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

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.

SUMOylation regulates nuclear accumulation and signaling activity of the soluble intracellular domain of the ErbB4 receptor tyrosine kinase.

Erb-B2 receptor tyrosine kinase 4 (ErbB4) is a kinase that can signal via a proteolytically released intracellular domain (ICD) in addition to classical receptor tyrosine kinase-activated signaling cascades. Previously, we have demonstrated that ErbB4 ICD is posttranslationally modified by the small ubiquitin-like modifier (SUMO) and functionally interacts with the PIAS3 SUMO E3 ligase. However, direct evidence of SUMO modification in ErbB4 signaling has remained elusive. Here, we report that the conserved lysine residue 714 in the ErbB4 ICD undergoes SUMO modification, which was reversed by sentrin-specific proteases (SENPs) 1, 2, and 5. Although ErbB4 kinase activity was not necessary for the SUMOylation, the SUMOylated ErbB4 ICD was tyrosine phosphorylated to a higher extent than unmodified ErbB4 ICD. Mutation of the SUMOylation site compromised neither ErbB4-induced phosphorylation of the canonical signaling pathway effectors Erk1/2, Akt, or STAT5 nor ErbB4 stability. In contrast, SUMOylation was required for nuclear accumulation of the ErbB4 ICD. We also found that Lys-714 was located within a leucine-rich stretch, which resembles a nuclear export signal, and could be inactivated by site-directed mutagenesis. Furthermore, SUMOylation modulated the interaction of ErbB4 with chromosomal region maintenance 1 (CRM1), the major nuclear export receptor for proteins. Finally, the SUMO acceptor lysine was functionally required for ErbB4 ICD-mediated inhibition of mammary epithelial cell differentiation in a three-dimensional cell culture model. Our findings indicate that a SUMOylation-mediated mechanism regulates nuclear localization and function of the ICD of ErbB4 receptor tyrosine kinase.

An Unbiased Functional Genetics Screen Identifies Rare Activating ERBB4 Mutations.

Despite the relatively high frequency of somatic ERBB4 mutations in various cancer types, only a few activating ERBB4 mutations have been characterized, primarily due to lack of mutational hotspots in the ERBB4 gene. Here, we utilized our previously published pipeline, an in vitro screen for activating mutations, to perform an unbiased functional screen to identify potential activating ERBB4 mutations from a randomly mutated ERBB4 expression library. Ten potentially activating ERBB4 mutations were identified and subjected to validation by functional and structural analyses. Two of the 10 ERBB4 mutants, E715K and R687K, demonstrated hyperactivity in all tested cell models and promoted cellular growth under two-dimensional and three-dimensional culture conditions. ERBB4 E715K also promoted tumor growth in in vivo Ba/F3 cell mouse allografts. Importantly, all tested ERBB4 mutants were sensitive to the pan-ERBB tyrosine kinase inhibitors afatinib, neratinib, and dacomitinib. Our data indicate that rare ERBB4 mutations are potential candidates for ERBB4-targeted therapy with pan-ERBB inhibitors. Statement of Significance: ERBB4 is a member of the ERBB family of oncogenes that is frequently mutated in different cancer types but the functional impact of its somatic mutations remains unknown. Here, we have analyzed the function of over 8,000 randomly mutated ERBB4 variants in an unbiased functional genetics screen. The data indicate the presence of rare activating ERBB4 mutations in cancer, with potential to be targeted with clinically approved pan-ERBB inhibitors.

Genome-wide screen of gamma-secretase-mediated intramembrane cleavage of receptor tyrosine kinases.

Receptor tyrosine kinases (RTKs) have been demonstrated to signal via regulated intramembrane proteolysis, in which ectodomain shedding and subsequent intramembrane cleavage by gamma-secretase leads to release of a soluble intracellular receptor fragment with functional activity. For most RTKs, however, it is unknown whether they can exploit this new signaling mechanism. Here we used a system-wide screen to address the frequency of susceptibility to gamma-secretase cleavage among human RTKs. The screen covering 45 of the 55 human RTKs identified 12 new as well as all nine previously published gamma-secretase substrates. We biochemically validated the screen by demonstrating that the release of a soluble intracellular fragment from endogenous AXL was dependent on the sheddase disintegrin and metalloprotease 10 (ADAM10) and the gamma-secretase component presenilin-1. Functional analysis of the cleavable RTKs indicated that proliferation promoted by overexpression of the TAM family members AXL or TYRO3 depends on gamma-secretase cleavage. Taken together, these data indicate that gamma-secretase-mediated cleavage provides an additional signaling mechanism for numerous human RTKs.

Time-resolved dissection of early phosphoproteome and ensuing proteome changes in response to TGF-ß.

Transforming growth factor-ß (TGF-ß) signaling promotes cell motility by inducing epithelial-to-mesenchymal transitions (EMTs) in normal physiology and development, as well as in pathological conditions, such as cancer. We performed a time-resolved analysis of the proteomic and phosphoproteomic changes of cultured human keratinocytes undergoing EMT and cell cycle arrest in response to stimulation with TGF-ß. We quantified significant changes in 2079 proteins and 2892 phosphorylation sites regulated by TGF-ß. We identified several proteins known to be involved in TGF-ß-induced cellular processes, such as the cytostatic response, extracellular matrix remodeling, and epithelial dedifferentiation. In addition, we identified proteins involved in other cellular functions, such as vesicle trafficking, that were not previously associated with TGF-ß signaling. Although many TGF-ß responses are mediated by phosphorylation of the transcriptional regulators of the SMAD family by the TGF-ß receptor complex, we observed rapid kinetics of changes in protein phosphorylation, indicating that many responses were mediated through SMAD-independent TGF-ß signaling. Combined analysis of changes in protein abundance and phosphorylation and knowledge of protein interactions and transcriptional regulation provided a comprehensive representation of the dynamic signaling events underlying TGF-ß-induced changes in cell behavior. Our data suggest that in epithelial cells stimulated with TGF-ß, early signaling is a mixture of both pro- and antiproliferative signals, whereas later signaling primarily inhibits proliferation.