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J. Biol. Chem., Vol. 277, Issue 37, 33922-33929, September 13, 2002
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From the Human immunodeficiency virus, type 1 (HIV-1), Tat
protein activates viral gene expression through promoting
transcriptional elongation by RNA polymerase II (RNAPII). In this
process Tat enhances phosphorylation of the C-terminal domain (CTD) of
RNAPII by activating cell cycle-dependent kinases (CDKs)
associated with general transcription factors of the promoter complex,
specifically CDK7 and CDK9. We reported a Tat-associated T-cell-derived
kinase, which contained CDK2. Here, we provide further evidence that
CDK2 is involved in Tat-mediated CTD phosphorylation and in HIV-1
transcription in vitro. Tat-mediated CTD phosphorylation by
CDK2 required cysteine 22 in the activation domain of Tat and amino
acids 42-72 of Tat. CDK2 phosphorylated Tat itself, apparently by
forming dynamic contacts with amino acids 15-24 and 36-49 of Tat.
Also, amino acids 24-36 and 45-72 of Tat interacted with CTD. CDK2
associated with RNAPII and was found in elongation complexes assembled
on HIV-1 long-terminal repeat template. Recombinant CDK2/cyclin E stimulated Tat-dependent HIV-1 transcription in
reconstituted transcription assay. Immunodepletion of CDK2/cyclin E in
HeLa nuclear extract blocked Tat-dependent transcription.
We suggest that CDK2 is part of a transcription complex that is
required for Tat-dependent transcription and that
interaction of Tat with CTD and a dynamic association of Tat with
CDK2/cyclin E stimulated CTD phosphorylation by CDK2.
HIV-1 Tat Interaction with RNA Polymerase II C-terminal Domain
(CTD) and a Dynamic Association with CDK2 Induce CTD Phosphorylation
and Transcription from HIV-1 Promoter*
,,, and§¶
Department of Biochemistry & Molecular
Biology, George Washington University Medical Center, Washington,
D. C. 20037, and the § Center for Sickle Cell Disease and
Department of Biochemistry, Howard University, Washington, D. C. 20059
*
This work was supported by National Institutes of Health
(NIH) Grants AI44357 and AI43894, an Alexandrine and Alexander
Sinsheimer Foundation grant, and a grant from George Washington
University (to F. K.) and by NHLBI, NIH Research Grant UH1 HL03679 and
The Office of Research on Minority Health (to T. A. and S. N.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
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