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.
|Number of pages||14|
|Journal||Molecular Biology of the Cell|
|State||Published - 15 May 2017|
Bibliographical noteFunding Information:
We thank Javier Bravo-Cordero for invaluable advice during the initial stages of this project and are grateful to Xianyun Ye for technical support. This work was funded by National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS) Grant T32GM007205 (B.J.R.), the Israel Science Foundation and Israel Cancer Research Fund (to H.G.H.), NIH/National Cancer Institute (NCI) Grants CA150344 and CA1154966 (J.C.), LLS Quest for CURES (K.M.), NIH/NIGMS Grant GM117061 (Y.I.W.), and NIH/NCI Grant CA133346 (A.J.K.).
© 2017 Rosenberg et al.