The guanine nucleotide exchange factor VAV3 participates in ERBB4-mediated cancer cell migration

Abstract

ERBB4 is a member of the epidermal growth factor receptor (EGFR)/ERBB subfamily of receptor tyrosine kinases that regulates cellular processes including proliferation, migration, and survival. ERBB4 signaling is involved in embryogenesis and homeostasis of healthy adult tissues, but also in human pathologies such as cancer, neurological disorders, and cardiovascular diseases. Here, an MS-based analysis revealed the Vav guanine nucleotide exchange factor 3 (VAV3), an activator of Rho family GTPases, as a critical ERBB4-interacting protein in breast cancer cells. We confirmed the ERBB4-VAV3 interaction by targeted MS and coimmunoprecipitation experiments and further defined it by demonstrating that kinase activity and Tyr-1022 and Tyr-1162 of ERBB4, as well as the intact phosphotyrosine-interacting SH2 domain of VAV3, are necessary for this interaction. We found that ERBB4 stimulates tyrosine phosphorylation of the VAV3 activation domain, known to be required for guanine nucleotide exchange factor (GEF) activity of VAV proteins. In addition to VAV3, the other members of the VAV family, VAV1 and VAV2, also coprecipitated with ERBB4. Analyses of the effects of overexpression of dominant-negative VAV3 constructs or shRNA-mediated down-regulation of VAV3 expression in breast cancer cells indicated that active VAV3 is involved in ERBB4-stimulated cell migration. These results define the VAV GEFs as effectors of ERBB4 activity in a signaling pathway relevant for cancer cell migration.

Keywords: ERBB4; HER4; Vav guanine nucleotide exchange factor 3 (VAV3); breast cancer; cancer; cell migration; guanine nucleotide exchange factor (GEF); phosphotyrosine signaling; protein-protein interaction; receptor tyrosine kinase.

Figure 1.

A, schematic structure of the ERBB4 isoforms JM-a CYT-1 and JM-a CYT-2. P, phosphorylated tyrosine residues in the cytoplasmic domains of activated ERBB4 isoforms. Red color in the CYT-1 cytoplasmic tail indicates the CYT-1–specific 16-amino acid insert. Whereas both isoforms have binding sites for Shc, the CYT-1–specific region has a binding site for P85, the regulatory subunit of PI3K. CYT-2 is more heavily phosphorylated than CYT-1. B, Strep-tagged ERBB4 JM-a CYT-1 and ERBB4 JM-a CYT-2 were expressed in MCF-7 cells, and the interacting proteins were purified using Strep-Tactin Superflow columns. The purified proteins were separated by SDS-PAGE and silver-stained. Each sample was loaded into two adjacent lanes of the gel. C, proteins copurifying with ERBB4 isoforms.

Figure 2.

Interaction of ERBB4 isoforms with VAV3. COS-7 cells were transfected with constructs encoding ERBB4 JM-a CYT-1 or ERBB4 JM-a CYT-2 and MYC-HIS–tagged VAV3. Lysates were incubated with anti-MYC (2272) to immunoprecipitate (IP) VAV3. Immunoprecipitation samples and total lysates were analyzed by Western blotting (W) with anti-ERBB4 (E200) and anti-MYC (2272).

Figure 3.

Molecular determinants of ERBB4-VAV3 interaction. A, COS-7 cells were transfected with ERBB4 (JM-a CYT-2 isoform) and WT or mutant MYC-HIS–tagged VAV3 constructs. Lysates were immunoprecipitated with anti-ERBB4 (sc-283). Immunoprecipitation (IP) samples and total lysates were analyzed by Western blotting (W) with anti-HIS and anti-ERBB4 (sc-283). R697L disrupts the SH2 domain of VAV3; the 1-PH construct lacks the SH2 domain. B, COS-7 cells were transfected with ERBB4 ICD and WT or mutant MYC-HIS–tagged VAV3 constructs and analyzed as in A. C, COS-7 cells were transfected with constructs encoding WT or kinase-dead K751R ERBB4 and MYC-HIS–tagged VAV3. Lysates were immunoprecipitated with anti-ERBB4 (HFR-1). Immunoprecipitation samples and total lysates were analyzed by Western blotting with anti-MYC (2272) and anti-ERBB4 (E200). D, COS-7 cells were transfected with constructs encoding WT or mutant ERBB4 and MYC-HIS–tagged VAV3 and analyzed as in C.

Figure 4.

