XMAP215 Phosphorylation and Function In Vivo.

微管是一种重要的亚细胞结构，是α-微管蛋白和β-微管蛋白的动态聚合物。MTs在真核细胞的生存中扮演着许多与结构和运动相关的角色。非洲爪蛙的卵和胚胎含有几个微管相关蛋白(MAP)，这些蛋白被认为调节微管的组织和动力学。其中之一，XMAP215，是进化上古老的MAP215/Dis1家族的成员，是真核生物MT组织和细胞分裂所必需的。XMAP215在体外促进MT的组装和动力学，并拮抗Kinesin家族成员XKCM1的不稳定作用。最近的研究结果表明，XMAP215在中心体和纺锤体极的结构和功能中发挥重要作用，从而在细胞分裂过程中纺锤体的组织和功能中发挥重要作用。XMAP215在M期被过度磷酸化，以往的研究结果表明，XMAP215的活性在体外受CDK1依赖的磷酸化调节。然而，磷酸化在调节XMAP215功能中的作用，从而调节MT在体内的组装和动力学还没有得到系统的研究。计算预测表明，XMAP215可能在多达100个不同的位点上被磷酸化。本项目将利用多种生化、生物物理和细胞生物学方法，研究体内外磷酸化对XMAP215活性的调节，重点研究三种已知的调节MT组织和细胞周期的激酶：CDK1、Aurora A和Mark。以下具体问题将被解决：1，XMAP215在体内是否被CDK1、Mark和/或Aurora A激酶磷酸化？质谱学将被用来对XMAP215M的磷酸肽进行定位和测序，提供第一个细胞中XMAP215上磷酸化的位点的“清单”；2.XMAP215中预测的CDK1靶点的磷酸化是否调节体内MT的结合和/或功能？定点突变将被用来探索在XMAP215中发现的两个预测的CDK1靶标在非洲爪哇组织培养细胞、卵母细胞和胚胎中的体内功能；3，Aurora A的磷酸化是否调节XMAP215的活性？将结合体外磷酸化、体外功能分析和定点突变来探讨Aurora A依赖的磷酸化在调节XMAP215体外和体内功能中的作用；以及4，预测的Mark和/或Aurora A靶标的磷酸化是否调节XMAP215中心体靶向和/或XMAP215在体内的功能？在非洲爪哇组织培养细胞、卵母细胞和胚胎中，定点突变技术将被用来探讨依赖Mark和Aurora A的磷酸化在XMAP215靶向中心体和MTOCs中的作用。拟议项目的智力优势：以上概述的实验结果应该回答了一些关于蛋白激酶和磷酸化调控XMAP215的悬而未决的问题，并应该提供体内磷酸化位点的图谱，以及这些位点在调节XMAP215定位和活性中所起作用的功能分析。这些信息反过来将为XMAP215和进化上古老的MAP215/Dis1微管相关蛋白家族的其他成员如何在细胞周期和发育期间调节MT的组装、组织和功能提供新的见解。拟议项目的更广泛影响：拟议项目将通过为至少一名研究生、两名或更多本科生以及一名或更多中学教师提供培训和教育(通过犹他大学理学院的中学教师理科硕士计划)，使教育和人力资源开发受益。加德博士还将通过对本科生和研究生教学以及大学外展计划的大量贡献来提供间接的教育利益。

Microtubules (MTs) are key subcellular structures that are dynamic polymers of alpha- and beta-tubulins. MTs play a number of structural and motility-related roles that are critical for the survival of eukaryotic cells. Eggs and embryos of the frog, Xenopus laevis, contain several microtubule (MT)-associated proteins (MAPs) that have been postulated to modulate MT organization and dynamics. One of these, XMAP215, is a member of the evolutionarily ancient MAP215/Dis1 family of MAPs required for MT organization and cell division in eukaryotes. XMAP215 promotes MT assembly and dynamics in vitro and antagonizes the destabilizing effects of the kinesin-family member XKCM1. Results from recent studies suggest that XMAP215 plays important roles in the structure and function of centrosomes and spindle poles, and thus in the organization and function of spindles during cell division. XMAP215 is hyperphosphorylated during M-phase, and previous results demonstrated that XMAP215 activity is regulated by CDK1-dependent phosphorylation in vitro. However, the role of phosphorylation in regulating XMAP215 function, and thereby MT assembly and dynamics, in vivo has not been systematically studied. Computational predictions suggest that XMAP215 might be phosphorylated on as many as one hundred different sites. In this project, a variety of biochemical, biophysical and cell biological methods will be used to study the regulation of XMAP215 activity in vitro and in vivo by phosphorylation, focusing on the roles of three kinases known to regulate MT organization and the cell cycle: CDK1, Aurora A, and MARK. The following specific questions will be addressed: 1, is XMAP215 phosphorylated by CDK1, MARK, and/or Aurora A kinases in vivo? Mass spectroscopy will be used to map and sequence phosphopeptides of XMAP215M, providing the first "inventory" of the sites phosphorylated on XMAP215 in cells; 2. does phosphorylation of the predicted CDK1 target sites in XMAP215 regulate MT binding and/or function in vivo? Site-directed mutagenesis will be used to probe the in vivo function of the two predicted CDK1 targets found in XMAP215, in Xenopus tissue culture cells, oocytes and embryos; 3, does phosphorylation by Aurora A modulate XMAP215 activity? A combination of in vitro phosphorylation, in vitro functional assays, and site-directed mutagenesis will be used to probe the role of Aurora A-dependent phosphorylation in regulating XMAP215 function in vitro and in vivo; and 4, does phosphorylation of the predicted MARK and/or Aurora A targets regulate XMAP215 centrosomal targeting and/or XMAP215 function in vivo? Site-directed mutagenesis will be used to probe the role of MARK and Aurora A-dependent phosphorylation in the targeting of XMAP215 to centrosomes and MTOCs in Xenopus tissue culture cells, oocytes and embryos. INTELLECTUAL MERIT OF THE PROPOSED PROJECT: Results from the experiments outlined above should answer a number of the outstanding questions regarding the regulation of XMAP215 by protein kinases and phosphorylation, and should provide a map of sites phosphorylated in vivo and functional analysis of the role those sites play in regulating XMAP215 localization and activity. This information will, in turn, provide new insight into how XMAP215, and other members of the evolutionally ancient MAP215/Dis1 family of microtubule-associated proteins, regulate MT assembly, organization, and function during the cell cycle and development. BROADER IMPACT OF THE PROPOSED PROJECT: The proposed project will benefit education and development of human resources by providing training and education for at least one graduate student, two or more undergraduate students, and one or more secondary school teachers (through the University of Utah College of Science's Masters of Science for Secondary School Teachers program). Indirect educational benefits will also be provided through Dr. Gard's substantial contributions to undergraduate and graduate teaching and University outreach programs.