Ookata K, Hisanaga S, Bulinski J C, Murofushi H, Aizawa H, Itoh T J, Hotani H, Okumura E, Tachibana K, Kishimoto T
Laboratory of Cell and Developmental Biology, Faculty of Biosciences, Tokyo Institute of Technology, Yokohama, Japan.
J Cell Biol. 1995 Mar;128(5):849-62. doi: 10.1083/jcb.128.5.849.
We previously demonstrated (Ookata et al., 1992, 1993) that the p34cdc2/cyclin B complex associates with microtubules in the mitotic spindle and premeiotic aster in starfish oocytes, and that microtubule-associated proteins (MAPs) might be responsible for this interaction. In this study, we have investigated the mechanism by which p34cdc2 kinase associates with the microtubule cytoskeleton in primate tissue culture cells whose major MAP is known to be MAP4. Double staining of primate cells with anti-cyclin B and anti-MAP4 antibodies demonstrated these two antigens were colocalized on microtubules and copartitioned following two treatments that altered MAP4 distribution. Detergent extraction before fixation removed cyclin B as well as MAP4 from the microtubules. Depolymerization of some of the cellular microtubules with nocodazole preferentially retained the microtubule localization of both cyclin B and MAP4. The association of p34cdc2/cyclin B kinase with microtubules was also shown biochemically to be mediated by MAP4. Cosedimentation of purified p34cdc2/cyclin B with purified microtubule proteins containing MAP4, but not with MAP-free microtubules, as well as binding of MAP4 to GST-cyclin B fusion proteins, demonstrated an interaction between cyclin B and MAP4. Using recombinant MAP4 fragments, we demonstrated that the Pro-rich C-terminal region of MAP4 is sufficient to mediate the cyclin B-MAP4 interaction. Since p34cdc2/cyclin B physically associated with MAP4, we examined the ability of the kinase complex to phosphorylate MAP4. Incubation of a ternary complex of p34cdc2, cyclin B, and the COOH-terminal domain of MAP4, PA4, with ATP resulted in intracomplex phosphorylation of PA4. Finally, we tested the effects of MAP4 phosphorylation on microtubule dynamics. Phosphorylation of MAP4 by p34cdc2 kinase did not prevent its binding to microtubules, but abolished its microtubule stabilizing activity. Thus, the cyclin B/MAP4 interaction we have described may be important in targeting the mitotic kinase to appropriate cytoskeletal substrates, for the regulation of spindle assembly and dynamics.
我们之前已经证明（Ookata等人，1992年，1993年），p34cdc2/细胞周期蛋白B复合物与海星卵母细胞有丝分裂纺锤体和减数分裂前星体中的微管相关，并且微管相关蛋白（MAPs）可能介导了这种相互作用。在本研究中，我们调查了在已知主要MAP为MAP4的灵长类组织培养细胞中，p34cdc2激酶与微管细胞骨架相关联的机制。用抗细胞周期蛋白B和抗MAP4抗体对灵长类细胞进行双重染色，结果显示这两种抗原在微管上共定位，并且在两种改变MAP4分布的处理后共同分配。固定前用去污剂提取可从微管上去除细胞周期蛋白B以及MAP4。用诺考达唑使部分细胞微管解聚，优先保留了细胞周期蛋白B和MAP4在微管上的定位。p34cdc2/细胞周期蛋白B激酶与微管的关联在生化水平上也显示是由MAP4介导的。纯化的p34cdc2/细胞周期蛋白B与含有MAP4的纯化微管蛋白共沉降，但不与不含MAP的微管共沉降，以及MAP4与GST-细胞周期蛋白B融合蛋白的结合，都证明了细胞周期蛋白B与MAP4之间存在相互作用。使用重组MAP4片段，我们证明MAP4富含脯氨酸的C末端区域足以介导细胞周期蛋白B-MAP4相互作用。由于p34cdc2/细胞周期蛋白B与MAP4存在物理关联，我们检测了该激酶复合物磷酸化MAP4的能力。将p34cdc2、细胞周期蛋白B和MAP4的COOH末端结构域PA4的三元复合物与ATP一起孵育，导致PA4在复合物内部被磷酸化。最后，我们测试了MAP4磷酸化对微管动力学的影响。p34cdc2激酶对MAP4的磷酸化并不阻止其与微管的结合，但消除了其微管稳定活性。因此，我们所描述的细胞周期蛋白B/MAP4相互作用可能在将有丝分裂激酶靶向合适的细胞骨架底物、调节纺锤体组装和动力学方面具有重要意义。
