Molecular Analysis of XMAP215 Structure and Function

微管（MTs）是一种动态的细胞内结构聚合物，在许多细胞功能中起着关键作用，包括细胞内运输、细胞和发育极性的规范和建立以及细胞分裂。微管动力学由一组不同的蛋白质调节，统称为微管相关蛋白（MAPs）。XMAP215是由Gard博士首先发现并从非洲爪蟾（Xenopus laevis）的卵中分离出来的一种MAP，它通过以末端特异性的方式增加MT伸长和缩短的速率，促进长而动态的MT的组装。最近，在植物（拟南芥）、酵母（S. cerevisiae和S. pombe）、无脊椎动物（Drosophila和C. elegans）和人类等多种生物中发现了相关蛋白，表明XMAP215是一个古老的MAPs家族的成员。根据该奖项进行的研究侧重于XMAP215结构和功能之间的关系。需要解决的具体问题包括：XMAP215结构和功能之间的关系是什么？A.序列比对确定的结构域是否与MT结合结构域相对应？B. HEAT重复对XMAP215函数至关重要吗？序列分析表明，XMAP215（以及其他生物中的同源物）包含四个结构域，每个结构域由多个HEAT重复组成，这是一种蛋白质基序，被认为是蛋白质相互作用的柔性支架。体外和体内实验的结合将用于检查XMAP215结构和功能之间的关系，解决XMAP215结构域和HEAT重复在XMAP215功能中的作用。细胞周期激酶CDK1和MARK激酶的磷酸化如何调节XMAP215的功能？CDK1对XMAP215的体外磷酸化减少了组装促进，但不减少MT结合，表明微管蛋白的多个结合位点可能由磷酸化独立调节。为了解决CDK1依赖性磷酸化调控XMAP215功能的机制，将使用位点定向诱变技术，用天冬氨酸（模拟磷酸化）或丙氨酸（一种不可磷酸化的氨基酸）取代XMAP215中两个预测CDK1靶序列中的苏氨酸残基。这些替换对XMAP215功能的影响将通过在转染细胞中表达携带突变的构建体在体外和体内进行检测。类似的研究将针对MARK的三个预测的靶标序列，MARK是一种mt相关的激酶，已知在其他细胞中调节MAP-MT相互作用。在哺乳动物（小鼠）发育过程中，XMAP215的多个同源物/异构体是否表达？编码XMAP215两种同工异构体的转录本在爪蟾发育过程中存在差异表达，在人类中存在多种转录本的证据以两种与XMAP215相关的cdna （KIAA0097和ch-TOG）的形式存在。然而，人们对XMAP215同源物在哺乳动物发育过程中的时空表达模式或作用知之甚少。在小鼠发育过程中表达的XMAP215同源物（MAP215）将使用各种技术在RNA和蛋白质水平上进行表征，包括Northern blots和逆转录酶聚合酶链反应（RT-PCR）来鉴定和表征mRNA转录物，原位杂交来定位mRNA表达，免疫印迹来表征蛋白质表达。最后，在小鼠中克隆和鉴定编码MAP215的基因将为进一步研究MAP215在小鼠发育中的作用奠定基础。该项目的结果将为XMAP215的结构和功能及其磷酸化调控提供新的见解，这也可能为这个古老的微管相关蛋白家族的其他成员的功能和调控提供线索。该项目还将支持本科生、研究生和博士后的培养。

Microtubules (MTs) are dynamic intracellular structural polymers that play critical roles in many cellular functions, including intracellular transport, the specification and establishment of cellular and developmental polarity, and cell division. Microtubule dynamics are modulated by a diverse group of proteins collectively referred to as microtubule-associated proteins (MAPs). XMAP215, a MAP first identified by Dr. Gard and isolated from eggs of the African frog Xenopus laevis, promotes the assembly of long, dynamic MTs by increasing the rates of both MT elongation and shortening in an end-specific manner. Recently, related proteins have been identified in taxonomically diverse organisms, including plants (Arabidopsis), yeast (S. cerevisiae and S. pombe), invertebrates (Drosophila and C. elegans), and humans, indicating that XMAP215 is a member of an ancient family of MAPs. The research to be performed under this award focuses on the relationship between XMAP215 structure and function. Specific questions to be addressed include: What is the relationship between XMAP215 structure and function? A. Do the domains identified by sequence alignment correspond to MT binding domains? B. Are HEAT repeats critical for XMAP215 function? Sequence analysis suggests that XMAP215 (and homologues in other organisms) contain four domains, each composed of multiple HEAT repeats, a protein motif thought to form a flexible scaffold for protein-protein interactions. A combination of in vitro and in vivo assays will be used to examine the relationship between XMAP215 structure and function, addressing the roles of XMAP215 domains and HEAT repeats in XMAP215 function. How does phosphorylation by the cell cycle kinase CDK1 and MARK kinase regulate XMAP215 function? Phosphorylation of XMAP215 by CDK1 in vitro reduces assembly promotion without reducing MT binding, suggesting that multiple binding sites for tubulin might be independently regulated by phosphorylation. To address the mechanism by which CDK1-dependent phosphorylation regulates XMAP215 function, site-directed mutagenesis will be used to replace threonine residues in the two predicted CDK1 target sequences in XMAP215 with aspartate (mimicking phosphorylation) or alanine (a non-phosphorylatable amino acid). The effects of these substitutions on XMAP215 function will be assayed in vitro and in vivo by expressing constructs bearing the mutations in transfected cells. Similar studies will target the three predicted target sequences for MARK, a MT-associated kinase known to regulate MAP-MT interactions in other cells. Are multiple homologs/isoforms of XMAP215 expressed during mammalian (mouse) development? Transcripts encoding two isoforms of XMAP215 are differentially expressed during Xenopus development, and evidence for multiple transcripts in humans exists in the form of two cDNAs related to XMAP215 (KIAA0097 and ch-TOG). However, little is known of the spatial and temporal patterns of expression or role(s) of XMAP215 homologues during mammalian development. XMAP215 homologs (MAP215) expressed during mouse development will be characterized at both RNA and protein levels using a variety of techniques, including Northern blots and reverse transcriptase polymerase chain reaction (RT-PCR) to identify and characterize mRNA transcripts, in situ hybridization to localize mRNA expression, and immunoblots to characterize protein expression. Finally, cloning and characterization of the gene encoding MAP215 in mice will lay the groundwork for future genetic studies of the role of MAP215 in mouse development. Results from this project will provide new insight into the structure and function of XMAP215 and its regulation by phosphorylation, which may also shed light on the function and regulation of other members of this ancient family of microtubule-associated proteins. The project will also support the training of undergraduate, graduate and post-doctoral students.