title: Phosphorylation of HEXIM1 at Tyr271 and Tyr274 Promotes Release of P-TEFb from the 7SK snRNP Complex and Enhances Proviral HIV Gene Expression.
abstract: Efficient HIV transcription requires P-TEFb, an essential co-factor for Tat. In actively replicating cells, P-TEFb is incorporated into the 7SK snRNP complex together with the repressor protein HEXIM1. Using an affinity purification-tandem mass spectrometry approach to identify modification sites on HEXIM1 that regulate the sequestration of P-TEFb by 7SK snRNP, we found that HEXIM1 can be phosphorylated on adjacent residues in a region immediately upstream of the coiled-coil dimerization domain (Ser268, Thr270, Tyr271, and Tyr274). Phosphomimetic mutations of Tyr271 and Tyr274 disrupted the assembly of P-TEFb and HEXIM1 into the 7SK snRNP complex. Although Y271E/Y274E did not adversely affect the nuclear localization pattern of HEXIM1, it induced the redistribution of the CDK9 subunit of P-TEFb into the cytoplasm. By contrast, the Y271F/Y274F HEXIM1 mutant assembled normally with P-TEFb within the 7SK snRNP complex but severely reduced proviral gene expression in T cells in response to activation signals and caused a severe growth defect of Jurkat T cells. Thus, Y271F/Y274F, which cannot be phosphorylated on these residues, appears to block the exchange of active P-TEFb from the 7SK complex, thereby limiting the level of P-TEFb below the threshold required to support transcription elongation of the HIV provirus and cellular genes.
authors.affiliation: Department of Molecular Biology and Microbiology, Case Western Reserve University School of Medicine, Cleveland, Ohio, USA.
authors.forename: Uri R
authors.initials: UR
authors.lastname: Mbonye
authors.affiliation: Center for Proteomics and Bioinformatics, Case Western Reserve University School of Medicine, Cleveland, Ohio, USA.
authors.forename: Benlian
authors.initials: B
authors.lastname: Wang
authors.affiliation: Center for Proteomics and Bioinformatics, Case Western Reserve University School of Medicine, Cleveland, Ohio, USA.
authors.forename: Giridharan
authors.initials: G
authors.lastname: Gokulrangan
authors.affiliation: Center for Proteomics and Bioinformatics, Case Western Reserve University School of Medicine, Cleveland, Ohio, USA.
authors.forename: Mark R
authors.initials: MR
authors.lastname: Chance
authors.affiliation: Department of Molecular Biology and Microbiology, Case Western Reserve University School of Medicine, Cleveland, Ohio, USA.
authors.forename: Jonathan
authors.initials: J
authors.lastname: Karn
mesh_terms: D000595:Amino Acid Sequence; D015153:Blotting, Western; D002478:Cells, Cultured; D002853:Chromatography, Liquid; D015967:Gene Expression Regulation, Viral; D057809:HEK293 Cells; D015658:HIV Infections / Q000235:genetics / Q000378:metabolism* / Q000821:virology; D015497:HIV-1 / Q000502:physiology*; D006801:Humans; D047468:Immunoprecipitation; D019169:Jurkat Cells; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D010766:Phosphorylation; D042925:Positive Transcriptional Elongation Factor B / Q000378:metabolism*; D011485:Protein Binding; D011499:Protein Processing, Post-Translational; D040901:Proteomics / Q000379:methods; D011533:Proviruses / Q000378:metabolism*; D012342:RNA, Small Nuclear / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D017411:Ribonucleoproteins, Small Nuclear / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D053719:Tandem Mass Spectrometry; D014157:Transcription Factors; D014764:Viral Proteins / Q000378:metabolism; D014779:Virus Replication
chemical_list: C476123:HEXIM1 protein, human; D012342:RNA, Small Nuclear; D016601:RNA-Binding Proteins; D017411:Ribonucleoproteins, Small Nuclear; D014157:Transcription Factors; D014764:Viral Proteins; D042925:Positive Transcriptional Elongation Factor B
keywords: 7SK RNP complex; HEXIM1 phosphorylation; HIV Tat; Microbiology; P-TEFb