title: Biochemical characterization of hyperactive beta2-chimaerin mutants revealed an enhanced exposure of C1 and Rac-GAP domains.
abstract: Recent studies established that the Rac-GAP beta2-chimaerin plays important roles in development, neuritogenesis, and cancer progression. A unique feature of beta2-chimaerin is that it can be activated by phorbol esters and the lipid second messenger diacylglycerol (DAG), which bind with high affinity to its C1 domain and promote beta2-chimaerin translocation to membranes, leading to the inactivation of the small G-protein Rac. Crystallographic evidence and cellular studies suggest that beta2-chimaerin remains in an inactive conformation in the cytosol with the C1 domain inaccessible to ligands. We developed a series of beta2-chimaerin point mutants in which intramolecular contacts that occlude the C1 domain have been disrupted. These mutants showed enhanced translocation in response to phorbol 12-myristate 13-acetate (PMA) in cells. Binding assays using [(3)H]phorbol 12,13-dibutyrate ([(3)H]PDBu) revealed that internal contact mutants have a reduced acidic phospholipid requirement for phorbol ester binding. Moreover, disruption of intramolecular contacts enhances binding of beta2-chimaerin to acidic phospholipid vesicles and confers enhanced Rac-GAP activity in vitro. These studies suggest that beta2-chimaerin must undergo a conformational rearrangement in order to expose its lipid binding sites and become activated.
authors.affiliation: Department of Pharmacology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104, USA.
authors.forename: Maria Soledad
authors.initials: MS
authors.lastname: Sosa
authors.forename: Nancy E
authors.initials: NE
authors.lastname: Lewin
authors.forename: Sung-Hee
authors.initials: SH
authors.lastname: Choi
authors.forename: Peter M
authors.initials: PM
authors.lastname: Blumberg
authors.forename: Marcelo G
authors.initials: MG
authors.lastname: Kazanietz
mesh_terms: D000818:Animals; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D004789:Enzyme Activation; D020690:GTPase-Activating Proteins / Q000378:metabolism*; D008024:Ligands; D008958:Models, Molecular; D009154:Mutation; D009363:Neoplasm Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D015240:Phorbol 12,13-Dibutyrate / Q000378:metabolism / Q000494:pharmacology; D010743:Phospholipids / Q000378:metabolism; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D021381:Protein Transport / Q000187:drug effects; D020744:rac GTP-Binding Proteins / Q000378:metabolism*
chemical_list: D020690:GTPase-Activating Proteins; D008024:Ligands; D009363:Neoplasm Proteins; D010743:Phospholipids; C079844:beta-chimaerin; D015240:Phorbol 12,13-Dibutyrate; D020744:rac GTP-Binding Proteins