An evolutionary conserved Hexim1 peptide binds to the Cdk9 catalytic site to inhibit P-TEFb

Abstract

The positive transcription elongation factor (P-TEFb) is required for the transcription of most genes by RNA polymerase II. Hexim proteins associated with 7SK RNA bind to P-TEFb and reversibly inhibit its activity. P-TEFb comprises the Cdk9 cyclin-dependent kinase and a cyclin T. Hexim proteins have been shown to bind the cyclin T subunit of P-TEFb. How this binding leads to inhibition of the kinase activity of Cdk9 has remained elusive, however. Using a photoreactive amino acid incorporated into proteins, we show that in live cells, cell extracts, and in vitro reconstituted complexes, Hexim1 cross-links and thus contacts Cdk9. Notably, replacement of a phenylalanine, F208, belonging to an evolutionary conserved Hexim1 peptide (²⁰²PYNTTQFLM²¹⁰) known as the "PYNT" sequence, cross-links a peptide within the activation segment that controls access to the Cdk9 catalytic cleft. Reciprocally, Hexim1 is cross-linked by a photoreactive amino acid replacing Cdk9 W193, a tryptophan within this activation segment. These findings provide evidence of a direct interaction between Cdk9 and its inhibitor, Hexim1. Based on similarities with Cdk2 3D structure, the Cdk9 peptide cross-linked by Hexim1 corresponds to the substrate binding-site. Accordingly, the Hexim1 PYNT sequence is proposed to interfere with substrate binding to Cdk9 and thereby to inhibit its kinase activity.

Keywords: benzoyl phenylalanine; cyclin-dependent kinase inhibition; protein–protein cross-linking; regulatory noncoding RNA; transcription factor regulation.

Fig. 1.

Coimmunoprecipitation of P-TEFb with Hexim1.Bpa. (A) Conserved functional domains in human Hexim1. The BR (blue) interacts with 7SK RNA, the AR (red) can interact with the BR, and the evolutionary conserved PYNT sequence (yellow) is located between the AR and BR regions. The coiled-coil dimerization domain (green) overlaps the cyclin T-binding domain. (B) HEK 293 cells were cotransfected with suppressor tRNA, Bpa synthetase, and a Hexim1 cDNA with a TAG stop codon replacing different amino acid codons (the corresponding amino acid position numbers are noted at the top). N-terminal Flag-tagged Hexim1 was immunoprecipitated from extracts of cells treated with (+) or without (−) 2 mM Bpa. Western blots were probed with antibodies directed against cyclin T1 (CycT1), Cdk9, or the N terminus of Hexim1 (HEX1) that detected both truncated (Tr) and FL Hexim1 proteins. Both Cdk9 (p42) and Cdk9 (p55) isoforms (39) are detected (even lanes).

Fig. 2.

Immunoprecipitation of Cdk9 × Hexim1.Bpa cross-linked species. (A and B) Flag immunoprecipitation of N-terminal Flag-tagged Hexim1 from UV irradiated (+) or not (−) cell extracts. (C) Cells expressing various Hexim.Bpa proteins were UV-irradiated alive (+) under ice-cold PBS. Flag immunoprecipitation was performed from cell extracts. (D) Cells were transfected with myc-tagged Cdk9, followed by myc immunoprecipitation of C-terminal myc-tagged Cdk9 from UV irradiated (+) or not (−) cell extracts. In A, C, and D, HEK 293-transfected cells were exposed to Bpa. In B, transfected cells were exposed to Azp. Stronger exposures of the cross-linked species are shown in the upper parts of A and C. Western blots were probed with antibodies directed against Cdk9 or the N terminus of Hexim1 (HEX1).

Fig. 3.

Mass spectrometry analysis of cross-linked in vitro reconstituted P-TEFb.Hexim1 F208Bpa.7SK complexes. (A) In vitro-reconstituted P-TEFb.Hexim1 208Bpa.Larp7.7SK complexes, both UV-irradiated (lane 2) and not (lane 1). Polyacrylamide gels were stained by Coomassie blue. (B) Histograms of MS/MS-peak XIC scores obtained from double-digested (trypsin and GluC V8 endopeptidase) gel bands. (C) Histograms of the top-seven validated PSMs for Hexim1 from band 1H (blue) or band 2X (red). (D) Histograms of the top-seven validated PSMs for Cdk9 peptides from band 1H (blue) or band 2X (red). Sequences of different charges or modification state but the same mass were averaged together over three LC-MS/MS runs. Lists of validated peptides are provided in Datasets S1–S3.

Fig. S1.

(A) In vitro-reconstituted P-TEFb.Hexim1 F208Bpa.Larp7.7SK complexes were UV-irradiated or not. Polyacrylamide gels stained by Coomassie blue. (B) Histograms of the emPAI for Hexim1and Cdk9 in bands X, L, H, and C digested by trypsin. Note the decrease in intensity of a 40-kDa band corresponding to Cdk9 (band C). (C) Histograms of the top-seven validated tryptic PSMs for Hexim1 from band 5C (blue) or band 6X (red). (D) Histograms of the top-eight validated tryptic PSMs for Cdk9 from band 5C (blue) or band 6X (red).

Fig. 4.

Localization of a Cdk9 peptide that cross-links Hexim1 F208Bpa on the P-TEFb 3D structure. (A) Surface representation of the Cdk9.cyclin T1 complex [Protein Data Bank (PDB) IDB code 3BLQ], where CDK9 is shown in cyan and cyclin T1 is in gray. Three views corresponding to 90° rotations are shown. (B) Cdk9 catalytic site peptide backbone corresponding to the activation segment (PDB ID code 3BLQ). (C) Cdk2 catalytic site peptide backbone corresponding to the activation segment (PDB ID code 1GMZ). (D) Cdk2 bound to p27^Kip (in pink) (PDB ID code 1JSU). (E) Cdk2 bound to the model peptide substrate HHASPRK (in green, except for the serine phosphate acceptor, which is in gray with its phosphorylable oxygen atom in red) (PDB ID code 1QMZ). The Cdk9 ¹⁸⁹VVTLWYRPPELLLGER²⁰⁴ peptide that cross-links Hexim1 F208Bpa is stained in yellow, except for W193, which is magenta. Color codes for Cdk2 sequences correspond to those in the homologous Cdk9 sequences. ATP (red spheres) and its associated Mg²⁺ ion (black sphere) mark the catalytic site. Phosphorylated T186, in the T-loop, is in orange, and Y287 is in deep blue.

Fig. 5.

Schemes for two Hexim1 functional conformations. (A) The BR (blue) interacts with the AR (red). (B) Binding of 7SK RNA (red) to the BR releases its association with the AR, thereby unmasking Cdk9- and cyclin T1-binding sites. The cyclin T-binding domain overlaps a Cdk9-binding site, as well as the coiled-coil dimerization domain (green).

Fig. S2.

Flag-Hexim1 mutants are expressed in HEK293 cells. (A) Flag immunoprecipitates were probed by Western blot with anti-Hexim1 or anti-Cdk9 antibodies. (B) Flag immunoprecipitates were incubated with a CTD4 peptide substrate in the presence of radioactive ATP as described previously (3). Incorporation of radioactive phosphate was followed as a function of reaction time. The addition of RNase to the beads degraded 7SK and released active P-TEFb in the reaction mixture.