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

Veera K Ojala,Anna M Knittle,Peppi Kirjalainen,Johannes A.M Merilahti,Maarit Kortesoja,Denis Tvorogov,Katri Vaparanta,Shujun Lin,Jürgen Kast,Arto T Pulliainen,Kari J Kurppa,Klaus Elenius

doi:10.1074/jbc.ra119.010925

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.

Highlights

ERBB receptors form the epidermal growth factor receptor (EGFR)/ERBB subfamily of receptor tyrosine kinases (RTK) that includes EGFR/ERBB1, ERBB2, ERBB3, and ERBB4
Whereas of the ERBB4-copurifying proteins were detected in both CYT-1 and CYT-2 samples, 7 proteins were identified in CYT-1 samples and in CYT-2 samples (Fig. 1C)
ERBB4 signaling is involved in embryogenesis and homeostasis of adult tissues, and in human pathologies including cancer [8, 9]

Summary

Introduction

ERBB receptors form the epidermal growth factor receptor (EGFR)/ERBB subfamily of receptor tyrosine kinases (RTK) that includes EGFR/ERBB1, ERBB2, ERBB3, and ERBB4. These findings indicate that VAV GTPases are novel effectors of ERBB4 activity in a signaling pathway relevant for cancer cell migration. To identify novel ERBB4-interacting proteins, Strep-tagged ERBB4 constructs encoding the cleavable JM-a isoform of the CYT-1 or CYT-2 type were generated and stably transfected to MCF-7 breast cancer cells (Fig. 1A).

Results

Conclusion