The human immunodeficiency virus Tat proteins specifically associate with TAK in vivo and require the carboxyl-terminal domain of RNA polymerase II for function.
Human immunodeficiency virus types 1 and 2 encode closely related proteins, Tat-1 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-1 protein, a 42-kDa polypeptide of unknown identity, and the carboxyl-terminal domain (CTD) of RNA polymerase II (RNAP II). We now show that the 42-kDa substrate of TAK cochromatographs with TAK activity, suggesting that this 42-kDa polypeptide is a subunit of TAK. We also show that the Tat proteins specifically associate with TAK in vivo, since wild-type Tat-1 and Tat-2 proteins expressed in mammalian cells, but not mutant Tat proteins containing 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 mutations at these sites suggests that phosphorylation is not essential for Tat-2 transactivation function. We further investigated whether the CTD of RNAP II is required for Tat function in vivo. Using plasmid constructs that express an alpha-amanitin-resistant RNAP II subunit with a truncated or full-length CTD, we found that an intact CTD is required 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.
Yang
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Division of Molecular Virology, Baylor College of Medicine, Houston, Texas 77030, USA.
Herrmann
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Rice
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AP
D000818:Animals; D001665:Binding Sites; D002461:Cell Line, Transformed; D002522:Chlorocebus aethiops; D015696:Gene Products, tat / Q000378:metabolism*; D015497:HIV-1 / Q000378:metabolism*; D015498:HIV-2 / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D012319:RNA Polymerase II / Q000378:metabolism*; D054322:tat Gene Products, Human Immunodeficiency Virus
D015696:Gene Products, tat; D054322:tat Gene Products, Human Immunodeficiency Virus; D017346:Protein Serine-Threonine Kinases; D012319:RNA Polymerase II