title: Phosphorylated cortactin recruits Vav2 guanine nucleotide exchange factor to activate Rac3 and promote invadopodial function in invasive breast cancer cells.
abstract: Breast carcinoma cells use specialized, actin-rich protrusions called invadopodia to degrade and invade through the extracellular matrix. Phosphorylation of the actin nucleation-promoting factor and actin-stabilizing protein cortactin downstream of the epidermal growth factor receptor-Src-Arg kinase cascade is known to be a critical trigger for invadopodium maturation and subsequent cell invasion in breast cancer cells. The functions of cortactin phosphorylation in this process, however, are not completely understood. We identify the Rho-family guanine nucleotide exchange factor Vav2 in a comprehensive screen for human SH2 domains that bind selectively to phosphorylated cortactin. We demonstrate that the Vav2 SH2 domain binds selectively to phosphotyrosine-containing peptides corresponding to cortactin tyrosines Y421 and Y466 but not to Y482. Mutation of the Vav2 SH2 domain disrupts its recruitment to invadopodia, and an SH2-domain mutant form of Vav2 cannot support efficient matrix degradation in invasive MDA-MB-231 breast cancer cells. We show that Vav2 function is required for promoting invadopodium maturation and consequent actin polymerization, matrix degradation, and invasive migratory behavior. Using biochemical assays and a novel Rac3 biosensor, we show that Vav2 promotes Rac3 activation at invadopodia. Rac3 knockdown reduces matrix degradation by invadopodia, whereas a constitutively active Rac3 can rescue the deficits in invadopodium function in Vav2-knockdown cells. Together these data indicate that phosphorylated cortactin recruits Vav2 to activate Rac3 and promote invadopodial maturation in invasive breast cancer cells.
authors.affiliation: Department of Cell Biology, Yale University, New Haven, CT 06520.
authors.forename: Brian J
authors.initials: BJ
authors.lastname: Rosenberg
authors.affiliation: Faculty of Medicine in the Galilee, Bar-Ilan University, Safed 1311520, Israel.
authors.forename: Hava
authors.initials: H
authors.lastname: Gil-Henn
authors.affiliation: Department of Cell Biology, Yale University, New Haven, CT 06520.
authors.forename: Christopher C
authors.initials: CC
authors.lastname: Mader
authors.affiliation: Department of Chemistry, Yale University, New Haven, CT 06520.
authors.forename: Tiffany
authors.initials: T
authors.lastname: Halo
authors.affiliation: Raymond and Beverly Sackler Laboratory of Genetics and Molecular Medicine, Department of Genetics and Genome Sciences and Center for Cell Analysis and Modeling, University of Connecticut School of Medicine, Farmington, CT 06030.
authors.forename: Taofei
authors.initials: T
authors.lastname: Yin
authors.affiliation: Department of Anatomy and Structural Biology and Gruss Lipper Biophotonics Center, Albert Einstein College of Medicine, Bronx, NY 10461.
authors.forename: John
authors.initials: J
authors.lastname: Condeelis
authors.affiliation: Raymond and Beverly Sackler Laboratory of Genetics and Molecular Medicine, Department of Genetics and Genome Sciences and Center for Cell Analysis and Modeling, University of Connecticut School of Medicine, Farmington, CT 06030.
authors.forename: Kazuya
authors.initials: K
authors.lastname: Machida
authors.affiliation: Raymond and Beverly Sackler Laboratory of Genetics and Molecular Medicine, Department of Genetics and Genome Sciences and Center for Cell Analysis and Modeling, University of Connecticut School of Medicine, Farmington, CT 06030.
authors.forename: Yi I
authors.initials: YI
authors.lastname: Wu
authors.affiliation: Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06520 anthony.koleske@yale.edu.|Department of Neuroscience, Yale University, New Haven, CT 06520.
authors.forename: Anthony J
authors.initials: AJ
authors.lastname: Koleske
mesh_terms: D000199:Actins / Q000378:metabolism; D001943:Breast Neoplasms / Q000378:metabolism* / Q000473:pathology*; D045744:Cell Line, Tumor; D051356:Cortactin / Q000378:metabolism*; D005109:Extracellular Matrix / Q000378:metabolism; D020662:Guanine Nucleotide Exchange Factors / Q000378:metabolism; D006801:Humans; D009361:Neoplasm Invasiveness; D010766:Phosphorylation; D019000:Phosphotyrosine / Q000378:metabolism; D000069261:Podosomes / Q000378:metabolism* / Q000502:physiology; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D051567:Proto-Oncogene Proteins c-vav / Q000378:metabolism*; D011554:Pseudopodia / Q000378:metabolism; D020744:rac GTP-Binding Proteins / Q000378:metabolism
chemical_list: D000199:Actins; D051356:Cortactin; D020662:Guanine Nucleotide Exchange Factors; D051567:Proto-Oncogene Proteins c-vav; C411563:RAC3 protein, human; C093651:VAV2 protein, human; D019000:Phosphotyrosine; D011505:Protein-Tyrosine Kinases; D020744:rac GTP-Binding Proteins