Modulation of Sp1 phosphorylation by human immunodeficiency virus type 1 Tat.
We previously reported (K. T. Jeang, R. Chun, N. H. Lin, A. Gatignol, C. G. Glabe, and H. Fan, J. Virol. 67: 6224-6233, 1993) that human immunodeficiency virus type 1 (HIV-1) Tat and Sp1 form a protein-protein complex. Here, we have characterized the physical interaction and a functional consequence of Tat-Sp1 contact. Using in vitro protein chromatography, we mapped the region in Tat that contacts Sp1 to amino acids 30 to 55. We found that in cell-free reactions, Tat augmented double-stranded DNA-dependent protein kinase (DNA-PK)-mediated Sp1 phosphorylation in a contact-dependent manner. Tat mutants that do not bind Sp1 failed to influence phosphorylation of the latter. In complementary experiments, we also found that Tat forms protein-protein contacts with DNA-PK. We confirmed that in HeLa and Jurkat cells, Tat expression indeed increased the intracellular amount of phosphorylated Sp1 in a manner consistent with the results of cell-free assays. Furthermore, using two phosphatase inhibitors and a kinase inhibitor, we demonstrated a modulation of reporter gene expression as a consequence of changes in Sp1 phosphorylation. Taken together, these findings suggest that activity at the HIV-1 promoter is influenced by phosphorylation of Sp1 which is affected by Tat and DNA-PK.
Chun
R F
RF
Molecular Virology Section, Laboratory of Molecular Microbiology, National Institutes of Allergy and Infectious Diseases, Bethesda, Maryland 20892-0460, USA.
Semmes
O J
OJ
Neuveut
C
C
Jeang
K T
KT
D000730:Androstadienes / Q000494:pharmacology; D001665:Binding Sites; D002467:Cell Nucleus / Q000378:metabolism; D051747:DNA-Activated Protein Kinase; D004268:DNA-Binding Proteins; D004791:Enzyme Inhibitors / Q000494:pharmacology; D015870:Gene Expression; D015696:Gene Products, tat / Q000235:genetics / Q000378:metabolism*; D016325:HIV Long Terminal Repeat; D015497:HIV-1 / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D019169:Jurkat Cells; D008387:Marine Toxins; D009687:Nuclear Proteins; D019319:Okadaic Acid / Q000494:pharmacology; D010080:Oxazoles / Q000494:pharmacology; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors; D010766:Phosphorylation; D017354:Point Mutation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics; D012694:Serine / Q000235:genetics; D016329:Sp1 Transcription Factor / Q000378:metabolism*; D014158:Transcription, Genetic; D000077191:Wortmannin; D054322:tat Gene Products, Human Immunodeficiency Virus
D000730:Androstadienes; D004268:DNA-Binding Proteins; D004791:Enzyme Inhibitors; D015696:Gene Products, tat; D008387:Marine Toxins; D009687:Nuclear Proteins; D010080:Oxazoles; D011993:Recombinant Fusion Proteins; D016329:Sp1 Transcription Factor; D054322:tat Gene Products, Human Immunodeficiency Virus; D019319:Okadaic Acid; D012694:Serine; C059041:calyculin A; D051747:DNA-Activated Protein Kinase; C497263:PRKDC protein, human; D017346:Protein Serine-Threonine Kinases; D010749:Phosphoprotein Phosphatases; D000077191:Wortmannin