title: Phosphorylation of CDK9 at Ser175 enhances HIV transcription and is a marker of activated P-TEFb in CD4(+) T lymphocytes.
abstract: The HIV transactivator protein, Tat, enhances HIV transcription by recruiting P-TEFb from the inactive 7SK snRNP complex and directing it to proviral elongation complexes. To test the hypothesis that T-cell receptor (TCR) signaling induces critical post-translational modifications leading to enhanced interactions between P-TEFb and Tat, we employed affinity purification-tandem mass spectrometry to analyze P-TEFb. TCR or phorbal ester (PMA) signaling strongly induced phosphorylation of the CDK9 kinase at Ser175. Molecular modeling studies based on the Tat/P-TEFb X-ray structure suggested that pSer175 strengthens the intermolecular interactions between CDK9 and Tat. Mutations in Ser175 confirm that this residue could mediate critical interactions with Tat and with the bromodomain protein BRD4. The S175A mutation reduced CDK9 interactions with Tat by an average of 1.7-fold, but also completely blocked CDK9 association with BRD4. The phosphomimetic S175D mutation modestly enhanced Tat association with CDK9 while causing a 2-fold disruption in BRD4 association with CDK9. Since BRD4 is unable to compete for binding to CDK9 carrying S175A, expression of CDK9 carrying the S175A mutation in latently infected cells resulted in a robust Tat-dependent reactivation of the provirus. Similarly, the stable knockdown of BRD4 led to a strong enhancement of proviral expression. Immunoprecipitation experiments show that CDK9 phosphorylated at Ser175 is excluded from the 7SK RNP complex. Immunofluorescence and flow cytometry studies carried out using a phospho-Ser175-specific antibody demonstrated that Ser175 phosphorylation occurs during TCR activation of primary resting memory CD4+ T cells together with upregulation of the Cyclin T1 regulatory subunit of P-TEFb, and Thr186 phosphorylation of CDK9. We conclude that the phosphorylation of CDK9 at Ser175 plays a critical role in altering the competitive binding of Tat and BRD4 to P-TEFb and provides an informative molecular marker for the identification of the transcriptionally active form of P-TEFb.
authors: Department of Molecular Biology and Microbiology, Case Western Reserve University School of Medicine, Cleveland, Ohio, USA.
authors: Uri R
authors: UR
authors: Mbonye
authors: Giridharan
authors: G
authors: Gokulrangan
authors: Manish
authors: M
authors: Datt
authors: Curtis
authors: C
authors: Dobrowolski
authors: Maxwell
authors: M
authors: Cooper
authors: Mark R
authors: MR
authors: Chance
authors: Jonathan
authors: J
authors: Karn
mesh_terms: D015415:Biomarkers / Q000378:metabolism; D015496:CD4-Positive T-Lymphocytes / Q000378:metabolism* / Q000821:virology; D018797:Cell Cycle Proteins; D056906:Cyclin T / Q000235:genetics / Q000378:metabolism; D042863:Cyclin-Dependent Kinase 9 / Q000235:genetics / Q000378:metabolism*; D005260:Female; D006678:HIV / Q000235:genetics / Q000378:metabolism*; D015658:HIV Infections / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008297:Male; D009154:Mutation; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation / Q000235:genetics; D042925:Positive Transcriptional Elongation Factor B / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding / Q000235:genetics; D011533:Proviruses / Q000235:genetics / Q000378:metabolism*; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism; D014158:Transcription, Genetic; D054322:tat Gene Products, Human Immunodeficiency Virus / Q000235:genetics / Q000378:metabolism; D000092468:Bromodomain Containing Proteins
chemical_list: D015415:Biomarkers; D018797:Cell Cycle Proteins; D056906:Cyclin T; D042863:Cyclin-Dependent Kinase 9; D009687:Nuclear Proteins; D042925:Positive Transcriptional Elongation Factor B; D014157:Transcription Factors; D054322:tat Gene Products, Human Immunodeficiency Virus; C441289:BRD4 protein, human; D000092468:Bromodomain Containing Proteins; C503684:CCNT1 protein, human; C467969:CDK9 protein, human
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