Gould K L, Feoktistova A, Fleig U
Howard Hughes Medical Institute and Department of Cell Biology, Vanderbilt University School of Medicine, Nashville, TN 37232, USA.
Mol Gen Genet. 1998 Sep;259(4):437-48. doi: 10.1007/s004380050834.
The protein kinase cdc2p is a key regulator of the G1-S and G2-M cell cycle transitions in the yeast Schizosaccharomyces pombe. Activation of cdc2p is regulated by its phosphorylation state and by interaction with other proteins. We have analyzed the consequences for cell cycle progression of altering the conserved threonine phosphorylation site, within the activation loop of cdc2p, to glutamic acid. This mutant, T167 E, promotes entry into mitosis, as judged by the accumulation of mitotic spindles and condensed chromosomes, despite the fact that it lacks demonstrable kinase activity both in vitro and in vivo. However, T167 E cannot promote the metaphase-anaphase transition. Since a component of the anaphase-promoting complex (APC) in S. pombe, cut9p, remains hypophosphorylated at the T167 E arrest point, the cell cycle block might be due to the inability of T167 E to activate the APC. T167 E is lethal when overexpressed, and overproduction also causes a mitotic arrest. Multicopy suppressors of the dominant negative phenotype were isolated, and identified as cdc13+ and suc1+. Overexpression of suc1+ suppresses the effects of T167 E overproduction by restoring sufficient amounts of suc1p to the cell to allow passage through mitosis.
蛋白激酶cdc2p是粟酒裂殖酵母中G1-S和G2-M细胞周期转换的关键调节因子。cdc2p的激活受其磷酸化状态以及与其他蛋白质相互作用的调控。我们分析了将cdc2p激活环内保守的苏氨酸磷酸化位点突变为谷氨酸后对细胞周期进程的影响。尽管该突变体T167E在体外和体内均缺乏可证实的激酶活性，但通过有丝分裂纺锤体和浓缩染色体的积累判断，它能促进细胞进入有丝分裂。然而，T167E不能促进中期-后期转换。由于粟酒裂殖酵母后期促进复合物（APC）的一个组分cut9p在T167E停滞点仍处于低磷酸化状态，细胞周期阻滞可能是由于T167E无法激活APC所致。T167E过表达时具有致死性，过量表达也会导致有丝分裂停滞。分离出了显性负性表型的多拷贝抑制子，并鉴定为cdc13+和suc1+。suc1+的过表达通过向细胞中恢复足够量的suc1p以允许细胞通过有丝分裂，从而抑制了T167E过量表达的影响。
