Biochemical characterization of hyperactive beta2-chimaerin mutants revealed an enhanced exposure of C1 and Rac-GAP domains.
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.
Sosa
Maria Soledad
MS
Department of Pharmacology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104, USA.
Lewin
Nancy E
NE
Choi
Sung-Hee
SH
Blumberg
Peter M
PM
Kazanietz
Marcelo G
MG
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*
D020690:GTPase-Activating Proteins; D008024:Ligands; D009363:Neoplasm Proteins; D010743:Phospholipids; C079844:beta-chimaerin; D015240:Phorbol 12,13-Dibutyrate; D020744:rac GTP-Binding Proteins