Tamemoto H, Kadowaki T, Tobe K, Ueki K, Izumi T, Chatani Y, Kohno M, Kasuga M, Yazaki Y, Akanuma Y
Institute for Diabetes Care and Research, Asahi Life Foundation, Tokyo, Japan.
J Biol Chem. 1992 Oct 5;267(28):20293-7.
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.
我们使用针对细胞外信号调节激酶-1（一种44 kDa的丝裂原活化蛋白激酶，MAPK）的位点特异性抗体，研究了中国仓鼠卵巢（CHO）细胞在细胞周期中的丝裂原活化蛋白激酶（MAPK）活性（Boulton，T.G.，Yancopoulos，G.D.，Gregory，J.S.，Slauer，C.，Moomaw，C.，Hsu，J.，和Cobb，M.H.（1990）《科学》249，64 - 67）。这些抗体在CHO细胞中检测到两种不同的MAPK（44 kDa和42 kDa的MAPK）。通过用诺考达唑处理，使CHO细胞在有丝分裂期的中期停滞，然后在解除停滞后的特定时间点分析MAPK的活性。免疫复合物激酶测定以及在含底物凝胶中的复性和磷酸化测定表明，44 kDa和42 kDa的MAPK在G1期至S期以及G2/M期均有活性，并且在G1期和M期左右被双相激活。在中期停滞的细胞中，MAPK失活。在细胞周期中，MAPK的量没有显著变化。在G1、S和G2/M期，MAPK的酪氨酸和苏氨酸残基均被磷酸化，而在中期停滞的细胞中则发生去磷酸化。我们的数据表明，MAPK可能在细胞周期中除G0/G1转换之外还发挥某些作用。
