Cdk9 T‐loop phosphorylation is regulated by the calcium signaling pathway

Abstract

Eukaryotic RNA polymerase II transcriptional elongation is a tightly regulated process and is dependent upon positive transcription elongation factor-b (P-TEFb). The core P-TEFb complex is composed of Cdk9 and Cyclin T and is essential for the expression of most protein coding genes. Cdk9 kinase function is dependent upon phosphorylation of Thr186 in its T-loop. In this study, we examined kinases and signaling pathways that influence Cdk9 T-loop phosphorylation. Using an RNAi screen in HeLa cells, we found that Cdk9 T-loop phosphorylation is regulated by Calcium/Calmodulin- dependent kinase 1D (CaMK1D). Using small molecules inhibitors in HeLa cells and primary CD4+ T lymphocytes, we found that the Ca2+ signaling pathway is required for Cdk9 T-loop phosphorylation. Inhibition of Ca2+ signaling led to dephosphorylation of Thr186 on Cdk9. In reporter plasmid assays, inhibition of the Ca2+ signaling pathway repressed the PCNA promoter and HIV-1 Tat transactivation of the HIV-1 LTR, but not HTLV-1 Tax transactivation of the HTLV-1 LTR, suggesting that perturbation of the Ca2+ pathway and reduction of Cdk9 T-loop phosphorylation inhibits transcription units that have a rigorous requirement for P-TEFb function.

Cyclin T1 and CDK9 T-Loop Phosphorylation Are Downregulated during Establishment of HIV-1 Latency in Primary Resting Memory CD4+T Cells

“…Upon the activation of these naïve cells, both Cyclin T1 and pCDK9 levels increase significantly. This is likely in response to extracellular cues such as Ca ϩ2 signaling, which, as we have shown previously, plays a part in CDK9 T-loop phosphorylation following the activation of resting CD4 ϩ T cells (45). Besides the increased availability of catalytically active P-TEFb, activation of CD4 ϩ T cells also increases the availability of other host cofactors, such as NF-B, thereby creating a more favorable environment for HIV-1 transcription (46).…”

“…The stability of total CDK9 protein is also dependent on T-loop phosphorylation, as we have previously shown that perturbation of T-loop phosphorylation affects total CDK9 levels (45). However, CDK9 T-loop phosphorylation does not affect Cyclin T1 protein stability.…”

Cyclin-dependent kinase 7 (CDK7)-mediated phosphorylation of the CDK9 activation loop promotes P-TEFb assembly with Tat and proviral HIV reactivation

“…CDK9 phosphorylation at the conserved T-loop residue Thr186 is inducible in quiescent CD4 + T lymphocytes, but is found to be constitutive in actively replicating T cell lines and expanding, fully activated, T cells (34)(35)(36)62,63). Consistent with molecular dynamics simulations, cell-based mutagenesis experiments demonstrated that the assembly of P-TEFb in T cells is critically dependent on the stabilization of the CDK9/CycT1 interface by phosphorylation of CDK9 at Thr186.…”

Role of Host Factors on the Regulation of Tat-Mediated HIV-1 Transcription

“…As mentioned previously, the CDK9 phosphorylation at Thr186 is required to sequester P-TEFb into 7SK snRNP [49, 50]. However, the same Thr186 phosphorylation is required during Tat-dependent transactivation [78, 79]. This led to the postulate that P-TEFb phosphorylated at Thr186, while inhibited by HEXIM1 on 7SK snRNP complex, is in a pre-activated state to facilitate a rapid transition to transcription elongation upon activation [49, 51, 65].…”