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.
Mesh Terms:
Benzoquinones, Cell Cycle Proteins, Chaperonins, Cyclin T, Cyclin-Dependent Kinase 9, Cyclin-Dependent Kinases, Cyclins, DNA Polymerase II, Dimerization, Drosophila Proteins, Enzyme Activation, Gene Products, tat, HIV-1, HSP70 Heat-Shock Proteins, HSP90 Heat-Shock Proteins, Humans, Lactams, Macrocyclic, Molecular Chaperones, Positive Transcriptional Elongation Factor B, Protein Binding, Protein-Serine-Threonine Kinases, Quinones, Species Specificity, Transcription, Genetic, tat Gene Products, Human Immunodeficiency Virus
Benzoquinones, Cell Cycle Proteins, Chaperonins, Cyclin T, Cyclin-Dependent Kinase 9, Cyclin-Dependent Kinases, Cyclins, DNA Polymerase II, Dimerization, Drosophila Proteins, Enzyme Activation, Gene Products, tat, HIV-1, HSP70 Heat-Shock Proteins, HSP90 Heat-Shock Proteins, Humans, Lactams, Macrocyclic, Molecular Chaperones, Positive Transcriptional Elongation Factor B, Protein Binding, Protein-Serine-Threonine Kinases, Quinones, Species Specificity, Transcription, Genetic, tat Gene Products, Human Immunodeficiency Virus
J. Biol. Chem.
Date: Jan. 07, 2000
PubMed ID: 10617616
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