Holmfeldt Per, Zhang Xin, Stenmark Sonja, Walczak Claire E, Gullberg Martin
Department of Molecular Biology, Umeå University, Umeå, Sweden.
EMBO J. 2005 Mar 23;24(6):1256-66. doi: 10.1038/sj.emboj.7600601. Epub 2005 Mar 3.
MCAK, a member of the kinesin-13 family, is a microtubule (MT) depolymerase that is necessary to ensure proper kinetochore MT attachment during spindle formation. Regulation of MCAK activity and localization is controlled in part by Aurora B kinase at the centromere. Here we analyzed human cells depleted of the ubiquitous Ca(2+)/calmodulin-dependent protein kinase IIgamma isoform (CaMKIIgamma) by RNA interference and found that CaMKIIgamma was necessary to suppress MCAK depolymerase activity in vivo. A functional overlap with TOGp, a MT regulator known to counteract MCAK, was suggested by similar CaMKIIgamma- and TOGp-depletion phenotypes, namely disorganized multipolar spindles. A replicating vector system, which permits inducible overexpression in cells that simultaneously synthesize interfering short hairpin RNAs, was used to dissect the functional interplay between CaMKIIgamma, TOGp, and MCAK. Our results revealed two distinct but functionally overlapping mechanisms for negative regulation of the cytosolic/centrosomal pool of MCAK. These two mechanisms, involving CaMKIIgamma and TOGp, respectively, are both essential for spindle bipolarity in a normal physiological context, but not in MCAK-depleted cells.
MCAK是驱动蛋白13家族的成员，是一种微管（MT）解聚酶，在纺锤体形成过程中确保动粒微管正确附着是必需的。MCAK活性和定位的调节部分受着丝粒处的极光激酶B控制。在这里，我们通过RNA干扰分析了缺失普遍存在的钙/钙调蛋白依赖性蛋白激酶IIγ亚型（CaMKIIγ）的人类细胞，发现CaMKIIγ在体内抑制MCAK解聚酶活性是必需的。已知可对抗MCAK的MT调节剂TOGp具有功能重叠，这是由相似的CaMKIIγ和TOGp缺失表型所表明的，即多极纺锤体紊乱。一种复制载体系统用于剖析CaMKIIγ、TOGp和MCAK之间的功能相互作用，该系统允许在同时合成干扰短发夹RNA的细胞中进行诱导性过表达。我们的结果揭示了两种不同但功能重叠的机制，用于负调控MCAK的胞质/中心体池。这两种机制分别涉及CaMKIIγ和TOGp，在正常生理环境中对纺锤体双极性都是必不可少的，但在MCAK缺失的细胞中并非如此。
