An evolutionary conserved Hexim1 peptide binds to the Cdk9 catalytic site to inhibit P-TEFb Lydia Kobbia, Emmanuelle Demey-Thomasb, Floriane Brayea, Florence Prouxa, Olga Kolesnikovac, Joelle Vinhb, Arnaud Poterszmanc, and Olivier Bensaudea,1 aEcole Normale Supérieure, Institut de Biologie de l’Ecole Normale Supérieure (IBENS), CNRS UMR 8197, INSERM U-1024, Université de Recherche Paris Sciences et Lettres, F-75230 Paris Cedex 05, France; bSpectrométrie de Masse Biologique et Protéomique, Ecole Supérieure de Physique et Chimie Industrielle de la ville de Paris (ESPCI), CNRS Unité de Services et de Recherche (USR) 3149, Université de Recherche Paris Sciences et Lettres, F-75231 Paris Cedex 05, France; and cDepartment of Integrated Structural Biology, Institut de Génétique et de Biologie Moléculaire et Cellulaire, INSERM U964, UMR 7104, CNRS/ Strasbourg University, 67404 Illkirch, France Edited by John T. Lis, Cornell University, Ithaca, NY, and approved September 29, 2016 (received for review July 26, 2016) The positive transcription elongation factor (P-TEFb) is required for the in the C-terminal part of Hexim1, overlapping a coiled-coil di- transcription of most genes by RNA polymerase II. Hexim proteins merizationdomain(24,25)(Fig.1A, green). Furthermore, an associated with 7SK RNA bind to P-TEFb and reversibly inhibit its ac- evolutionary conserved motif in the center of the protein, the tivity. P-TEFb comprises the Cdk9 cyclin-dependent kinase and a cyclin “PYNT” sequence, is critical for P-TEFb binding and inhibition (20, T. Hexim proteins have been showntobindthecyclinTsubunitof 21,26).Thismotif,distinctfrom the cyclin T-binding domain, is P-TEFb. How this binding leads to inhibition of the kinase activity of located between a basic region (BR) involved in 7SK RNA binding Cdk9 has remained elusive, however. Using a photoreactive amino andanacidicregion(AR)(Fig.1A). acid incorporated into proteins, we show that in live cells, cell extracts, Despite these insights, how Hexim1 binding inhibits Cdk9 kinase and in vitro reconstituted complexes, Hexim1 cross-links and thus con- activity remains unclear. Hypotheses might be formulated based on tacts Cdk9. Notably, replacement of a phenylalanine, F208, belonging our knowledge of other Cdk inhibitors. Hexim1 might compete with to an evolutionary conserved Hexim1 peptide (202PYNTTQFLM210) ATP to prevent it from interacting with the kinase active site like the known as the “PYNT” sequence, cross-links a peptide within the acti- p27Kip1 protein inhibitor of Cdk2/cyclin A (27). Hexim1 might in- vation segment that controls access to the Cdk9 catalytic cleft. Recipro- duce conformational changes weakening cyclin binding to the kinase cally, Hexim1 is cross-linked by a photoreactive amino acid replacing subunit, distorting the ATP binding site and/or misaligning catalytic Cdk9 W193, a tryptophan within this activation segment. These find- residues like the Ink4 inhibitors of Cdk4/6/cyclin D (28, 29). ings provide evidence of a direct interaction between Cdk9 and its in- To characterize the mechanism underlying the inhibition of hibitor, Hexim1. Based on similarities with Cdk2 3D structure, the Cdk9 P-TEFb activity by Hexim1, we investigated possible direct inter- peptide cross-linked by Hexim1 corresponds to the substrate binding- actions with Cdk9. Protein–protein contacts were probed by co- site. Accordingly, the Hexim1 PYNT sequence is proposed to interfere valent protein–protein coupling using a photoreactive amino acid with substrate binding to Cdk9 and thereby to inhibit its kinase activity. incorporated at defined positions into protein partners (30–32). When photoreactive amino acids are used, proteins in close contact cyclin-dependent kinase inhibition | transcription factor regulation | can be covalently photocross-linked (33). In the present study, we regulatory noncoding RNA | benzoyl phenylalanine | protein–protein demonstrate that the same Hexim1 residues cross-link Cdk9 in live cross-linking cells, in cell extracts, and in in vitro reconstituted complexes. Cross- linked peptides generated by proteolytic digestion can be charac- he positive transcription elongationfactor(P-TEFb)isrequired terized by mass spectrometry (34–36). Hexim1 cross-links a Cdk9 Tfor the transcription of most genes by RNA polymerase II (1, 2). peptide overlapping the activation segment that controls access to In particular, it antagonizes the negative elongation factor (NELF) the catalytic cleft (37). These findings support the model that and DRB sensitivity-inducing factor (DSIF), which promote tran- Hexim1 inhibits P-TEFb activity through interference with the BIOCHEMISTRY scriptional pausing shortly after the initiation of transcription. P-TEFb substrate-binding surface in the catalytic site of Cdk9. comprises a kinase subunit, Cdk9, and a cyclin T. The activity of P-TEFb is regulated. In metazoan cells, 7SK RNA and Hexim pro- Significance teins are dynamically associated with an inactive form of P-TEFb (3–6). 