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

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.

P-TEFb is degraded by Siah1/2 in quiescent cells

“…Also, we employed a mutant CycT1 protein (CycT14MUT (E137D, T143A, T149A and T155A), Figure 1B ) that does not interact with CDK9. This mutant CycT14MUT protein recapitulates the degradation of free CycT1, which occurs in resting, terminally differentiated, anergic and exhausted cells, in growing, proliferating cells, such as human embryonic kidney (HEK) 293T cell line ( 27 ).…”

“…Levels of P-TEFb are reduced greatly due to the absence of CycT1 in quiescent and terminally differentiated cells ( 23 , 29–30 ). Nevertheless, mRNA levels of CycT1 and CDK9 remain high in these cells, indicating that post-transcriptional steps are responsible ( 27 , 31 ). Consistent with previous studies, we also confirmed that CycT1 is stabilized by the proteasomal inhibitor bortezomib (100 nM, 24 h) in resting T cells to levels equivalent to those in activated T cells that were treated with PMA (5 nM) and PHA (15 ng/ml) for 24 h (Figure 1A , upper panels, panel 1, compare lanes 2 and 3 to lane 1, ∼14.1-fold and ∼18.2-fold increase) ( 27 ).…”

“…Recently, we demonstrated that PKCs phosphorylate CycT1 on two threonines (Thr143 and Thr149), which increases its binding to CDK9. Indeed, the stability of CycT1 is determined by its binding to CDK9 (P-TEFb assembly) ( 27 ). We sought to clarify the mechanism by which free CycT1 is degraded in cells.…”

“…In these cells, transcripts for CycT1 and CDK9 remain high ( 21 , 26 ), indicating that CycT1 protein levels are regulated post-transcriptionally. Recently, we demonstrated that CycT1 unbound to CDK9 (free CycT1) is rapidly degraded by the proteasome, which occurs in resting T cells as well as in unresponsive T cells, which include anergic and exhausted T cells ( 27 ). Whereas, P-TEFb assembly is promoted by the phosphorylation of CycT1 by PKC, its dephosphorylation by protein phosphatase 1 (PP1) causes its dissociation from CDK9 and subsequent degradation ( 27 ).…”

“…Recently, we demonstrated that CycT1 unbound to CDK9 (free CycT1) is rapidly degraded by the proteasome, which occurs in resting T cells as well as in unresponsive T cells, which include anergic and exhausted T cells ( 27 ). Whereas, P-TEFb assembly is promoted by the phosphorylation of CycT1 by PKC, its dephosphorylation by protein phosphatase 1 (PP1) causes its dissociation from CDK9 and subsequent degradation ( 27 ). To further clarify the mechanism of CycT1 degradation, we first mapped its degron.…”