Transcription of human immunodeficiency virus (HIV)-1 genesis activated by HIV-1 Tat protein, which induces phosphorylation of the C-terminal domain of RNA polymerase-II by CDK9/cyclin T1. We previously showed that Tat-induced HIV-1 transcription is regulated by protein phosphatase-1 (PP1). In the present study we demonstrate that Tat interacts with PP1 and that disruption of this interaction prevents induction of HIV-1 transcription. We show that PP1 interacts with Tat in part through the binding of Val 36 and Phe 38 of Tat to PP1 and that Tat is involved in the nuclear and subnuclear targeting of PP1. The PP1 binding mutant Tat-V36A/ F38A displayed a decreased affinity for PP1 and was a poor activator of HIV-1 transcription. Surprisingly, Tat-Q35R mutant that had a higher affinity for PP1 was also a poor activator of HIV-1 transcription, because strong PP1 binding competed out binding of Tat to CDK9/cyclin T1. Our results suggest that Tat might function as a nuclear regulator of PP1 and that interaction of Tat with PP1 is critical for activation of HIV-1 transcription by Tat. Human immunodeficiency virus type 1 (HIV-1)3 is a retrovirus that encodes transcriptional activator (Tat) protein. The activation domain of Tat (amino acids 1-48) interacts with host cell factors, whereas the positively charged RNA-binding domain (amino acids 49 -57) interacts with HIV-1 transactivation response (TAR) RNA (1). In cell-free transcription assays Tat induces elongation of transcription (2, 3). In vivo, Tat also induces initiation of transcription from the integrated HIV-1 promoter (4 -6). In a recent study Tat was shown to stimulate formation of a transcription complex containing TATA box-binding protein but not TATA box-binding protein-associated factors, thus indicating that Tat may enhance initiation of transcription (4). This finding apparently agrees with the early observation that Tat binds directly to the TATA box-binding protein-containing basal transcription factor TFIID (7). Tat activates HIV-1 transcription by recruiting transcriptional coactivators that include positive transcription elongation factor b, containing CDK9/cyclin T1, an RNA polymerase II CTD kinase (3,8,9) and histone acetyltransferases (10 -12). Whereas positive transcription elongation factor b induces HIV-1 transcription from non-integrated HIV-1 template (3,8,9), histone acetyltransferases allow induction of integrated HIV-1 provirus (10 -12). In contrast to the well defined role of protein kinases, the role of protein phosphatases in Tat-mediated HIV-1 transcription is less well understood. FCP1, a CTD phosphatase that dephosphorylates Ser-2 during elongation of transcription (13), is inhibited by Tat and this inhibition may alleviate FCP1-mediated pausing of transcription (14,15). In addition to FCP1, PP2A and PP1 were also shown to be involved in the regulation of HIV-1 transcription. Disregulation of cellular enzymatic activity of PP2A inhibited Tat-induced HIV-1 transcription (16). Expression of the catalytic subunit of PP2A enhanced activa...

Background: Transcription of HIV-1 genes is activated by HIV-1 Tat protein, which induces phosphorylation of RNA polymerase II (RNAPII) C-terminal domain (CTD) by CDK9/cyclin T1. Earlier we showed that CDK2/cyclin E phosphorylates HIV-1 Tat in vitro. We also showed that CDK2 induces HIV-1 transcription in vitro and that inhibition of CDK2 expression by RNA interference inhibits HIV-1 transcription and viral replication in cultured cells. In the present study, we analyzed whether Tat is phosphorylated in cultured cells by CDK2 and whether Tat phosphorylation has a regulatory effect on HIV-1 transcription.

Head and neck squamous cell carcinoma (HNSCC) development is closely associated with inflammation. Cyclooxygenase-2 (COX-2) is an important mediator of inflammation. Therefore, celecoxib, a selective inhibitor of COX-2, was hailed as a promising chemopreventive agent for HNSCC. Dose-dependent cardiac toxicity limits long-term use of celecoxib, but it seems likely that this may be diminished by lowering its dose. We found that salvianolic acid B (Sal-B), isolated from Salvia miltiorrhiza Bge, can effectively suppress COX-2 expression and induce apoptosis in a variety of cancer cell lines. In this study, we report that combination of Sal-B with low-dose celecoxib results in a more pronounced anticancer effect in HNSCC than either agent alone. The combination effects were assessed in four HNSCC cell lines (JHU-06, JHU-011, JHU-013, and JHU-022) by evaluating cell viability, proliferation, and tumor xenograft growth. Cell viability and proliferation were significantly inhibited by both the combined and single-agent treatments. However, the combination treatment significantly enhanced anticancer efficacy in JHU-013 and JHU-022 cell lines compared with the single treatment regimens. A half-dose of daily Sal-B (40 mg/kg/d) and celecoxib (2.5 mg/kg/d) significantly inhibited JHU-013 xenograft growth relative to mice treated with a full dose of Sal-B or celecoxib alone. The combination was associated with profound inhibition of COX-2 and enhanced induction of apoptosis. Taken together, these results strongly suggest that combination of Sal-B, a multifunctional anticancer agent, with low-dose celecoxib holds potential as a new preventive strategy in targeting inflammatory-associated tumor development. Cancer Prev Res; 3(6); 787-96. ©2010 AACR.