Abstract
Y-27632 [(+)-(R)-trans-4-(1-aminoethyl)-N-(4-pyridyl)cyclohexanecarboxamide dihydrochloride] is widely used as a specific inhibitor of the Rho-associated coiled-coil forming protein serine/threonine kinase (ROCK) family of protein kinases. This study examined the inhibition mechanism and profile of actions of Y-27632 and a related compound, Y-30141 [(+)-(R)-trans- 4-(1-aminoethyl)-N-(1H-pyrrolo[2,3-b]pyridin-4-yl)cyclohexan-ecarboxamide dihydrochloride]. Y-27632 and Y-30141 inhibited the kinase activity of both ROCK-I and ROCK-II in vitro, and this inhibition was reversed by ATP in a competitive manner. This suggests that these compounds inhibit the kinases by binding to the catalytic site. Their affinities for ROCK kinases as determined by Ki values were at least 20 to 30 times higher than those for two other Rho effector kinases, citron kinase and protein kinase PKN. [3H]Y-30141 was taken up by cells in a temperature- and time-dependent and saturable manner, and this uptake was competed with unlabeled Y-27632. No concentrated accumulation was found, suggesting that the uptake is a carrier-mediated facilitated diffusion. Y-27632 abolished stress fibers in Swiss 3T3 cells at 10 μM, but the G1-S phase transition of the cell cycle and cytokinesis were little affected at this concentration. Y-30141 was 10 times more potent than Y-27632 in inhibiting the kinase activity and stress fiber formation, and it caused significant delay in the G1-S transition and inhibition of cytokinesis at 10 μM.
Footnotes
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Send reprint requests to: Shuh Narumiya, Department of Pharmacology, Kyoto University Faculty of Medicine, Yoshida, Sakyo-ku, Kyoto 606-8501, Japan. E-mail:snaru{at}mfour.med.kyoto-u.ac.jp
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This work was supported in part by Grants-in-Aid for Specially Promoted Research (08102007) and for Scientific Research (C)(2) (10670120) from the Ministry of Education, Science, and Culture of Japan, and grants from Searle Fellowship, the Japan Foundation for Applied Enzymology, the Tanabe Medical Frontier Conference, and the Human Frontier Science Program.
- Abbreviations:
- ROCK
- Rho-associated coiled-coil forming protein serine/threonine kinase
- BrdU
- bromodeoxyuridine
- DMEM
- Dulbecco's modified Eagle's medium
- FBS
- fetal bovine serum
- LPA
- lysophosphatidic acid
- PBS
- phosphate-buffered saline
- PKC
- protein kinase C
- Y-27632
- (+)-(R)-trans-4-(1-aminoethyl)-N-(4-pyridyl)cyclohexanecarboxamide dihydrochloride
- Y-30141
- (+)-(R)-trans-4-(1-aminoethyl)-N-(1H-pyrrolo[2,3-b]pyridin-4-yl)cyclohexanecarboxamide dihydrochloride
- Received August 10, 1999.
- Accepted January 12, 2000.
- The American Society for Pharmacology and Experimental Therapeutics
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