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.
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.
Deng
Longwen
L
Department of Biochemistry & Molecular Biology, George Washington University Medical Center, Washington, D.C. 20037, USA.
Ammosova
Tatyana
T
Pumfery
Anne
A
Kashanchi
Fatah
F
Nekhai
Sergei
S
D000595:Amino Acid Sequence; D042846:CDC2-CDC28 Kinases; D019927:Cyclin E / Q000502:physiology; D051357:Cyclin-Dependent Kinase 2; D018844:Cyclin-Dependent Kinases / Q000502:physiology*; D015696:Gene Products, tat / Q000737:chemistry / Q000502:physiology*; D015497:HIV-1 / Q000235:genetics*; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D010766:Phosphorylation; D011401:Promoter Regions, Genetic; D017346:Protein Serine-Threonine Kinases / Q000502:physiology*; D012319:RNA Polymerase II / Q000737:chemistry*; D020449:Repetitive Sequences, Amino Acid; D014158:Transcription, Genetic; D054322:tat Gene Products, Human Immunodeficiency Virus
D019927:Cyclin E; D015696:Gene Products, tat; D054322:tat Gene Products, Human Immunodeficiency Virus; D017346:Protein Serine-Threonine Kinases; D042846:CDC2-CDC28 Kinases; C495894:CDK2 protein, human; D051357:Cyclin-Dependent Kinase 2; D018844:Cyclin-Dependent Kinases; D012319:RNA Polymerase II