Sawin K E, Mitchison T J
Department of Pharmacology, University of California, San Francisco 94143-0450, USA.
Proc Natl Acad Sci U S A. 1995 May 9;92(10):4289-93. doi: 10.1073/pnas.92.10.4289.
Eg5, a member of the bimC subfamily of kinesin-like microtubule motor proteins, localizes to spindle microtubules in mitosis but not to interphase microtubules. We investigated the molecular basis for spindle localization by transient transfection of Xenopus A6 cells with myc-tagged derivatives of Eg5. Expressed at constitutively high levels from a cytomegalovirus promoter, mycEg5 protein is cytoplasmic throughout interphase, begins to bind microtubules in early prophase, and remains localized to spindle and/or midbody microtubules through mitosis to the end of telophase. Both N- and C-terminal regions of Eg5 are required for this cell-cycle-regulated targeting. Eg5 also contains within its C-terminal domain a sequence conserved among bimC subfamily proteins that includes a potential p34cdc2 phosphorylation site. We show that mutation of a single threonine (T937) within this site to nonphosphorylatable alanine abolishes localization of the mutant protein to the spindle, whereas mutation of T937 to serine preserves spindle localization. We hypothesize that phosphorylation of Eg5 may regulate its localization to the spindle in the cell cycle.
驱动蛋白样微管运动蛋白bimC亚家族成员Eg5，在有丝分裂时定位于纺锤体微管，而在间期微管中则不会定位。我们通过用myc标签的Eg5衍生物瞬时转染非洲爪蟾A6细胞，研究了纺锤体定位的分子基础。从巨细胞病毒启动子组成性高水平表达的mycEg5蛋白，在整个间期都位于细胞质中，在前期早期开始结合微管，并在有丝分裂过程中一直定位于纺锤体和/或中体微管，直至末期结束。Eg5的N端和C端区域对于这种细胞周期调节的靶向定位都是必需的。Eg5在其C端结构域中还包含一个在bimC亚家族蛋白中保守的序列，其中包括一个潜在的p34cdc2磷酸化位点。我们发现，该位点内的单个苏氨酸（T937）突变为不可磷酸化的丙氨酸会消除突变蛋白在纺锤体上的定位，而将T937突变为丝氨酸则保留纺锤体定位。我们推测，Eg5的磷酸化可能在细胞周期中调节其在纺锤体上的定位。
