Fong Ka-Wing, Hau Shiu-Yeung, Kho Yik-Shing, Jia Yue, He Lisheng, Qi Robert Z
Department of Biochemistry, The Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong, China.
Mol Biol Cell. 2009 Aug;20(16):3660-70. doi: 10.1091/mbc.e09-01-0009. Epub 2009 Jun 24.
Mutations in cdk5rap2 are linked to autosomal recessive primary microcephaly, and attention has been paid to its function at centrosomes. In this report, we demonstrate that CDK5RAP2 localizes to microtubules and concentrates at the distal tips in addition to centrosomal localization. CDK5RAP2 interacts directly with EB1, a prototypic member of microtubule plus-end tracking proteins, and contains the basic and Ser-rich motif responsible for EB1 binding. The EB1-binding motif is conserved in the CDK5RAP2 sequences of chimpanzee, bovine, and dog but not in those of rat and mouse, suggesting a function gained during the evolution of mammals. The mutation of the Ile/Leu-Pro dipeptide within the motif abolishes EB1 interaction and plus-end attachment. In agreement with the mutational analysis, suppression of EB1 expression inhibits microtubule tip-tracking of CDK5RAP2. We have also found that the CDK5RAP2-EB1 complex regulates microtubule dynamics and stability. CDK5RAP2 depletion by RNA interference impacts the dynamic behaviors of microtubules. The CDK5RAP2-EB1 complex induces microtubule bundling and acetylation when expressed in cell cultures and stimulates microtubule assembly and bundle formation in vitro. Collectively, these results show that CDK5RAP2 targets growing microtubule tips in association with EB1 to regulate microtubule dynamics.
细胞周期蛋白依赖性激酶5调节相关蛋白2（CDK5RAP2）的突变与常染色体隐性原发性小头畸形有关，人们已关注到它在中心体的功能。在本报告中，我们证明CDK5RAP2定位于微管，除了定位于中心体外，还集中在微管远端。CDK5RAP2直接与EB1相互作用，EB1是微管正端追踪蛋白的原型成员，并且包含负责与EB1结合的富含碱性氨基酸和丝氨酸的基序。EB1结合基序在黑猩猩、牛和狗的CDK5RAP2序列中保守，但在大鼠和小鼠中不保守，这表明该功能是在哺乳动物进化过程中获得的。该基序内异亮氨酸/亮氨酸 - 脯氨酸二肽的突变消除了EB1相互作用和正端附着。与突变分析一致，抑制EB1表达会抑制CDK5RAP2的微管末端追踪。我们还发现CDK5RAP2 - EB1复合物调节微管动力学和稳定性。RNA干扰导致的CDK5RAP2缺失影响微管的动态行为。CDK5RAP2 - EB1复合物在细胞培养物中表达时会诱导微管束集和乙酰化，并在体外刺激微管组装和成束。总的来说，这些结果表明CDK5RAP2与EB1结合靶向生长中的微管末端以调节微管动力学。
