β3 Tyrosine Phosphorylation and αvβ3-mediated Adhesion Are Required for Vav1 Association and Rho Activation in Leukocytes

Scott D. Blystone,Erik Schaefer,Chunlei Gao,Montaha Lakkis

doi:10.1074/jbc.m414457200

Scott D. Blystone, Erik Schaefer + Show 2 more

Open Access

https://doi.org/10.1074/jbc.m414457200

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Abstract

Integrin alpha(v)beta(3)-mediated adhesion of hematopoietic cells to vitronectin results in activation of the Rho GTPases. Mutation of beta(3) tyrosine residue 747, previously shown to disrupt cell adhesion, results in sustained activation of Cdc42 and diminished Rac and Rho activity. We investigated the role of the hematopoietically restricted guanine nucleotide exchange factor Vav1 in alpha(v)beta(3)-mediated adhesion. We find that Vav1, a guanine nucleotide exchange factor for Rac and Rho, associates with alpha(v)beta(3) upon cell adhesion to vitronectin and that this association requires beta(3) tyrosine phosphorylation. Expression of exogenous Vav1 demonstrates that Y160F, but not wild type or the Vav1Y174F mutant, inhibits Rac and Rho activation during alpha(v)beta(3)-mediated cell adhesion to vitronectin. Cells expressing Vav1Y160F exhibit a sustained Cdc42 activation similar to nonphosphorylatable beta(3) mutants. In addition, cytoskeletal reorganization and cell adhesion are severely suppressed in Vav1Y160F-transfected cells, and Vav1Y160F fails to associate with beta(3) integrins. Furthermore, Vav1 itself is selectively phosphorylated upon tyrosine 160 after alpha(v)beta(3)-mediated adhesion, and the association between Vav1 and beta(3) occurs in specific response to adhesion to substrate. These studies describe a phosphorylation-dependent association between beta(3) integrin and Vav1 which is essential for cell progression to a Rho-dominant phenotype during cell adhesion.

Highlights

The ultimate fate of integrin receptor complexes in adherent cells is their association with the actin cytoskeleton
We find that Vav1, a guanine nucleotide exchange factor for Rac and Rho, associates with ␣v␤3 upon cell adhesion to vitronectin and that this association requires ␤3 tyrosine phosphorylation
Rho Family GTPases Activation and ␤3 Integrin Phosphorylation—Integrin engagement has been shown previously to regulate Rho GTPase activation, and ␤3 integrin phosphorylation plays a critical role in Rho activation during ␣v␤3 integrinmediated cell adhesion to Vn (1)

Summary

EXPERIMENTAL PROCEDURES

Cells and Materials—K562 cells were stably transfected with cDNA encoding wild type ␤3, Y747F ␤3, Y759F ␤3, and Y747F/Y759F ␤3 together with the ␣V subunit and maintained as described previously (2). ␣V␤3 expression in K562 cells (K␣v␤3, K␣v␤3 Y747F, K␣v␤3Y759F, K␣v␤3Y747F/Y759F) was equivalent between cell lines and monitored by flow cytometry. For Vav and GFP immunoprecipitation, cell lysates were precleared with protein A-Sepharose followed by incubation with anti-Vav and anti-GFP antibodies for 1 h at 4 °C and precipitated with protein A-Sepharose. Total Rho, Rac, and Cdc were determined by loading 1/10 total volume of cell lysate on a 12% SDS-polyacrylamide gel under reducing conditions. Antibodies used for Western blotting were polyclonal anti-Rho, anti-Rac, and anti-Cdc. K␣V␤3, K␣V␤3Y747F/Y759F, or GFP-Vav1-, GFP-Vav1Y160F-, or GFP-Vav1Y174F-transfected K␣V␤3 cells were added at 1 ϫ105 cells/well in Hanks’ buffered saline solution containing 1.0 mM Ca2ϩ and Mg2ϩ and allowed to adhere for 1 h at 37 °C. Wells were rinsed three times in Hanks’ buffered saline solution without Ca2ϩ or Mg2ϩ, and adherent cells were fixed in 3.7% formaldehyde for 1 h at 4 °C and stained with 0.05% crystal violet for 30 min at room temperature. Actin was detected by Western blotting with anti-actin monoclonal antibody

In Vitro Tyrosine Phosphorylation and Guanine Nucleotide Exchange

RESULTS

DISCUSSION