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J Virol, April 1998, p. 2615-2629, Vol. 72, No. 4
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Modulation of Sp1 Phosphorylation by Human
Immunodeficiency Virus Type 1 Tat
Rene F.
Chun,
O. J.
Semmes,
Christine
Neuveut, and
Kuan-Teh
Jeang*
Molecular Virology Section, Laboratory of
Molecular Microbiology, National Institutes of Allergy and
Infectious Diseases, Bethesda, Maryland 20892-0460
Received 8 August 1997/Accepted 19 December 1997
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.
*
Corresponding author. Mailing address: Molecular
Virology Section, Laboratory of Molecular Microbiology, National
Institutes of Allergy and Infectious Diseases, Building 4, Room 306, 9000 Rockville Pike, Bethesda, MD 20892-0460. Phone: (301) 496-6680. Fax: (301) 402-0226. E-mail:
kjeang{at}atlas.niaid.nih.gov.
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