Site-specific phosphorylation of the human immunodeficiency virus type-1 Rev protein accelerates formation of an efficient RNA-binding conformation.
Phosphorylation is important in the regulation of many cellular processes, yet the precise role of protein phosphorylation for many RNA-binding protein substrates remains obscure. In this report, we demonstrate that phosphorylation of a recombinant human immunodeficiency virus type-1 Rev protein promotes rapid formation of an efficient RNA-binding state. The apparent dissociation constant for ligand binding is enhanced 7-fold for the protein following phosphorylation; however, phosphate addition leads to a 1. 6-fold decrease in RNA ligand-protein complex stability. RNA ligand binding stimulates slow formation of an equally competent binding state for the unphosphorylated protein, indicating that the addition of phosphate or ligand binding promotes a similar conformational change in Rev. Phosphorylation directly alters the conformation of Rev, as revealed by modification experiments that monitor the solvent accessibility of cysteines in the protein. These biochemical properties are attributed to the addition of phosphate at one of two serine residues (Ser-54 or Ser-56) that lie within the multimerization domain adjacent to the RNA-binding helix. Glutaraldehyde-mediated cross-linking experiments revealed that phosphorylation of Rev does not affect Rev multimerization activity. The Rev protein from the less pathogenic HIV-2 isolate lacks this phosphorylation site in the amino acid sequence; thus, the described biochemical properties of the phosphorylated protein may contribute to Rev activity and possibly to HIV-1 virulence during natural infection.
Fouts
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DE
Department of Microbiology and College of Medicine, University of Illinois at Urbana-Champaign, USA.
True
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HL
Cengel
K A
KA
Celander
D W
DW
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002417:Cattle; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D015694:Gene Products, rev / Q000737:chemistry* / Q000378:metabolism*; D015497:HIV-1 / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D010766:Phosphorylation; D011487:Protein Conformation; D011493:Protein Kinase C / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism*; D012694:Serine / Q000378:metabolism; D054321:rev Gene Products, Human Immunodeficiency Virus
D015694:Gene Products, rev; D016601:RNA-Binding Proteins; D054321:rev Gene Products, Human Immunodeficiency Virus; D012694:Serine; D017868:Cyclic AMP-Dependent Protein Kinases; D011493:Protein Kinase C