Characterization of Cdk9 T-loop phosphorylation in resting and activated CD4(+) T lymphocytes

Department of Molecular Virology and Microbiology, Baylor College of Medicine, Houston, Texas 77030, USA.
Journal of leukocyte biology (Impact Factor: 4.29). 09/2009; 86(6):1345-50. DOI: 10.1189/jlb.0509309
Source: PubMed


The cellular kinase complex P-TEFb is composed of Cdk9 and cyclin T, and it is required for expression of most protein-coding genes by RNAP II. Cdk9 has been shown recently to be activated in cis by autophosphorylation of Thr186 in its T-loop. Using a phosphospecific Cdk9 antibody, we examined the level of Cdk9 T-loop phosphorylation in resting and activated CD4(+) T lymphocytes. Cdk9 T-loop phosphorylation was found to be low-to-undetectable in resting CD4(+) T lymphocytes, and upon activation by distinct stimuli, there is a rapid (<1 h) increase in pCdk9 that does not require protein synthesis. The low level of Cdk9 T-loop phosphorylation was not to be a result of the absence of an associated regulatory cyclin partner. These observations suggest that autophosphorylation of the Cdk9 T-loop is repressed in resting CD4(+) T lymphocytes. The low level of T-loop phosphorylation in resting cells is also reflected in a low level of phosphorylation of Ser2 in the carboxyl terminal domain of RNAP II, suggesting that lack of Cdk9 T-loop autophosphorylation may limit RNAP II elongation in quiescent CD4(+) T lymphocytes.

