Structure and Function of the Dbl Homology Domain of Human Beta-PIX, a Guanine Nucleotide Exchange Factor for the Rho GTPases

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RosenMCB 98173761. TechnicalGTPases in the Rho family regulate cytoskeletal structure,adhesion, motility, and gene expression in cells. A key unresolved issuein Rho-family signaling is how the GTPases are activated by upstreamguanine nucleotide exchange factors (GEFs) in the Dbl-family. Thisproposal describes structural and functional analysis of a recentlydiscovered Dbl-family GEF, human PIXB (hPIXB). Initial stages of the workwill focus on structure determination of the Dbl-homology (DH) domain ofhPIXB by NMR spectroscopy. The large size and poor aliphatic chemicalshift dispersion of the domain will necessitate use of selective methylgroup and aromatic labeling strategies to obtain sidechain chemical shiftassignments and a global fold of the domain. The work will represent thefirst application of these strategies to a protein of unknown fold, and assuch will provide a critical test of their utility in structuredetermination of larger proteins by NMR. The structure will facilitateamino acid sequence analyses across the Dbl family, providing importantclues regarding mechanisms of catalysis and GTPase recognition by DHdomains. The structure, along with NMR mapping of the GTPase interface,will then guide mutagenesis experiments to identify the roles of specificamino acids in mediating GEF catalysis. Comparisons with structural andfunctional data on other GEF domains will then reveal general principles ofnucleotide exchange catalysis. Finally, the role of the plekstrin homology(PH) domain, found immediately C-terminal to all DH domains, in regulatingGTPase binding and GEF activity will be examined biochemically andspectroscopically. The results will explain whether the hPIXB PH domainfunctions simply as a membrane-targeting sequence or as an allostericregulator of DH domain GEF activity. This information will haveimplications regarding the regulation of DH proteins and mechanisms ofGTPase activation in the cell.2. Non-technicalThe Rho GTPases represent an important class of proteins thattransmit signals controlling cell shape, adhesion and movement. Thesemolecules cycle between an inactive GDP-bound state and an active GTP-boundstate. When complexed with GTP, but not GDP, they have strong affinity forselected target proteins, and binding to these molecules constitutestransmission of information within the cell. Guanine nucleotide exchangefactors (GEFs) in the Dbl family are enzymes that catalyze release of GDPand rebinding of GTP, thus serving to increase the cellular population ofactivated Rho proteins. This study focuses on understanding the molecularmechanisms of Dbl protein activity toward the Rho GTPases. Initial workdeals with determination of the three-dimensional structure of one Dblfamily member, hPIXB, using nuclear magnetic resonance spectroscopy (NMR).In addition to providing important functional information, these studieswill also provide a platform for development of new strategies forstructure determination by NMR. This work will be followed by biochemicalstudies of hPIXB mutants that will enable identification of individualamino acids in the protein that are important for activity. Finally, thereis evidence that the activities of the Dbl proteins may be regulated in anintramolecular fashion through interactions of the catalytic domain with anadjacent regulatory module. Spectroscopic and biochemical assays will besued to probe the nature and function of this interaction. Together, thesestudies will explain many of the biochemical and structural mechanisms usedto regulate the level of activated Rho family GTPases in the cell.