Lim H H, Loy C J, Zaman S, Surana U
Institute of Molecular and Cell Biology, National University of Singapore.
Mol Cell Biol. 1996 Aug;16(8):4573-83. doi: 10.1128/MCB.16.8.4573.
Entry into mitosis requires activation of cdc2 kinase brought on by its association with cyclin B, phosphorylation of the conserved threonine (Thr-167 in Schizosaccharomyces pombe) in the T loop, and dephosphorylation of the tyrosine residue at position 15. Exit from mitosis, on the other hand, is induced by inactivation of cdc2 activity via cyclin destruction. It has been suggested that in addition to cyclin degradation, dephosphorylation of Thr-167 may also be required for exit from the M phase. Here we show that Saccharomyces cerevisiae cells expressing cdc28-E169 (a CDC28 allele in which the equivalent threonine, Thr-169, has been replaced by glutamic acid) are able to degrade mitotic cyclin Clb2, inactivate the Cdc28/Clb2 kinase, and disassemble the anaphase spindles, suggesting that they exit mitosis normally. The cdc28-E169 allele is active with respect to its mitotic functions, since it complements the mitosis-defective cdc28-1N allele. Whereas replacement of Thr-169 with serine affects neither Start nor the mitotic activity of Cdc28, replacement with glutamic acid or alanine renders Cdc28 inactive for Start-related functions. Coimmunoprecipitation experiments show that although Cdc28-E169 associates with mitotic cyclin Clb2, it fails to associate with the G1 cyclin Cln2. Thus, an unmodified threonine at position 169 in Cdc28 is important for interaction with G1 cyclins. We propose that in S. cerevisiae, dephosphorylation of Thr-169 is not required for exit from mitosis but may be necessary for commitment to the subsequent division cycle.
进入有丝分裂需要通过细胞周期蛋白B与cdc2激酶结合来激活cdc2激酶，T环中保守的苏氨酸（在粟酒裂殖酵母中为Thr-167）磷酸化，以及第15位酪氨酸残基的去磷酸化。另一方面，有丝分裂的退出是由细胞周期蛋白破坏导致cdc2活性失活所诱导的。有人提出，除了细胞周期蛋白降解外，Thr-167的去磷酸化可能也是退出M期所必需的。在这里，我们表明，表达cdc28-E169（一种CDC28等位基因，其中等效的苏氨酸Thr-169被谷氨酸取代）的酿酒酵母细胞能够降解有丝分裂细胞周期蛋白Clb2，使Cdc28/Clb2激酶失活，并拆解后期纺锤体，这表明它们正常退出有丝分裂。cdc28-E169等位基因在其有丝分裂功能方面是活跃的，因为它能弥补有丝分裂缺陷的cdc28-1N等位基因。虽然将Thr-169替换为丝氨酸既不影响起始也不影响Cdc28的有丝分裂活性，但替换为谷氨酸或丙氨酸会使Cdc28在与起始相关的功能中失活。免疫共沉淀实验表明，虽然Cdc28-E169与有丝分裂细胞周期蛋白Clb2结合，但它不能与G1细胞周期蛋白Cln2结合。因此，Cdc28中第169位未修饰的苏氨酸对于与G1细胞周期蛋白的相互作用很重要。我们提出，在酿酒酵母中，Thr-169的去磷酸化不是退出有丝分裂所必需的，但可能是进入后续分裂周期所必需的。
