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: 4.99). 09/2009; 86(6):1345-50. DOI: 10.1189/jlb.0509309
Source: PubMed

ABSTRACT 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, Aug 12, 2015
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    • "A recent study suggests that cyclin T1 acetylation also triggers dissociation of HEXIM1 and 7SK RNA from the inactive 7SK snRNP complex and activates the transcriptional activity of P-TEFb (Cho et al. 2009). In contrast to Jurkat T-cells both primary resting central memory T-cells (Ramakrishnan et al. 2009) and primary monocytes (Sung and Rice 2009) show highly restricted levels of CycT1. Activation of P-TEFb in these cells therefore requires multiple steps involving both the initial assembly of the 7SK RNP complex and its relocalization to nuclear speckles where it becomes accessible to Tat and the rest of the transcription machinery. "
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    • "In addition, in resting T cells or undifferentiated monocytes, phosphorylation of the CDK9 T loop is barely detectable, while it is rapidly induced upon T cell activation and monocyte differentiation (Ramakrishnan et al., 2009). Besides autophosphorylation , the precise mechanism responsible for T-loop phosphorylation in CDK9 remains unknown, but calcium/calmodulindependent kinase 1D and Csk1 are candidate kinases in HeLa cells and in fission yeast, respectively (Pei et al., 2006; Ramakrishnan and Rice, 2011). "
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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.