Biochemical and cellular effects of roscovitine, a potent and selective inhibitor of the cyclin-dependent kinases cdc2, cdk2 and cdk5.
Cyclin-dependent kinases (cdk) play an essential role in the intracellular control of the cell division cycle (cdc). These kinases and their regulators are frequently deregulated in human tumours. Enzymatic screening has recently led to the discovery of specific inhibitors of cyclin-dependent kinases, such as butyrolactone I, flavopiridol and the purine olomoucine. Among a series of C2, N6, N9-substituted adenines tested on purified cdc2/cyclin B, 2-(1-ethyl-2-hydroxyethylamino)-6-benzylamino-9-isopropylpurine (roscovitine) displays high efficiency and high selectivity towards some cyclin-dependent kinases. The kinase specificity of roscovitine was investigated with 25 highly purified kinases (including protein kinase A, G and C isoforms, myosin light-chain kinase, casein kinase 2, insulin receptor tyrosine kinase, c-src, v-abl). Most kinases are not significantly inhibited by roscovitine. cdc2/cyclin B, cdk2/cyclin A, cdk2/cyclin E and cdk5/p35 only are substantially inhibited (IC50 values of 0.65, 0.7, 0.7 and 0.2 microM, respectively). cdk4/cyclin D1 and cdk6/cyclin D2 are very poorly inhibited by roscovitine (IC50 > 100 microM). Extracellular regulated kinases erk1 and erk2 are inhibited with an IC50 of 34 microM and 14 microM, respectively. Roscovitine reversibly arrests starfish oocytes and sea urchin embryos in late prophase. Roscovitine inhibits in vitro M-phase-promoting factor activity and in vitro DNA synthesis in Xenopus egg extracts. It blocks progesterone-induced oocyte maturation of Xenopus oocytes and in vivo phosphorylation of the elongation factor eEF-1. Roscovitine inhibits the proliferation of mammalian cell lines with an average IC50 of 16 microM. In the presence of roscovitine L1210 cells arrest in G1 and accumulate in G2. In vivo phosphorylation of vimentin on Ser55 by cdc2/cyclin B is inhibited by roscovitine. Through its unique selectivity for some cyclin-dependent kinases, roscovitine provides a useful antimitotic reagent for cell cycle studies and may prove interesting to control cells with deregulated cdc2, cdk2 or cdk5 kinase activities.
Meijer
L
L
CNRS, Station Biologique, Roscoff, France. meijer@sb-roscoff.fr
Borgne
A
A
Mulner
O
O
Chong
J P
JP
Blow
J J
JJ
Inagaki
N
N
Inagaki
M
M
Delcros
J G
JG
Moulinoux
J P
JP
D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects; D001665:Binding Sites; D016203:CDC2 Protein Kinase / Q000037:antagonists & inhibitors*; D042846:CDC2-CDC28 Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism; D002453:Cell Cycle / Q000187:drug effects*; D051357:Cyclin-Dependent Kinase 2; D051360:Cyclin-Dependent Kinase 5; D018844:Cyclin-Dependent Kinases / Q000037:antagonists & inhibitors*; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D006131:Growth Inhibitors / Q000494:pharmacology; D006801:Humans; D007701:Kinetin; D007939:Leukemia L1210 / Q000201:enzymology; D016200:Maturation-Promoting Factor / Q000378:metabolism; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases; D008969:Molecular Sequence Data; D009866:Oogenesis; D010766:Phosphorylation / Q000187:drug effects; D019000:Phosphotyrosine / Q000378:metabolism; D047428:Protein Kinase Inhibitors; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors*; D011687:Purines / Q000494:pharmacology*; D000077546:Roscovitine; D012617:Sea Urchins; D013215:Starfish; D013379:Substrate Specificity; D014746:Vimentin / Q000378:metabolism; D029867:Xenopus Proteins; D014982:Xenopus laevis
D000970:Antineoplastic Agents; D004791:Enzyme Inhibitors; D006131:Growth Inhibitors; D047428:Protein Kinase Inhibitors; D011687:Purines; D014746:Vimentin; D029867:Xenopus Proteins; D000077546:Roscovitine; D019000:Phosphotyrosine; C090046:olomoucine; D000255:Adenosine Triphosphate; D051360:Cyclin-Dependent Kinase 5; D017346:Protein Serine-Threonine Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D016203:CDC2 Protein Kinase; D042846:CDC2-CDC28 Kinases; C495894:CDK2 protein, human; C495905:CDK5 protein, human; C495891:Cdk2 protein, Xenopus; C495908:Cdk5 protein, Xenopus; D051357:Cyclin-Dependent Kinase 2; D018844:Cyclin-Dependent Kinases; D016200:Maturation-Promoting Factor; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases; D007701:Kinetin