The multi-tasking P-TEFb complex

Abstract

P-TEFb (CycT1:Cdk9), the metazoan RNA polymerase II Ser2 C-terminal domain (CTD) kinase, regulates transcription elongation at many genes and integrates mRNA synthesis with histone modification, pre-mRNA processing, and mRNA export. Recruitment of P-TEFb to target genes requires deubiquitination of H2Bub, phosphorylation of H3S10, and the bromodomain protein, Brd4. Brd4 activates growth-related genes in the G1 phase of the cell cycle and can also tether P-TEFb to mitotic chromosomes, possibly to mark sites of active transcription throughout cell division. P-TEFb co-operates with c-Myc during transactivation and cell transformation, and also requires SKIP (cSki-interacting protein), an mRNA elongation and splicing factor. Some functions of the P-TEFb/ Ser2P CTD are executed by the Spt6 transcription elongation factor, which binds directly to the phosphorylated CTD and recruits the Iws1 ('interacts with Spt6') protein. Iws1, in turn, interacts with the REF1/Aly nuclear export adaptor and stimulates the kinetics of mRNA export. Given the prominent role of Spt6 in regulating chromatin structure, the CTD-bound Spt6:Iws1 complex may also control histone modifications during elongation. Following transcription, P-TEFb accompanies the mature mRNA to the cytoplasm to promote translation elongation.

Chemoproteomics-based kinome profiling and target deconvolution of clinical multi-kinase inhibitors in primary chronic lymphocytic leukemia cells

“…The p-TEFb complex is thought to be comprised of CDK9 as the catalytic subunit and one of three cyclins T1, T2 or K, as the regulatory subunit. 31,42 Of note, we detected two additional proteins with inhibition profiles closely matching those of CDK9 and cyclin T1, the Table S1) and normal B cells. Kinases and associated proteins were isolated from CLL cell samples and from a pool of B cells from six healthy donors, and were subjected to quantitative LC-MS/MS profiling.…”

“…For both flavopiridol and BMS-387032 the most potently inhibited kinase is CDK9, with IC 50 values of 30 nM for flavopiridol and 50 nM for BMS-387032 consistent with the slightly more pronounced effect of flavopiridol on cell viability. CDK9 is known to be part of the p-TEFb complex, which has a key role in RNA polymerase II transcriptional elongation, and in human cells consists of the CDK9 catalytic subunit and one of three cyclins, T1, T2 or K. 31 The proteomics approach used in this study can identify intact protein complexes because the conditions used for lysate preparation and drug binding largely preserve intact protein complexes. 9 Consequently, the binding of the drugs to the p-TEFb complex was detected by matching dose-response binding curves for the catalytic subunit CDK9 and the regulatory subunit cyclin T1 (CCNT1).…”

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

“…[56][57][58] A major mechanism of P-TEFb regulation is through association with a large inhibitory complex containing 7SK-RNA, MEPCE, LARP7 and HEXIM1 (7SK/HEXIM/P-TEFb complex) that inhibits recruitment of P-TEFb to promoters. 8,15,59 Several additional P-TEFb containing complexes have also been described including a super elongation complex (SEC), 58 which contains P-TEFb, the elongation factors ELL1 to ELL3 and several other chromosomal translocation partners of the mixed lineage leukemia (MLL) gene, including AFF1, AFF4, ENL and AF9. SEC localizes at Hsp70 gene upon stress, and this complex is also involved in HIV proviral transcription.…”

“…The transition of paused RNA PolII into productive elongation involves an exchange of associated factors that favors polymerase processivity and mRNA maturation, orchestrated in part by the phosphorylation of specific residues in the RNA Pol II C-terminal domain (CTD). This transition involves multiple steps and is regulated both positively and negatively by a large number of trans-acting factors, [6][7][8] providing an opportunity for gene-specific regulation.…”

“…Probably the best-described mechanism for gene-specific and signaldependent regulation of pausing and elongation is through the recruitment of P-TEFb by interaction with transcriptional activators such as Myc, NFκB and p53, [23][24][25] or specific chromatin-binding factors such as Brd4. 8 We investigated whether P-TEFb binding at Sp3-repressed target genes was affected by depletion of Sp3. We found that loss of Sp3-dependent repression did not increase P-TEFb binding at p21 CIP1 or sod2 or other promoters examined 11 (Fig.…”