thr residues near the CDK9 C terminus (27Garber M.E. Mayall T.P. Suess E.M. Meisenhelder J. Thompson N.E. Jones K.A. Mol. Cell Biol. 2000; 20: 6958-6969Crossref PubMed Scopus (142) Google Scholar), substitution of 4 (C4S/T-A: Ser-347, Ser-353, Thr-354, and Ser-357) or 8 (C8S/T-A: Ser-329, Thr-330, Thr-333, Ser-334, Ser-347, Thr-350, Ser-353, and Thr-354) Ser/Thr in and around this region with Ala had no or only minor effect on CDK9 binding to 7SK and CycT1 (Fig. 2C). Similarly, the Ser-175 to Ala (S175A) mutation in the T-loop only slightly decreased the binding to 7SK, which was fully restored when Ser-175 was changed to Asp (S175D). In contrast, mutation of Thr-186 to either Ala (T186A) or Glu (T186E) completely abolished the binding to 7SK, although neither mutation significantly affected the CycT1-CDK9 interaction. Thus, among all the known phosphorylation sites in CDK9, Thr-186 was the only one essential for the 7SK·P-TEFb binding. Since this conserved threonine is invariably phosphorylated in all activated CDKs, the phosphorylation-dependent 7SK·P-TEFb binding could potentially be attributed to the phosphorylation of Thr-186. The Phosphorylation Critical for 7SK·P-TEFb Binding Is Not Caused by CDK9 Autophosphorylation—We next performed kinase reactions to examine whether the T-loop mutations could affect P-TEFb ability to phosphorylate GST-CTD. Prior to the reactions, the immunoprecipitated CycT1/CDK9-FLAG were washed with a high salt (0.8 m KCl) buffer, which removed the associated 7SK and HEXIM1 without disrupting the CycT1-CDK9 binding (see below). In kinase reactions, the T186A, T186E, and S175A mutations all inactivated P-TEFb, whereas S175D fully restored P-TEFb kinase activity (Fig. 2D), revealing a requirement for a negatively charged amino acid at position 175. More importantly, the ability of S175A to inactivate the P-TEFb kinase without significantly affecting P-TEFb binding to 7SK suggests that the CDK9 kinase activity is not required for the binding. This notion was further supported by the observation that another P-TEFb complex containing the D167N mutant CDK9 was completely inactive as a kinase (Fig. 2D), but fully capable of binding to 7SK (Fig. 2E). Moreover, as with the wild-type 7SK-bound P-TEFb, treatment of the D167N-containing complex with phosphatase also released 7SK (Fig. 2F), indicating that phosphorylation was required for this kinase-defective P-TEFb to bind to 7SK. Because D167N cannot phosphorylate itself or any associated proteins, it is possible that the phosphorylation, possibly on Thr-186, was caused by another unrelated kinase. This issue will be revisited later. In Vitro Reconstitution of the 7SK·P-TEFb Interaction—To further study the control of the 7SK·P-TEFb interaction, we decided to reconstitute this interaction in vitro. Since CDK9 phosphorylation has been implicated as critical for the interaction, we tried to assemble the 7SK·P-TEFb complex from P-TEFb that had acquired the key phosphorylation in vivo. We took advantage of an observation (35Shore S.M. Byers S.A. Maury W. Price D.H. Gene (Amst.). 2003; 307: 175-182Crossref PubMed Scopus (81) Google Scholar) that 7SK could be washed off the immobilized P-TEFb with buffers containing greater than 0.6 m KCl (e.g. 0.8 m KCl in buffer D0.8M), whereas the CycT1/CDK9-FLAG dimer was mostly unaffected and remained tightly bound to the beads (Fig. 3A). HEXIM1, which bound to P-TEFb through 7SK, was also stripped off P-TEFb by the same D0.8M buffer (17Yik J.H.N. Chen R. Nishimura R. Jennings J.L. Link A.J. Zhou Q. Mol. Cell. 2003; 12: 971-983Abstract Full Text Full Text PDF PubMed Scopus (382) Google Scholar). The 7SK/HEXIM1-free P-TEFb thus obtained was then incubated with HeLa NE, protein-free total RNA extracted from NE, purified native 7SK RNA or heat-denatured (Δ) 7SK to reconstitute the 7SK·P-TEFb interaction (Fig. 3B). The purified 7SK was from a fraction (termed 7SK fraction) that was stripped off the immobilized P-TEFb by buffer D0.8M (Fig. 3B). After the incubation and washing with buffer D0.4M, the reconstituted 7SK·P-TEFb complex was eluted with FLAG peptide and analyzed by Western and Northern blotting. The control reactions in lanes 6–10, Fig. 3C used HeLa NE in anti-FLAG immunoprecipitation, which retained no P-TEFb on the beads. Compared with these reactions, the 7SK-free CycT1/CDK9-FLAG derived from F1C2 NE interacted with 7SK from all four sources (Fig. 3C, lanes 2–5), which contained a similar level of 7SK (lanes 11–14). Even the heat-denatured 7SK interacted with P-TEFb, probably because the RNA quickly adopted an active conformation once in the reaction. Thus, no additional protein from HeLa NE was required for the reconstituted 7SK·P-TEFb interaction. Dependence on Thr-186 for Reconstituted 7SK·P-TEFb Interaction—To test the specificity of the reconstituted 7SK·P-TEFb interaction, we examined whether Thr-186 in the CDK9 T-loop was required. P-TEFb containing wild-type or mutant (T186A or D167N) CDK9-FLAG was immunoprecipitated from the transfected HeLa cells and then washed with D0.8M to remove 7SK/HEXIM1. While both wild-type and the D167N P-TEFb bound to 7SK present in total RNA and HeLa NE, the T186A P-TEFb failed (Fig. 4A). Thus, like the situation in vivo (Fig. 2E), Thr-186 was also required for the 7SK·P-TEFb binding in vitro. Moreover, a Thr-186-dependent binding could be detected between P-TEFb and the in vitro transcribed 7SK by T7 RNA polymerase (Fig. 4A). This binding was as efficient as the one involving the native HeLa 7SK (data not shown), ruling out a significant role of any potential 7SK modification in the binding. Specific Reconstituted Intera

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