Receptor tyrosine kinases (RTKs) can signal via regulated intramembrane proteolysis, leading to release of an intracellular receptor fragment with functional activity. A system-wide screen covering 45 of the 55 human RTKs identified 12 novel gamma-secretase substrates and indicated that at least half of all RTKs are susceptible to cleavage.

Transforming growth factor-b (TGF-b) 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-b. We quantified significant changes in 2079 proteins and 2892 phosphorylation sites regulated by TGF-b. We identified several proteins known to be involved in TGF-b-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-b signaling. Although many TGF-b responses are mediated by phosphorylation of the transcriptional regulators of the SMAD family by the TGF-b receptor complex, we observed rapid kinetics of changes in protein phosphorylation, indicating that many responses were mediated through SMADindependent TGF-b 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-b-induced changes in cell behavior. Our data suggest that in epithelial cells stimulated with TGF-b, early signaling is a mixture of both pro-and antiproliferative signals, whereas later signaling primarily inhibits proliferation.

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.