Mechanism of H‐8 inhibition of Cyclin‐dependent kinase 9: study using inhibitor‐immobilized matrices

Background : Positive transcription elongation factor b (P-TEFb), which phosphorylates the carboxylterminal 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-dichloro1-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-9binding region. These biochemical data support previously reported observations which were based on crystallographic data.