Abstract
Positive transcription elongation factor b (P-TEFb) regulates eukaryotic gene expression at the level of elongation, and is itself controlled by the reversible association of 7SK RNA and an RNA-binding protein, HEXIM1 or HEXIM2. To further understand how P-TEFb is regulated, we analyzed the stoichiometry of all the known components of the large, inactive P-TEFb complex. Mutational analyses of a putative coiled coil region in the carboxyl-terminal portion of HEXIM1 revealed that the protein is a dimer in solution and remains a dimer after binding to 7SK. Although a HEXIM1 dimer contains two potential RNA binding motifs and ultimately recruits two P-TEFb molecules, it associates with only one molecule of RNA. The first 172 nucleotides of the 330-nucleotide 7SK are sufficient to bind HEXIM1 or HEXIM2, and then recruit and inhibit P-TEFb. Deletion of the first 121 amino acids of HEXIM1 allowed it to inhibit P-TEFb partially in the absence of 7SK RNA. Mutation of a conserved tyrosine (Tyr(271) in HEXIM1) to alanine or glutamate or mutation of a conserved phenylalanine (Phe(208)) to alanine, aspartate, or lysine, resulted in loss of inhibition of P-TEFb, but did not affect formation of the 7SK.HEXIM.P-TEFb complex. Analysis of T-loop phosphorylation in Cdk9 indicated that phosphorylation of Thr(186), but not Ser(175), was essential for kinase activity and for recruitment of P-TEFb to the 7SK.HEXIM complex. A model illustrates what is currently known about how HEXIM proteins, 7SK, and P-TEFb assemble to maintain an activated kinase in a readily available, but inactive form.
Highlights
Cyclin-dependent kinases (Cdks)1 are key regulators of a variety of cellular processes, such as cell cycle progression, transcription, and neuronal differentiation
We identified conserved residues Tyr271 and Phe208 as critical for the inhibition of Positive transcription elongation factor b (P-TEFb) kinase activity, but not the formation of the 7SK1⁄7HEXIM11⁄7P-TEFb complex
We demonstrated that phosphorylation of Thr186 in the T-loop of Cdk9 is required for the P-TEFb kinase activity, and for P-TEFb to associate with the 7SK1⁄7HEXIM1 complex
Summary
Cyclin-dependent kinases; EMSA, electrophoretic mobility shift assay; Ni-NTA, nickel-nitrilotriacetic acid; MS, mass spectrometry. When cells are treated with P-TEFb inhibitors, such as 5,6-dichloro-1--Dribofuranosylbenzimidazole, or other agents that block transcription elongation, the large form is converted into the small active form [21] This form of P-TEFb regulation is physiologically significant, because it has been shown that all signals that trigger cardiac hypertrophy converge at the critical step of activating P-TEFb through dissociation of 7SK and HEXIM. We identified residues critical for inhibition of the kinase activity of P-TEFb, and formation of the 7SK1⁄7HEXIM11⁄7P-TEFb complex Functional Interactions in the Large P-TEFb Complex mechanistic insight into how P-TEFb kinase activity is controlled by 7SK RNA and HEXIM proteins
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