Reversible phosphorylation of cyclin T1 promotes assembly and stability of P-TEFb.

Fang Huang,Boris Matija Peterlin,Andrej Sali,Daniele C Cary,Ramachandran Rakesh,Trang TT Nguyen,Koh Fujinaga,Arthur Weiss,Hana Paculova,Ignacia Echeverria

doi:10.7554/elife.68473

Abstract

The positive transcription elongation factor b (P-TEFb) is a critical coactivator for transcription of most cellular and viral genes, including those of HIV. While P-TEFb is regulated by 7SK snRNA in proliferating cells, P-TEFb is absent due to diminished levels of CycT1 in quiescent and terminally differentiated cells, which has remained unexplored. In these cells, we found that CycT1 not bound to CDK9 is rapidly degraded. Moreover, productive CycT1:CDK9 interactions are increased by PKC-mediated phosphorylation of CycT1 in human cells. Conversely, dephosphorylation of CycT1 by PP1 reverses this process. Thus, PKC inhibitors or removal of PKC by chronic activation results in P-TEFb disassembly and CycT1 degradation. This finding not only recapitulates P-TEFb depletion in resting CD4+ T cells but also in anergic T cells. Importantly, our studies reveal mechanisms of P-TEFb inactivation underlying T cell quiescence, anergy, and exhaustion as well as proviral latency and terminally differentiated cells.

Highlights

In eukaryotic cells, coding gene expression starts with transcription of DNA to RNA by RNA polymerase II (RNAPII) in the nucleus
Interactions between mutant CycT1 proteins and the endogenous CDK9 protein were analyzed by co-immunoprecipitation (Figures 1B to 1D)
We conclude that two threonine residues (Thr[143] and Thr149) in CycT1 are critical for its binding to CDK9 to form positive transcription elongation factor b (P-TEFb), and that mutant CycT1 proteins that do not interact or poorly interact with CDK9 are rapidly degraded by the proteasome. 166 Phosphorylation of Thr[143] and Thr[149] in CycT1 contributes to its binding to 167 CDK9 Since threonine residues are potential phosphorylation sites, we examined whether phosphorylation of Thr[143] and/or Thr[149] in CycT1 contributes to P-TEFb assembly. 293T cells ectopically expressing CycT1 (Figure 2A) or CycT1(280) (Figure 2B) were treated with the potent protein phosphatase inhibitors okadaic acid for 1.5 h or calyculin A for 1 h prior to cell lysis

Summary

Introduction

In eukaryotic cells, coding gene expression starts with transcription of DNA to RNA by RNA polymerase II (RNAPII) in the nucleus. This process consists of initiation, promoter clearance, capping, elongation and termination, upon which the transcribed single strand RNA is cleaved and poly-adenylated before transport to the cytoplasm (Lis, 2019; Peterlin & Price, 2006; Proudfoot, 2016; Zhou, Li, & Price, 2012). Clearance of RNAP II from promoters requires the phosphorylation of its C-terminal domain (CTD) at position 5 (Ser5) in tandemly repeated heptapeptide (52 repeats of Tyr1-Ser2-Pro3-Thr4-Ser[549] Pro6-Ser7) by cyclin-dependent kinase 7 (CDK7) from the transcription factor-II H (TFIIH) (Chapman, Heidemann, Hintermair, & Eick, 2008). N-terminus of CycT1 contains two highly conserved cyclin boxes for CDK9 binding, followed by the Tat-TAR recognition motif (TRM), a coil-coiled motif, the histidine (His) rich motif that binds to the CTD and a

Methods

Results

Conclusion