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

Department of Molecular Virology and Microbiology, Baylor College of Medicine, Houston, Texas 77030, USA.
Journal of Cellular Physiology (Impact Factor: 3.84). 02/2012; 227(2):609-17. DOI: 10.1002/jcp.22760
Source: PubMed


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 Ca(2+)/calmodulin-dependent kinase 1D (CaMK1D). Using small molecules inhibitors in HeLa cells and primary CD4(+) T lymphocytes, we found that the Ca(2+) signaling pathway is required for Cdk9 T-loop phosphorylation. Inhibition of Ca(2+) signaling led to dephosphorylation of Thr186 on Cdk9. In reporter plasmid assays, inhibition of the Ca(2+) 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 Ca(2+) pathway and reduction of Cdk9 T-loop phosphorylation inhibits transcription units that have a rigorous requirement for P-TEFb function.

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Available from: Rajesh Ramakrishnan,
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    • "Thus, CDK7/cyclin H which was previously identified as CDK-activating kinase (CAK) for the CDKs involved in the cell cycle such as CDK1, 2, and 4 also functions as CAK for CDKs involved in the regulation of transcription that include CDK8, 9, 12, and 13 (reviewed in [61]). Global analysis of kinases that may phosphorylate CDK9 T-loop using siRNA identified Ca(2+)/calmodulin-dependent kinase 1D (CaMK1D) knock down by siRNA decreased Thr-186 phosphorylation [62]. Accordingly, small molecule inhibition of Ca(2+) signaling pathway decreased Thr-186 phosphorylation and inhibited Tat-induced HIV-1 transcription but not Tax-mediated HTLV-1 transcription [62]. "
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    • "Interestingly, activation of resting CD4+ T cells rapidly and dramatically induced Cdk9 T-loop phosphorylation in the absence of new protein synthesis [67], while monocyte differentiation also appeared to increase levels of T-loop phosphorylation [68]. This implies that in resting cells and monocytes, Cdk9 T-loop phosphorylation is repressed either by the action of phosphatases, a yet unidentified repressor, or low concentrations of other co-factors [69]. For example, Cdk9 T-loop phosphorylation appears to be dependent on Ca2+signaling, with calcium/calmodulin-dependent kinase 1D (CAMK1D) acting upstream of the pathway [69]. "
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    • "However, as a previous study indicated in PBLs (peripheral blood lymphocytes) as opposed to resting CD4+T cells examined in this study, it is possible that under certain conditions, CDK7 protein levels are up-regulated [40]. We had reported that Ca2+ signalling is required for CDK9 T-loop phosphorylation [41] and it is possible that a Ca2+ influx following T cell activation might modulate PPM1A activity. This implies that in resting CD4+T cells, an equilibrium exists between the T-loop phosphorylating mechanism (possibly an upstream activating kinase) and phosphatase activity to meet the low metabolic requirements of quiescent cells. "
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