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.
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 chaperone-Cdk9 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.
O'Keeffe
B
B
Department of Molecular Biology, University of California at Berkeley, Berkeley, California 94720-3206, USA.
Fong
Y
Y
Chen
D
D
Zhou
S
S
Zhou
Q
Q
D016227:Benzoquinones; D018797:Cell Cycle Proteins / Q000378:metabolism; D018833:Chaperonins; D056906:Cyclin T; D042863:Cyclin-Dependent Kinase 9; D018844:Cyclin-Dependent Kinases / Q000378:metabolism; D016213:Cyclins / Q000378:metabolism*; D004257:DNA Polymerase II / Q000378:metabolism; D019281:Dimerization; D029721:Drosophila Proteins; D004789:Enzyme Activation; D015696:Gene Products, tat / Q000378:metabolism*; D015497:HIV-1 / Q000235:genetics*; D018840:HSP70 Heat-Shock Proteins / Q000378:metabolism; D018841:HSP90 Heat-Shock Proteins / Q000378:metabolism; D006801:Humans; D047029:Lactams, Macrocyclic; D018832:Molecular Chaperones / Q000378:metabolism*; D042925:Positive Transcriptional Elongation Factor B; D011485:Protein Binding / Q000187:drug effects; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D011809:Quinones / Q000494:pharmacology; D013045:Species Specificity; D014158:Transcription, Genetic; D054322:tat Gene Products, Human Immunodeficiency Virus
D016227:Benzoquinones; C503684:CCNT1 protein, human; C498362:CDC37 protein, human; D018797:Cell Cycle Proteins; C503687:CycT protein, Drosophila; D056906:Cyclin T; D016213:Cyclins; D029721:Drosophila Proteins; D015696:Gene Products, tat; D018840:HSP70 Heat-Shock Proteins; D018841:HSP90 Heat-Shock Proteins; D047029:Lactams, Macrocyclic; D018832:Molecular Chaperones; D011809:Quinones; D054322:tat Gene Products, Human Immunodeficiency Virus; D042925:Positive Transcriptional Elongation Factor B; D017346:Protein Serine-Threonine Kinases; C467969:CDK9 protein, human; D042863:Cyclin-Dependent Kinase 9; D018844:Cyclin-Dependent Kinases; D004257:DNA Polymerase II; D018833:Chaperonins; C001277:geldanamycin