Requirement for a Kinase-specific Chaperone Pathway in the Production of a Cdk9/Cyclin T1 Heterodimer Responsible for P-TEFb-mediated Tat Stimulation of HIV-1 Transcription

Abstract

Tat activation of HIV-1 transcription is mediated by human transcription elongation factor P-TEFb, which interacts with Tat and phosphorylates the C-terminal domain of RNA polymerase II. The catalytic subunit of the P-TEFb complex, Cdk9, has been shown to interact with cyclin T and several other proteins of unknown identity. Consequently, the exact subunit composition of active P-TEFb has not been determined. Here we report the affinity purification and identification of the Cdk9-associated proteins. In addition to forming a heterodimer with cyclin T1, Cdk9 interacted with the molecular chaperone Hsp70 or a kinase-specific chaperone complex, Hsp90/Cdc37, to form two separate chaperoneCdk9 complexes. Although the Cdk9/cyclin T1 dimer was exceptionally stable and produced slowly in the cell, free and unprotected Cdk9 appeared to be degraded rapidly. Several lines of evidence indicate the heterodimer of Cdk9/cyclin T1 to be the mature, active form of P-TEFb responsible for phosphorylation of the C-terminal domain of RNA polymerase II interaction with the Tat activation domain, and mediation of Tat activation of HIV-1 transcription. Pharmacological inactivation of Hsp90/Cdc37 function by geldanamycin revealed an essential role for the chaperone-Cdk9 complexes in generation of Cdk9/cyclin T1. Our data suggest a previously unrecognized chaperone-dependent pathway involving the sequential actions of Hsp70 and Hsp90/Cdc37 in the stabilization/folding of Cdk9 as well as the assembly of an active Cdk9/cyclin T1 complex responsible for P-TEFb-mediated Tat transactivation.The efficient transcription of the full-length proviral DNA depends on the viral transactivator protein Tat. Tat stimulation of HIV-1 transcription is unique because of its ability to stimulate the processivity of RNA polymerase II and its specificity for an RNA stem-loop structure known as TAR, located at the 5Ј end of the nascent viral transcripts (for reviews see Refs. 1 and 2). Tat contains two regions that are important for its function: an arginine-rich region that mediates the binding to TAR RNA and an activation domain that mediates the interaction with the cellular transcription apparatus.Tat stimulation of HIV-1 elongation requires specific cellular cofactors. Recent studies indicate that Tat recruits the human positive transcription elongation factor P-TEFb (also called TAK; Ref.3) to the HIV-1 promoter through a Tat-TAR interaction (4 -7). P-TEFb was originally identified in Drosophila nuclear extract as a general elongation factor with a kinase activity that can be inhibited by the nucleoside analog 5,6-dichloro-1-␤-D-ribofuranosylbenzimidazole (DRB), 1 a known inhibitor of transcription elongation (8, 9). Cloning and sequence analysis of the small subunit of the Drosophila P-TEFb complex (10) revealed its extensive homology to a previously identified Cdc2-related human kinase Cdk9 (previously called PITALRE; Ref. 11). In a search for cellular proteins associated with Tat, a novel cyclin C-related protein called cyclin T1 was identified (12). ...

Cyclin T1 Expression Is Regulated by Multiple Signaling Pathways and Mechanisms during Activation of Human Peripheral Blood Lymphocytes

“…CDK9 protein up-regulation mirrored that of cyclin T1 (Fig. 4C), which is consistent with stabilization of CDK9 by the increased availability of the newly synthesized, limiting, cyclin T1 subunit (43,44). Up-regulation of cyclin H, the regulatory subunit of CDK7, which phosphorylates the CTD of RNAP II during transcriptional initiation, was also prevented.…”

“…1C). CDK9 levels mirrored the increase in cyclin T1 expression, which is consistent with CDK9 stabilization dependence on binding to cyclin T1 (43)(44)(45). As a control, we measured cyclin A expression, which is only up-regulated by PHA, as well as ERK 1/2 phosphorylation, which is only induced by PMA.…”

Direct inhibition of CDK9 blocks HIV-1 replication without preventing T-cell activation in primary human peripheral blood lymphocytes

“…Expression of HA-dnCDK9 leads to clear downregulation of endogenous CDK9 protein expression, an effect previously observed when wild type CDK9 is ectopically expressed (Garriga et al, 1996a). Downregulation is due to rapid degradation of monomeric CDK9, which cannot form stable complexes with rate-limiting cyclin T subunits (O'Keeffe et al, 2000;Garriga et al, 2003;Chiu et al, 2004). To determine if HA-dnCDK9 reduces cellular CDK9 activity, anti-CDK9 immunoprecipitates from puro and HA-dnCDK9 whole lysates were incubated with bacterially expressed GST-RNAPII-CTD in in vitro kinase reactions.…”

Flavopiridol induces phosphorylation of AKT in a human glioblastoma cell line, in contrast to siRNA-mediated silencing of Cdk9: Implications for drug design and development

“…[1][2][3][4][5][6][21][22][23][24][25][26][27][28][29][30][31][32][33][34] For instance, the Cdk9-interacting cellular factors comprise MyoD, retinoblastoma protein (pRb), p53, c-Myc, hSPT5 and gp130. 5 In these cases, the Cdk9-related pathway controls the phosphorylation of the aforementioned cellular factors.…”