The structure of P-TEFb (CDK9/cyclin T1), its complex with flavopiridol and regulation by phosphorylation

Abstract

The positive transcription elongation factor b (P-TEFb) (CDK9/cyclin T (CycT)) promotes mRNA transcriptional elongation through phosphorylation of elongation repressors and RNA polymerase II. To understand the regulation of a transcriptional CDK by its cognate cyclin, we have determined the structures of the CDK9/CycT1 and free cyclin T2. There are distinct differences between CDK9/ CycT1 and the cell cycle CDK CDK2/CycA manifested by a relative rotation of 261 of CycT1 with respect to the CDK, showing for the first time plasticity in CDK cyclin interactions. The CDK9/CycT1 interface is relatively sparse but retains some core CDK-cyclin interactions. The CycT1 C-terminal helix shows flexibility that may be important for the interaction of this region with HIV TAT and HEXIM. Flavopiridol, an anticancer drug in phase II clinical trials, binds to the ATP site of CDK9 inducing unanticipated structural changes that bury the inhibitor. CDK9 activity and recognition of regulatory proteins are governed by autophosphorylation. We show that CDK9/CycT1 autophosphorylates on Thr186 in the activation segment and three C-terminal phosphorylation sites. Autophosphorylation on all sites occurs in cis.

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

“…4A, yellow) that binds ATP (red) and the catalytic magnesium atom (black sphere). It overlaps with the activation segment (residues 167-198) that controls the accessibility of substrates to the catalytic cleft (40,42). Our attention was attracted to tryptophan W193 in Cdk9 (Fig.…”

Enforcing the pause: Transcription factor Sp3 limits productive elongation by RNA polymerase II

“…64 However, phosphorylation of CDK9 at this same residue, Thr186 and other residues is required for full enzymatic activity at promoters. [64][65][66][67][68][69] These studies suggest dynamic cycles of phosphorylation and dephosphorylation regulate P-TEFb activity to control the release of the RNA Pol II into productive elongation.…”

Orphan kinases turn eccentric

“…Three-dimensional structures are available for many CDK monomers (CDKs 2, 4, 5, 8 and 9) as well as cyclin/CDK dimers (cyclins A, B, C, H, T and p25/Pho80 in complex with their CDKs), [36][37][38][39][40][41][42] which provided a molecular understanding of how cyclins activate CDKs. 24,43,44 Like all kinases, cyclin-dependent kinases have a two-lobed structure.…”

“…[50][51][52] Via their C-terminal cyclin fold, "transcriptional" cyclins make additional interactions with the catalytic subunit, resulting in a more open conformation suitable to accommodate additional proteins. 41,42 The formation of these complexes usually stabilizes one of the partners, like the CDK subunit in the cyclin T1/CDK9 53 or the cyclin subunit in the cyclin C/CDK8 complex. 54 Thus, this class of cyclin/CDK pairs appears to be regulated by complex formation-induced stabilization of subunits.…”

“…In contrast to most of the CDKs, which require phosphorylation of a conserved threonine in the T-loop, 42,50,55 CDK8 is proposed to be solely activated by interaction with its cyclin. 39 Additional mechanisms of regulation of this subclass of CDKs include autophosphorylation (CDK9, 11) 41 and activation-dependent dimerization (CDK11). 56 Another class of CDKs appears to function close to the cell membrane.…”