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- Title
Mechanism of H-8 inhibition of Cyclin-dependent kinase 9: study using inhibitor-immobilized matrices.
- Authors
Shima, Daisuke; Yugami, Masato; Tatsuno, Michiko; Wada, Tadashi; Yamaguchi, Yuki; Handa, Hiroshi
- Abstract
Abstract Background: Positive transcription elongation factor b (P-TEFb), which phosphorylates the carboxyl-terminal domain (CTD) of RNA polymerase II (RNAPII), is comprised of the catalytic subunit cyclin-dependent kinase 9 (CDK9) and the regulatory subunit cyclin T. The kinase activity and transcriptional activation potential of P-TEFb is sensitive to various compounds, including H-8, 5,6-dichloro-1-β-d-ribofuranosylbenzimidazole (DRB), and flavopiridol. Results: We investigated the molecular mechanism of the H-8 inhibition of CDK9 using matrices to which H-9, an amino derivative of H-8, was immobilized. CDK9 bound specifically to H-9, and this interaction was competitively inhibited by ATP and DRB, but not by flavopiridol. Mutational analyses demonstrated that the central region of CDK9, which encompasses the T-loop region, was important for its binding to H-9. Conclusions: H-9-immobilized latex beads are useful for trapping CDK9 and a subset of kinases from crude cell extracts. The flavopiridol-binding region of CDK9 is most likely different from its H-9-binding region. These biochemical data support previously reported observations which were based on crystallographic data.
- Subjects
CYCLIN-dependent kinases; GENETIC transcription
- Publication
Genes to Cells, 2003, Vol 8, Issue 3, p215
- ISSN
1356-9597
- Publication type
Article
- DOI
10.1046/j.1365-2443.2003.00627.x