Phosphorylation of HEXIM1 at Tyr271 and Tyr274 Promotes Release of P-TEFb from the 7SK snRNP Complex and Enhances Proviral HIV Gene Expression

Proteomics. 2015 Jun;15(12):2078-86. doi: 10.1002/pmic.201500038. Epub 2015 May 15.

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.

Keywords: 7SK RNP complex; HEXIM1 phosphorylation; HIV Tat; Microbiology; P-TEFb.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Blotting, Western
  • Cells, Cultured
  • Chromatography, Liquid
  • Gene Expression Regulation, Viral
  • HEK293 Cells
  • HIV Infections / genetics
  • HIV Infections / metabolism*
  • HIV Infections / virology
  • HIV-1 / physiology*
  • Humans
  • Immunoprecipitation
  • Jurkat Cells
  • Molecular Sequence Data
  • Mutation / genetics
  • Phosphorylation
  • Positive Transcriptional Elongation Factor B / metabolism*
  • Protein Binding
  • Protein Processing, Post-Translational
  • Proteomics / methods
  • Proviruses / metabolism*
  • RNA, Small Nuclear / metabolism*
  • RNA-Binding Proteins / genetics
  • RNA-Binding Proteins / metabolism*
  • Ribonucleoproteins, Small Nuclear / metabolism*
  • Sequence Homology, Amino Acid
  • Tandem Mass Spectrometry
  • Viral Proteins / metabolism
  • Virus Replication

Substances

  • HEXIM1 protein, human
  • RNA, Small Nuclear
  • RNA-Binding Proteins
  • Ribonucleoproteins, Small Nuclear
  • Viral Proteins
  • Positive Transcriptional Elongation Factor B