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 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-beta-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.
Shima
Daisuke
D
Faculty of Bioscience and Biotechnology, Tokyo Institute of Technology, 4259 Nagatsuta-cho, Midori-ku, Yokohama 226-8501, Japan.
Yugami
Masato
M
Tatsuno
Michiko
M
Wada
Tadashi
T
Yamaguchi
Yuki
Y
Handa
Hiroshi
H
D000255:Adenosine Triphosphate / Q000378:metabolism; D042863:Cyclin-Dependent Kinase 9; D018844:Cyclin-Dependent Kinases / Q000037:antagonists & inhibitors*; D004004:Dichlororibofuranosylbenzimidazole / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D006801:Humans; D007546:Isoquinolines / Q000494:pharmacology*; D007700:Kinetics; D008863:Microspheres; D013449:Sulfonamides
D004791:Enzyme Inhibitors; D007546:Isoquinolines; D013449:Sulfonamides; D004004:Dichlororibofuranosylbenzimidazole; C044388:N-(2-aminoethyl)-5-isoquinolinesulfonamide; C053177:N-(2-(methylamino)ethyl)-5-isoquinolinesulfonamide; D000255:Adenosine Triphosphate; C467969:CDK9 protein, human; D042863:Cyclin-Dependent Kinase 9; D018844:Cyclin-Dependent Kinases