CDK9 Autophosphorylation Regulates High-Affinity Binding of the Human Immunodeficiency Virus Type 1 Tat–P-TEFb Complex to TAR RNA

Abstract

, and full-length CycT1 (amino acids 728) [CycT1(1-728)], but not truncated CycT1(1-303), was also phosphorylated by CDK9. P-TEFb complexes containing a catalytically inactive CDK9 mutant (D167N) bound TAR RNA weakly and independently of ATP, as did a C-terminal truncated CDK9 mutant that was catalytically active but unable to undergo autophosphorylation. Analysis of different Tat proteins revealed that the 101-amino-acid SF2 HIV-1 Tat was unable to bind TAR with CycT1(1-303) in the absence of phosphorylated CDK9, whereas unphosphorylated CDK9 strongly blocked binding of HIV-2 Tat to TAR RNA in a manner that was reversed upon autophosphorylation. Replacement of CDK9 phosphorylation sites with negatively charged residues restored binding of CycT1(1-303)-D167N-Tat, and rendered D167N a more potent inhibitor of transcription in vitro. Taken together, these results demonstrate that CDK9 phosphorylation is required for high-affinity binding of Tat-P-TEFb to TAR RNA and that the state of P-TEFb phosphorylation may regulate Tat transactivation in vivo.Activation of human immunodeficiency virus type-1 (HIV-1) transcription by the virus-encoded transcription factor, Tat, provides an important paradigm for understanding the mechanisms that regulate transcription elongation by RNA polymerase II (RNAPII). Transcription complexes that form at the HIV-1 promoter in the absence of Tat are competent to initiate transcription but elongate inefficiently, due to the effects of negative general elongation factors (22,50,51,55,57; reviewed in references 16 and 56) and an inhibitory RNA structure that induces pausing of RNAPII complexes (38). Tat functions as a promoter-specific transcription elongation factor through binding to the transactivation response element (TAR) in the 5Ј-untranslated leader of viral transcripts to stimulate processive transcription by RNAPII (for a review, see references 29 and 30).Tat regulates an early step in transcription elongation that requires cyclin T1 (CycT1) and CDK9 (21,35,40,52,(58)(59)(60), which are subunits of the positive transcription elongation factor P-TEFb (36) and Tat-associated kinase (21, 23, 24) complexes. CDK9 is a Cdc2-related kinase (20) that promotes general elongation of transcription at many promoters in vitro and can phosphorylate the C-terminal domain (CTD) of the largest subunit of RNAPII (9,35,60). We previously cloned CycT1 as a protein that interacts strongly with the 48-amino-

Studies of Nematode TFIIE Function Reveal a Link between Ser-5 Phosphorylation of RNA Polymerase II and the Transition from Transcription Initiation to Elongation

“…(ii) Two novel studies using the chromatin immunoprecipitation method have demonstrated that Ser-5, but not Ser-2, phosphorylation was dependent on transcription and that Ser-5-phosphorylated Pol II was primarily localized at promoter regions (23,54). In addition, two studies on human immunodeficiency virus type 1 gene transcription have reported that the transcriptional activator of this gene, Tat, stimulates both Ser-5 phosphorylation of the CTD of Pol II by Cdk9 and Pol II processivity for transcription elongation (12,73). These results all support the idea that Ser-5 phosphorylation makes Pol II processive in transcription.…”

Characterization of the Schizosaccharomyces pombe Cdk9/Pch1 Protein Kinase

“…The unlabeled phosphoamino acid standards were visualized by staining the plate with ninhydrin; 32 P-labeled material was visualized by autoradiography. 32 P-Labeled phosphoamino acid analysis of autophosphorylated SpCdk9 and Pch1 was performed as follows. Reaction mixtures (200 l) containing 50 mM Tris acetate (pH 6.0), 1 mM DTT, 2.5 mM MnCl 2 , 50 M [␥-32 P]ATP, and 10 g of SpCdk9/Pch1 complex were incubated for 2 h at 22°C, then supplemented with 300 l of binding buffer B (50 mM NaH 2 PO 4 (pH 8.0), 50 mM NaCl, 7.5 mM imidazole, and 0.0025% Tween 20) and 50 l of nickel-agarose beads.…”

“…Analysis of the recombinant SpCdk9/Pch1 complex produced in baculovirus-infected insect cells showed that the S. pombe proteins comprise a bona fide protein kinase with a heterodimeric quaternary structure (30). The capacity of SpCdk9/Pch1 to phosphorylate the CTD arrays of both pol II and Spt5 in vitro echoed the substrate specificity of metazoan P-TEFb (11,12,31,32). These findings suggested a model whereby Spt5-induced arrest of early elongation ensures a temporal window for recruitment of the capping enzymes, which in turn attract Cdk9 to alleviate the arrest via phosphorylation of one or more components of the pol II elongation complex (30).…”

Enforcing the pause: Transcription factor Sp3 limits productive elongation by RNA polymerase II

“…64 However, phosphorylation of CDK9 at this same residue, Thr186 and other residues is required for full enzymatic activity at promoters. [64][65][66][67][68][69] These studies suggest dynamic cycles of phosphorylation and dephosphorylation regulate P-TEFb activity to control the release of the RNA Pol II into productive elongation.…”