Molecular Analysis of XMAP215 Structure and Function

University Of Utah, Salt Lake City UT

Investigators

Abstract

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.