Specificity of Rho insert-mediated activation of phospholipase D1.
Members of the Rho subfamily of GTP-binding proteins regulate phospholipase D1 (PLD1) activity and signaling. In previous work, we demonstrated that binding of the Rho family member Cdc42 to PLD1 and the subsequent stimulation of its enzymatic activity are distinct events. Deletion of the insert helix from Cdc42 does not interfere with its switch I-mediated, GTP-dependent binding to PLD1 but inhibits Cdc42-stimulated PLD1 activity. To understand the mechanism of the insert-mediated activation of PLD1 by Cdc42 and to develop reagents to study Cdc42-activated PLD1 in cellular signaling events, we have undertaken a mutational analysis of the Rho insert region of Cdc42 and examined the specificity of the insert helix requirement in the other Rho family members, RhoA and Rac1. Here, we identify a critical residue, serine 124, in the Cdc42 insert helix central to its activation mechanism. Further, we examine this activation mechanism with respect to other members of the Rho family and demonstrate that each Rho protein activates PLD by distinct mechanisms, potentially allowing for unique signaling outcomes in the cell.
Walker
Stephanie J
SJ
Department of Molecular Medicine, Veterinary Medical Center, Field of Biochemistry, Molecular, and Cell Biology, Cornell University, Ithaca, New York 14853.
Brown
H Alex
HA
D019943:Amino Acid Substitution; D004252:DNA Mutational Analysis; D004789:Enzyme Activation; D006160:Guanosine Triphosphate / Q000378:metabolism; D018257:Helix-Loop-Helix Motifs; D006801:Humans; D016297:Mutagenesis, Site-Directed; D010739:Phospholipase D / Q000378:metabolism*; D012694:Serine / Q000378:metabolism; D015398:Signal Transduction; D013329:Structure-Activity Relationship; D020764:cdc42 GTP-Binding Protein / Q000235:genetics / Q000378:metabolism*; D020830:rac1 GTP-Binding Protein / Q000235:genetics / Q000378:metabolism*; D020742:rhoA GTP-Binding Protein / Q000235:genetics / Q000378:metabolism*
D012694:Serine; D006160:Guanosine Triphosphate; D010739:Phospholipase D; C106801:phospholipase D1; D020764:cdc42 GTP-Binding Protein; D020830:rac1 GTP-Binding Protein; D020742:rhoA GTP-Binding Protein