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 immu nodeficiency virus type 1 (HIV-1) Tat and Sp1 form a protein-protein c omplex. Here, we have characterized the physical interaction and a fun ctional consequence of Tat-Sp1 contact. Using in vitro protein chromat ography, 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-s tranded DNA-dependent protein kinase (DNA-PK)-mediated Sp1 phosphoryla tion in a contact-dependent manner. Tat mutants that do not bind Sp1 f ailed to influence phosphorylation of the latter. In complementary exp eriments, we also found that Tat forms protein-protein contacts with D NA-PK. We confirmed that in HeLa and Jurkat cells, Tat expression inde ed increased the intracellular amount of phosphorylated Sp1 in a manne r consistent with the results of cell-free assays. Furthermore, using two phosphatase inhibitors and a kinase inhibitor, we demonstrated a m odulation of reporter gene expression as a consequence of changes in S p1 phosphorylation. Taken together, these findings suggest that activi ty at the HIV-1 promoter is influenced by phosphorylation of Sp1 which is affected by Tat and DNA-PK.