A Cdc42 mutant specifically activated by intersectin.
The Rho family GTPase Cdc42 functions as a molecular switch and controls many fundamental cellular processes such as cytoskeletal regulation, cell polarity, and vesicular trafficking. Guanine nucleotide exchange factors of the Dbl family activate Cdc42 and other Rho GTPases by catalyzing the removal of bound GDP, allowing for GTP loading, and subsequent effector recognition ultimately leading to downstream signaling events. Analysis of existing structural data reveals that the Dbl exchange factor intersectin engages a strictly conserved GTPase residue of Cdc42 (tyrosine 32) in a unique mode with respect to all other visualized exchange factor-Rho GTPase interfaces. To investigate this differential binding architecture, we analyzed the role of tyrosine 32 of Cdc42 in binding, and stimulation by Dbl family exchange factors. Deletion of the hydroxyl side chain of tyrosine 32 substantially increases the affinity of Cdc42 for intersectin, yet severely cripples interaction with Dbs, a normally potent exchange factor of Cdc42. Moreover, Cdc42(Y32F) is exclusively activated by intersectin, while virtually unresponsive to other Cdc42-activating exchange factors in vitro and in vivo. Further, the structural determinants unique to intersectin, which permit selective recognition and concomitant stimulation of Cdc42(Y32F), have been defined. Cdc42 and other individual Rho GTPases receive input stimulatory signals from a multitude of Dbl exchange factors, and therefore, Cdc42(Y32F) could act as a valuable reagent for understanding the specific influence of ITSN on Cdc42-mediated signaling phenomena.
Smith
William J
WJ
Department of Chemistry and Chemical Biology, Cornell University, Ithaca, New York 14853, USA.
Hamel
Brant
B
Yohe
Marielle E
ME
Sondek
John
J
Cerione
Richard A
RA
Snyder
Jason T
JT
D033942:Adaptor Proteins, Vesicular Transport / Q000737:chemistry* / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D002384:Catalysis; D018360:Crystallography, X-Ray; D003599:Cytoskeleton / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006147:Guanine / Q000737:chemistry; D020662:Guanine Nucleotide Exchange Factors / Q000737:chemistry; D006153:Guanosine Diphosphate / Q000737:chemistry; D006160:Guanosine Triphosphate / Q000737:chemistry; D006367:HeLa Cells; D006801:Humans; D007700:Kinetics; D051379:Mice; D008856:Microscopy, Fluorescence; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D010957:Plasmids / Q000378:metabolism; D017354:Point Mutation; D011485:Protein Binding; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D051567:Proto-Oncogene Proteins c-vav / Q000737:chemistry; D064067:Rho Guanine Nucleotide Exchange Factors; D015398:Signal Transduction; D020349:Surface Plasmon Resonance; D013997:Time Factors; D014443:Tyrosine / Q000737:chemistry; D020764:cdc42 GTP-Binding Protein / Q000235:genetics*; D020741:rho GTP-Binding Proteins / Q000737:chemistry
D033942:Adaptor Proteins, Vesicular Transport; D018076:DNA, Complementary; D020662:Guanine Nucleotide Exchange Factors; C092132:Mcf2l protein, mouse; D051567:Proto-Oncogene Proteins c-vav; D064067:Rho Guanine Nucleotide Exchange Factors; C496619:Vav1 protein, mouse; C493893:WGEF protein, mouse; C115919:intersectin 1; D006153:Guanosine Diphosphate; D049452:Green Fluorescent Proteins; D014443:Tyrosine; D006147:Guanine; D006160:Guanosine Triphosphate; D020764:cdc42 GTP-Binding Protein; D020741:rho GTP-Binding Proteins