Interaction of ERBB4 with VAV1, VAV2, and VAV3. COS-7 cells were transfected with constructs encoding ERBB4 (JM-a CYT-2 isoform) and HA-tagged VAV1, VAV2, or VAV. Lysates were immunoprecipitated with anti-ERBB4 (sc-283). Immunoprecipitation (IP) samples and total lysates were analyzed by Western blotting (W) with anti-HA and anti-ERBB4 (sc-283).

Figure 5.

Phosphorylation of VAV3. A, lysates from COS-7 cells expressing MYC-HIS–tagged VAV3 were immunoprecipitated (IP) with anti-MYC (9E10), and precipitates were incubated without or with ERBB4 ICD (CYT-2 isoform, including the kinase domain) in the presence of ATP. Samples were analyzed by Western blotting (W) with anti-phosphotyrosine 4G10 and anti-MYC (9E10). B, COS-7 cells were transfected with ERBB4 (JM-a CYT-2 isoform) and MYC-HIS–tagged VAV3. Lysates were immunoprecipitated with anti-MYC (9E10), and samples were analyzed by Western with anti-phosphotyrosine 4G10, anti-HIS, and anti-ERBB4 (sc-283). Total lysates were analyzed by Western with anti-MYC (9E10) and anti-ERBB4 (sc-283). C, COS-7 cells were transfected with ERBB4 (JM-a CYT-2 isoform) and WT or mutant MYC-HIS-tagged VAV3. Lysates were immunoprecipitated with anti-MYC (9E10), and samples were analyzed by Western with anti-phosphotyrosine 4G10, anti-HIS, and anti-ERBB4 (E200). Total lysates were analyzed by Western blotting with anti-HIS and anti-ERBB4 (E200). R697L disrupts the SH2 domain of VAV3; Tyr-160, -164, and -173 are predicted phosphorylation sites of VAV3. D, lysates from COS-7 cells expressing ERBB4 (JM-a CYT-2 isoform) and WT or mutant MYC-HIS–tagged VAV3 were immunoprecipitated with anti-MYC (9E10) and analyzed by Western blotting with anti-phosphotyrosine 4G10, anti-MYC (9E10), and anti-ERBB4 (E200). Total lysates were analyzed by Western blotting with anti-MYC (9E10) and anti-ERBB4 (E200), and loading was controlled with anti-actin. E, MCF-7 cells expressing ERBB4 (JM-a CYT-2 isoform) and WT or mutant MYC-HIS–tagged VAV3 were stimulated with 50 ng/ml NRG-1 for 10 min. Lysates were analyzed by Western blotting with anti-phospho-MLC, anti-MLC, and anti-actin.

Figure 6.

Role of VAV3 in ERBB4-dependent migration. A, NIH3T3-7d cells stably expressing ERBB4 (JM-b CYT-1 isoform) were transfected without or with a construct encoding MYC-HIS–tagged dominant-negative VAV3 construct (DN-VAV3). Expression of ERBB4 and VAV3 was analyzed by Western blotting (W) with anti-ERBB4 (sc-283) and anti-HIS. B, NIH3T3 transfectants were plated in Transwell cell culture inserts with or without 25 ng/ml NRG-1 or 1% fetal calf serum in the bottom well and allowed to migrate for 8 h before fixation and crystal violet staining. Representative images of cells migrated through the Transwell membrane are shown. Bar, 100 μm. C, quantification of migrated cells. Data from three experiments are presented as a box plot, with horizontal lines indicating the median, boxes indicating the second and third quartile, and whiskers indicating the minimum and maximum values of the distribution. Individual data points are indicated as dots. For statistical testing, negative binomial regression analysis was used. The p values were Benjamini–Hochberg corrected for multiple testing error. D, MCF-7 cells stably expressing scrambled control shRNA or shRNAs targeting ERBB4 or VAV3 were analyzed for ERBB4 and VAV3 expression by Western with anti-ERBB4 (E200) and anti-VAV3. Anti-lamin B was used to control loading. E, MCF-7 cells stably expressing the indicated shRNAs were plated in Transwell cell culture inserts with 0 or 10 ng/ml NRG-1 in the bottom well. The cells were allowed to migrate for 8 h and analyzed as in B. Bar, 100 μm. F, quantification of migrated cells. Data from 10–18 experiments are presented as a box plot with statistical analysis as in C, except that the whiskers indicate 1.5 × interquartile range. G, quantification of migrated MCF-7 cells expressing WT or Y160F/Y164F/Y173F mutant VAV3. Migration was analyzed in the presence or absence of 10 ng/ml NRG-1 as in E. Data from four experiments are presented as a box plot with statistical analysis as in C.