The activity of the GTPase-activating protein CdGAP is regulated by the endocytic protein intersectin.
The Rho GTPases RhoA, Rac1, and Cdc42 play a major role in regulating the reorganization of the actin cytoskeleton. We recently identified CdGAP, a novel GTPase-activating protein with activity toward Rac1 and Cdc42. CdGAP consists of a N-terminal GAP domain, a central domain, and a C-terminal proline-rich domain. Here we show that through a subset of its Src homology 3 domains, the endocytic protein intersectin interacts with CdGAP. In platelet-derived growth factor-stimulated Swiss 3T3 cells, intersectin co-localizes with CdGAP and inhibits its GAP activity toward Rac1. Intersectin-Src homology 3 also inhibits CdGAP activity in GAP assays in vitro. Although the C-terminal proline-rich domain of CdGAP is required for the regulation of its GAP activity by intersectin both in vivo and in vitro, it is not necessary for CdGAP-intersectin interaction. Our data suggest that the central domain of CdGAP is required for CdGAP-intersectin interaction. Thus, we propose a model in which intersectin binding results in a change of CdGAP conformation involving the proline-rich domain that leads to the inhibition of its GAP activity. These observations provide the first demonstration of a direct regulation of RhoGAP activity through a protein-protein interaction and suggest a function for intersectin in Rac1 regulation and actin dynamics.
Jenna
Sarah
S
Department of Anatomy and Cell Biology, McGill University, Montreal, QC H3A 2B2, Canada.
Hussain
Natasha K
NK
Danek
Eric I
EI
Triki
Ibtissem
I
Wasiak
Sylwia
S
McPherson
Peter S
PS
Lamarche-Vane
Nathalie
N
D016475:3T3 Cells; D033942:Adaptor Proteins, Vesicular Transport; D000818:Animals; D001665:Binding Sites; D019921:Biotinylation; D019556:COS Cells; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D006224:Cricetinae; D004705:Endocytosis / Q000502:physiology*; D020690:GTPase-Activating Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D051379:Mice; D016133:Polymerase Chain Reaction; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D014162:Transfection; D020741:rho GTP-Binding Proteins / Q000378:metabolism; D018909:src Homology Domains
D033942:Adaptor Proteins, Vesicular Transport; C115491:Arhgap31 protein, mouse; D002352:Carrier Proteins; D020690:GTPase-Activating Proteins; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; C115919:intersectin 1; D020741:rho GTP-Binding Proteins