7SK RNA is a noncoding RNA that stably associates Larp7 and – The positive transcription elongation factor (P-TEFb) is required for Mepce proteins to form a core 7SK snRNP (7 9). Although the total transcription of most genes by RNA polymerase II. Hexim proteins amount of 7SK RNA does not vary in live cells, the level of 7SK – associated to 7SK, a noncoding RNA, bind to P-TEFb and reversibly RNA available for Hexim binding is modulated (10 12). As a con- inhibit its activity. This transcription factor comprises the kinase sequence, the overall P-TEFb activity increases on inhibition of Cdk9 and a cyclin T. Using a photoreactive amino acid incorporated transcription (3, 4). Thus, in addition to other more specific functions – into proteins, we provide the first evidence of a direct interaction (13 15), 7SK RNA serves as a sensor of transcriptional activity between Cdk9 and Hexim1, in live cells, in cell extracts and in through a feedback loop, fine-tuning P-TEFb activity (10). Un- in vitro assembled complexes. An evolutionary conserved Hexim1 derstanding how this noncoding RNA mechanistically regulates the peptide, the “PYNT” sequence, cross-links to the Cdk9 activation activity of P-TEFb, a central transcription factor, remains challenging. segment that controls access to the catalytic cleft. Interference with Hexim proteins have been conserved throughout evolution from binding of substrates accounts for kinase inhibition. mammals to nematodes (16–19). Most mammals have two distinct genes coding for the cognate Hexim1 and Hexim2 proteins. Both Author contributions: J.V., A.P., and O.B. designed research; L.K., E.D.-T., F.B., and F.P. performed proteins share similar properties and functions, but most previous research; O.K. contributed new reagents/analytic tools; L.K., E.D.-T., F.B., and F.P. analyzed data; studies have been performed with human Hexim1. The binding and O.B. wrote the paper. of 7SK snRNP to a Hexim1 dimer creates an inactive P-TEFb The authors declare no conflict of interest. complex comprising two P-TEFb modules (19–21). Studies using This article is a PNAS Direct Submission. yeast two-hybrid analysis, GST pull-downs, analytical gel filtration, 1To whom correspondence should be addressed. Email: [email protected]. and isothermal calorimetry indicate that Hexim1 interacts directly This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. with cyclin T (5, 22, 23). The cyclin T-binding domain is located 1073/pnas.1612331113/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1612331113 PNAS | November 8, 2016 | vol. 113 | no. 45 | 12721–12726 Downloaded by guest on September 24, 2021 weak suppression resulting in synthesis of some full-length Flag- A PYNT BR AR Coiled Coil HEXIM1 protein. 211 211 249 284 313 319 348 359 200 162 150 Position-dependent cross-links in live cells between Cdk9 and Hexim1. To – Dimerization generate protein protein cross-links, transfected cell lysates were 7SK Cyclin T irradiated and immunoprecipitated with anti-Flag beads. Immuno- precipitates were probed by Western blot analysis. After irradiation, B high molecular weight bands (>130 kDa) were detected with a F Y F Y F F F Y Y A Upper Flag HEX1 Hexim1 antibody (Fig. 2 , ). The most significant cross-links 194 203 208 225 241 262 267 271 274 are observed upon Bpa replacement of Hexim1 Y167 or W172 or Bpa - + - + - + - + - + - + - + - + - + F262 or F267 or Y274. Two types of profiles are distinguished. - 70 Replacement of Y167 and W172 leads to a smear between 130 and FL- 170 kDa apparent molecular weight, whereas F262, F267, and Y274 - 55 > HEX1 Tr- replacement results mainly in bands 170 kDa. { - 40 Anti-Cdk9 antibodies detect UV generated cross-linked spe- 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 Mw cies between 130 and 170 kDa when Hexim1 residues between W164, Y167, and F208 were replaced with Bpa (Fig. 2A, Lower). CycT1 - 100 p55- - 55 CDK9 A Bpa p42- - 40 Flag HEX1 W Y W F F Y F Y F F F Y Y 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 Mw WT 164 167 172 178 194 203 208 225 241 262 267 271 274 UV - + - + - + - + - + - + - + - + - + - + - + - + - + - + Fig. 1. Coimmunoprecipitation of P-TEFb with Hexim1.Bpa. (A) Conserved - 170 functional domains in human Hexim1. The BR (blue) interacts with 7SK RNA, - 130 the AR (red) can interact with the BR, and the evolutionary conserved PYNT - 170 sequence (yellow) is located between the AR and BR regions. The coiled-coil - 130 dimerization domain (green) overlaps the cyclin T-binding domain. (B) HEK HEX1 - 100 293 cells were cotransfected with suppressor tRNA, Bpa synthetase, and a FL- - 70 Hexim1 cDNA with a TAG stop codon replacing different amino acid codons Tr-{ - 55 (the corresponding amino acid position numbers are noted at the top). N-terminal Flag-tagged Hexim1 was immunoprecipitated from extracts of W Y W F F Y F Y F F F Y Y + − - WT 164 167 172 178 194 203 208 225 241 262 267 271 274 cells treated with ( ) or without ( ) 2 mM Bpa.