Phosphorylated cortactin recruits Vav2 guanine nucleotide exchange factor to activate Rac3 and promote invadopodial function in invasive breast cancer cells

Brian J. Rosenberg, Hava Gil-Henn, Christopher C. Mader, Tiffany Halo, Taofei Yin, John S. Condeelis, Kazuya Machida, Yi I. Wu, Anthony J. Koleske

Research output: Contribution to journalArticle

15 Citations (Scopus)

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.

Original languageEnglish (US)
Pages (from-to)1347-1360
Number of pages14
JournalMolecular Biology of the Cell
Volume28
Issue number10
DOIs
StatePublished - May 15 2017

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Cortactin
Guanine Nucleotide Exchange Factors
Breast Neoplasms
src Homology Domains
Actins
Rho Guanine Nucleotide Exchange Factors
Phosphorylation
Phosphotyrosine
src-Family Kinases
Biosensing Techniques
Podosomes
Epidermal Growth Factor Receptor
Polymerization
Extracellular Matrix
Tyrosine
Peptides
Mutation

ASJC Scopus subject areas

  • Molecular Biology
  • Cell Biology

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Phosphorylated cortactin recruits Vav2 guanine nucleotide exchange factor to activate Rac3 and promote invadopodial function in invasive breast cancer cells. / Rosenberg, Brian J.; Gil-Henn, Hava; Mader, Christopher C.; Halo, Tiffany; Yin, Taofei; Condeelis, John S.; Machida, Kazuya; Wu, Yi I.; Koleske, Anthony J.

In: Molecular Biology of the Cell, Vol. 28, No. 10, 15.05.2017, p. 1347-1360.

Research output: Contribution to journalArticle

Rosenberg, Brian J. ; Gil-Henn, Hava ; Mader, Christopher C. ; Halo, Tiffany ; Yin, Taofei ; Condeelis, John S. ; Machida, Kazuya ; Wu, Yi I. ; Koleske, Anthony J. / Phosphorylated cortactin recruits Vav2 guanine nucleotide exchange factor to activate Rac3 and promote invadopodial function in invasive breast cancer cells. In: Molecular Biology of the Cell. 2017 ; Vol. 28, No. 10. pp. 1347-1360.
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AU - Rosenberg, Brian J.

AU - Gil-Henn, Hava

AU - Mader, Christopher C.

AU - Halo, Tiffany

AU - Yin, Taofei

AU - Condeelis, John S.

AU - Machida, Kazuya

AU - Wu, Yi I.

AU - Koleske, Anthony J.

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AB - 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.

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