NSF Award
9817376
Rosen<br/>MCB 9817376<br/><br/>1. Technical<br/><br/>GTPases in the Rho family regulate cytoskeletal structure,<br/>adhesion, motility, and gene expression in cells. A key unresolved issue<br/>in Rho-family signaling is how the GTPases are activated by upstream<br/>guanine nucleotide exchange factors (GEFs) in the Dbl-family. This<br/>proposal describes structural and functional analysis of a recently<br/>discovered Dbl-family GEF, human PIXB (hPIXB). Initial stages of the work<br/>will focus on structure determination of the Dbl-homology (DH) domain of<br/>hPIXB by NMR spectroscopy. The large size and poor aliphatic chemical<br/>shift dispersion of the domain will necessitate use of selective methyl<br/>group and aromatic labeling strategies to obtain sidechain chemical shift<br/>assignments and a global fold of the domain. The work will represent the<br/>first application of these strategies to a protein of unknown fold, and as<br/>such will provide a critical test of their utility in structure<br/>determination of larger proteins by NMR. The structure will facilitate<br/>amino acid sequence analyses across the Dbl family, providing important<br/>clues regarding mechanisms of catalysis and GTPase recognition by DH<br/>domains. The structure, along with NMR mapping of the GTPase interface,<br/>will then guide mutagenesis experiments to identify the roles of specific<br/>amino acids in mediating GEF catalysis. Comparisons with structural and<br/>functional data on other GEF domains will then reveal general principles of<br/>nucleotide exchange catalysis. Finally, the role of the plekstrin homology<br/>(PH) domain, found immediately C-terminal to all DH domains, in regulating<br/>GTPase binding and GEF activity will be examined biochemically and<br/>spectroscopically. The results will explain whether the hPIXB PH domain<br/>functions simply as a membrane-targeting sequence or as an allosteric<br/>regulator of DH domain GEF activity. This information will have<br/>implications regarding the regulation of DH proteins and mechanisms of<br/>GTPase activation in the cell.<br/><br/><br/>2. Non-technical<br/><br/>The Rho GTPases represent an important class of proteins that<br/>transmit signals controlling cell shape, adhesion and movement. These<br/>molecules cycle between an inactive GDP-bound state and an active GTP-bound<br/>state. When complexed with GTP, but not GDP, they have strong affinity for<br/>selected target proteins, and binding to these molecules constitutes<br/>transmission of information within the cell. Guanine nucleotide exchange<br/>factors (GEFs) in the Dbl family are enzymes that catalyze release of GDP<br/>and rebinding of GTP, thus serving to increase the cellular population of<br/>activated Rho proteins. This study focuses on understanding the molecular<br/>mechanisms of Dbl protein activity toward the Rho GTPases. Initial work<br/>deals with determination of the three-dimensional structure of one Dbl<br/>family member, hPIXB, using nuclear magnetic resonance spectroscopy (NMR).<br/>In addition to providing important functional information, these studies<br/>will also provide a platform for development of new strategies for<br/>structure determination by NMR. This work will be followed by biochemical<br/>studies of hPIXB mutants that will enable identification of individual<br/>amino acids in the protein that are important for activity. Finally, there<br/>is evidence that the activities of the Dbl proteins may be regulated in an<br/>intramolecular fashion through interactions of the catalytic domain with an<br/>adjacent regulatory module. Spectroscopic and biochemical assays will be<br/>sued to probe the nature and function of this interaction. Together, these<br/>studies will explain many of the biochemical and structural mechanisms used<br/>to regulate the level of activated Rho family GTPases in the cell.