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Available from: Rajesh Ramakrishnan, Oct 07, 2015
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    • "Of these factors, only NUCKS1, as a positively Tat-regulating factor, was expressed at a lower level in latently infected cells than in normal parent cells (Figure 6A and B). It has been reported that HIV-1 Tat regulating factors such as Cyclin T1 and CDK9 were regulated by T cell receptor activation, which were expressed at an extremely lower level and exhibited restricted activity in primary resting cells [52,53]. Although the expression profile of these factors from our latency model using quickly dividing cell lines was quite different from that of tightly regulated primary resting memory T cells, our data may suggest that a low expression level of NUCKS1 seems to be important for maintaining latent infection characterized by restricted HIV-1 gene expression in the cell line model. "
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    ABSTRACT: Background Human immunodeficiency virus-1 (HIV-1) Tat protein plays an essential role in HIV gene transcription from the HIV-1 long terminal repeat (LTR) and replication. Transcriptional activity of Tat is modulated by several host factors, but the mechanism responsible for Tat regulation by host factors is not understood fully.ResultsUsing a yeast two-hybrid screening system, we identified Nuclear ubiquitous casein and cyclin-dependent kinase substrate 1 (NUCKS1) as a novel Tat-interacting partner. Here, we report its function as a positive regulator of Tat. In a coimmunoprecipitation assay, HIV-1 Tat interacted sufficiently with both endogenous and ectopically expressed NUCKS1. In a reporter assay, ectopic expression of NUCKS1 significantly increased Tat-mediated transcription of the HIV-1 LTR, whereas knockdown of NUCKS1 by small interfering RNA diminished Tat-mediated transcription of the HIV-1 LTR. We also investigated which mechanism contributes to NUCKS1-mediated Tat activation. In a chromatin immunoprecipitation assay (ChIP), knockdown of NUCKS1 interrupted the accumulation of Tat in the transactivation-responsive (TAR) region on the LTR, which then led to suppression of viral replication. However, NUCKS1 expression did not increase Tat nuclear localization and interaction with Cyclin T1. Interestingly, the NUCKS1 expression level was lower in latently HIV-1-infected cells than in uninfected parent cells. Besides, expression level of NUCKS1 was markedly induced, which then facilitated HIV-1 reactivation in latently infected cells.Conclusion Taken together, our data demonstrate clearly that NUCKS1 is a novel Tat coactivator that is required for Tat-mediated HIV-1 transcription and replication, and that it may contribute to HIV-1 reactivation in latently HIV-1 infected cells.
    Retrovirology 08/2014; 11(1):67. DOI:10.1186/PREACCEPT-1444760432121123
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    • "However, in resting cells the CDK9 kinase is inactive as a consequence of dephosphorylation of its regulatory activation loop (T-loop) at the highly conserved CDK residue Thr186 (Budhiraja et al., 2012a). Activation of T cells rapidly induces Thr186 phosphorylation of CDK9 concomitant with the elevation in hCycT1 expression (Mbonye et al., 2013; Ramakrishnan et al., 2009). Using an elegant chemical genetic approach Larochelle et al. (2012) have recently identified the CDK7 subunit of TFIIH to be the nuclear CDK9- activating kinase. "
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    ABSTRACT: Replication-competent latent HIV-1 proviruses that persist in the genomes of a very small subset of resting memory T cells in infected individuals under life-long antiretroviral therapy present a major barrier towards viral eradication. Multiple molecular mechanisms are required to repress the viral trans-activating factor Tat and disrupt the regulatory Tat feedback circuit leading to the establishment of the latent viral reservoir. In particular, latency is due to a combination of transcriptional silencing of proviruses via host epigenetic mechanisms and restrictions on the expression of P-TEFb, an essential co-factor for Tat. Induction of latent proviruses in the presence of antiretroviral therapy is expected to enable clearance of latently infected cells by viral cytopathic effects and host antiviral immune responses. An in-depth comprehensive understanding of the molecular control of HIV-1 transcription should inform the development of optimal combinatorial reactivation strategies that are intended to purge the latent viral reservoir.
    Virology 02/2014; 454-455(1). DOI:10.1016/j.virol.2014.02.008
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    • "In resting CD4+ T cells, Cyclin T1 and Cyclin T2 associate with Cdk9 even in the absence or at low levels of T-loop phosphorylation [67]. Cyclin T2 has two splice variants, T2a and T2b [94]. "
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    ABSTRACT: Productive transcription of the integrated HIV-1 provirus is restricted by cellular factors that inhibit RNA polymerase II elongation. The viral Tat protein overcomes this by recruiting a general elongation factor, P-TEFb, to the TAR RNA element that forms at the 5' end of nascent viral transcripts. P-TEFb exists in multiple complexes in cells, and its core consists of a kinase, Cdk9, and a regulatory subunit, either Cyclin T1 or Cyclin T2. Tat binds directly to Cyclin T1 and thereby targets the Cyclin T1/P-TEFb complex that phosphorylates the CTD of RNA polymerase II and the negative factors that inhibit elongation, resulting in efficient transcriptional elongation. P-TEFb is tightly regulated in cells infected by HIV-1-CD4+ T lymphocytes and monocytes/macrophages. A number of mechanisms have been identified that inhibit P-TEFb in resting CD4+ T lymphocytes and monocytes, including miRNAs that repress Cyclin T1 protein expression and dephosphorylation of residue Thr186 in the Cdk9 T-loop. These repressive mechanisms are overcome upon T cell activation and macrophage differentiation when the permissivity for HIV-1 replication is greatly increased. This review will summarize what is currently known about mechanisms that regulate P-TEFb and how this regulation impacts HIV-1 replication and latency.
    Biology 12/2012; 1(1):94-115. DOI:10.3390/biology1010094
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Questions & Answers about this publication

  • Rajesh Ramakrishnan added an answer in Protein Purification:
    Suggestions for immunoprecipitation kits/protocols?
    I'm somewhat new to IP and am looking for a good kit (or bead type) to isolate my proteins (large family of related proteins; we have an antibody specific for a conserved region).
    My antibody is a polyclonal IgG from rabbit. My proteins of interest range from 20kD-200kD in size (I don't know if that matters). The antibody has a decent number of primary amines.
    I'm looking to elute my proteins without the antibody, and would prefer not to denature my proteins.
    I don't have any experience with commercial IP kits, so any suggestions would be appreciated.