THE HUMAN-IMMUNODEFICIENCY-VIRUS TAT PROTEINS SPECIFICALLY ASSOCIATE WITH TAK IN-VIVO AND REQUIRE THE CARBOXY-TERMINAL DOMAIN OF RNA-POLYMERASE-II FOR FUNCTION

Human immunodeficiency virus types I and 2 encode closely related prot eins, Tat-l and Tat-2, that stimulate viral transcription. previously, we showed that the activation domains of these proteins specifically interact in vitro with a cellular protein kinase named TAK. In vitro, TAK phosphorylates the Tat-2 but not the Tat-l protein, a 42-kDa polyp eptide of unknown identity and the carboxyl-terminal domain (CTD) of R NA polymerase EI (RNAP II), We now show that the 42-kDa substrate of T AK cochromatographs with TAK activity, suggesting that this 42-kDa pol ypeptide is a subunit of TAK. We also show that tile Tat proteins spec ifically associate with TAK in vivo, since wild-type Tat-l and Tat-2 p roteins expressed in mammalian cells, but not mutant Tar proteins cont aining a nonfunctional activation domain, can be coimmunoprecipitated with TAK. We also mapped the in vivo phosphorylation sites of Tat-2 to the carboxyl terminus of the protein, but analysis of proteins with m utations at these sites suggests that phosphorylation is not essential fur Tat-2 transactivation function, We further investigated whether t he CTD of RNAP II is required for Tat function in vivo. Using plasmid constructs that express an alpha-amanitin-resistant RNAP II subunit wi th a truncated or full-length CTD, we found that an intact CTD is requ ired for Tat function. These observations strengthen the proposal that the mechanism of action of Tat involves the recruitment or activation of TAK, resulting in activated transcription through phosphorylation of the CTD.