Biphasic activation of two mitogen-activated protein kinases during the cell cycle in mammalian cells.

H Tamemoto,T Kadowaki,K Tobe,K Ueki,T Izumi,Y Chatani,M Kohno,M Kasuga,Y Yazaki,Y Akanuma

doi:10.1016/s0021-9258(19)88700-8

Abstract

We studied mitogen-activated protein kinase (MAPK) activities during the cell cycle of Chinese hamster ovary (CHO) cells using site-specific antibodies against extracellular signal-regulated kinase-1, a 44-kDa MAPK (Boulton, T.G., Yancopoulos, G.D., Gregory, J.S., Slauer, C., Moomaw, C., Hsu, J., and Cobb, M.H. (1990) Science 249, 64-67). These antibodies detected two distinct MAPKs (44- and 42-kDa MAPKs) in CHO cells. CHO cells were arrested at metaphase in the M phase by treatment with nocodazole, and activities of MAPKs were analyzed at specific time points after release from arrest. Immune complex kinase assay and renaturation and phosphorylation assay in substrate-containing gel revealed that both 44- and 42-kDa MAPKs had activities in the G1 through S and G2/M phases and were activated biphasically, in the G1 phase and around the M phase. MAPKs were inactivated in metaphase-arrested cells. The amount of MAPKs did not change significantly in the cell cycle. In the G1, S, and G2/M phases, MAPKs were phosphorylated on both tyrosine and threonine residues and dephosphorylated in metaphase-arrested cells. Our data suggest that MAPKs may play some role in the cell cycle other than G0/G1 transition.

Highlights

We studied mitogen-activated protein kinase FUS3 or KSS1, which is a yeast gene involved in cell cycle (MAPK)activities during the cell cycle Cohfinese hamster ovary (CHO) cellsusingsite-specific antibodies against extracellular signal-regulated kinase-1, a 44
MAPK activitywas measured in thecell cycle in proliferating Chinese hamster ovary (CHO) cells
We have found that both 44- and 42-kDa MAPKs have activities in thGe1through S and G2/M phases and are activatedbiphasically: in GI and around tMhephase

Summary

RESULTS

At the point when nocodazole-arrested cells by adding 30 pl of kinase buffer containing 50 mM Tris-HC1, pH 7.4, were collected, immunoprecipitated cdc kinase activity was. 16-18 h after release from M phase arrest by nocodazole, histone H1 kinase activity of immunoprecipitated cdc kinase was reactivated This second peak of histone H1 kinase activity ranged from 14to 20 h with its maximum at 16h (Fig. 2B). At the indicated times after replating, cells were washed oncewithserum-free medium and incubated in the same medium containing 5-6 X lo cpm of [methyl-3H]thymidine for 1 h a t 37 "C. At the min on ice, the lysate was centrifuged a t 15,000 rpm for 15 min at indicated time after release from M phase arrest, cells were pulse. The lysate was incubated with 5 plof Radioactivityincorporated intothe trichloroacetic acid-insoluble immunoaffinity-purified anti-MAPK antibody (aY91) for 2 h on ice. fraction was counted

BAicpthivaasticon of Two MAP Kinases

Biphasic Activationof Two MAPKinases

DISCUSSION