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Src homology 3 (SH3) domains play a critical role in mediating protein-protein interactions (PPIs) involved in cell proliferation, migration, and the cytoskeleton. Despite their abundance in the human proteome, the functions and molecular interactions of many SH3 domains remain unknown, and this is in part due to the lack of SH3-domain-specific reagents available for their study. Affimer proteins have been developed as affinity reagents targeting a diverse range of targets, including those involved in PPIs. In this study, Affimer proteins were isolated against both the N- and C-terminal SH3 domains (NSH3 and CSH3) of growth-factor-receptor-bound protein 2 (Grb2), an adapter protein that provides a critical link between cell surface receptors and Ras signalling pathways. Targeting the CSH3 alone for the inhibition of PPIs appeared sufficient for curtailing Ras signalling in mammalian cell lines stimulated with human epidermal growth factor (EGF), which conflicts with the notion that the predominant interactions with Ras activating Son of sevenless (SOS) occur via the NSH3 domain. This result supports a model in which allosteric mechanisms involved in Grb2-SOS1 interaction modulate Ras activation.

Vascular endothelial growth factor A (VEGF-A) regulates many aspects of vascular function. VEGF-A binding to vascular endothelial growth factor receptor 2 (VEGFR2) stimulates endothelial signal transduction and regulates multiple cellular responses. Activated VEGFR2 undergoes ubiquitination but the enzymes that regulate this post-translational modification are unclear. In this study, the de-ubiquitinating enzyme, USP8, is shown to regulate VEGFR2 trafficking, de-ubiquitination, proteolysis and signal transduction. USP8-depleted endothelial cells displayed altered VEGFR2 ubiquitination and production of a unique VEGFR2 extracellular domain proteolytic fragment caused by VEGFR2 accumulation in the endosome-lysosome system. In addition, perturbed VEGFR2 trafficking impaired VEGF-A-stimulated signal transduction in USP8-depleted cells. Thus, regulation of VEGFR2 ubiquitination and de-ubiquitination has important consequences for the endothelial cell response and vascular physiology., (© 2015 The Authors. Traffic published by John Wiley & Sons Ltd.)

The vascular endothelial growth factor A (VEGF-A) is a multifunctional cytokine that stimulates blood vessel sprouting, vascular repair, and regeneration. VEGF-A binds to VEGF receptor tyrosine kinases (VEGFRs) and stimulates intracellular signaling leading to changes in vascular physiology. An important aspect of this phenomenon is the spatiotemporal coordination of VEGFR trafficking and intracellular signaling to ensure that VEGFR residence in different organelles is linked to downstream cellular outputs. Here, we describe a series of assays to evaluate the effects of VEGF-A-stimulated intracellular signaling from intracellular compartments such as the endosome-lysosome system. These assays include the initial isolation and characterization of primary human endothelial cells, performing reverse genetics for analyzing protein function; methods used to study receptor trafficking, signaling, and proteolysis; and assays used to measure changes in cell migration, proliferation, and tubulogenesis. Each of these assays has been exemplified with studies performed in our laboratories. In conclusion, we describe necessary techniques for studying the role of VEGF-A in endothelial cell function., (© 2014 Elsevier Inc. All rights reserved.)

Tumour invasion and metastasis is the most common cause of death from cancer. For epithelial cells to invade surrounding tissues and metastasise, an epithelial-mesenchymal transition (EMT) is required. We have demonstrated that FGFR1 expression is increased in bladder cancer and that activation of FGFR1 induces an EMT in urothelial carcinoma (UC) cell lines. Here, we created an in vitro FGFR1-inducible model of EMT, and used this model to identify regulators of urothelial EMT. FGFR1 activation promoted EMT over a period of 72 hours. Initially a rapid increase in actin stress fibres occurred, followed by an increase in cell size, altered morphology and increased migration and invasion. By using site-directed mutagenesis and small molecule inhibitors we demonstrated that combined activation of the mitogen activated protein kinase (MAPK) and phospholipase C gamma (PLCγ) pathways regulated this EMT. Actin stress fibre formation was regulated by PLCγ activation, and was also important for the increase in cell size, migration and altered morphology. MAPK activation regulated migration and E-cadherin expression, indicating that combined activation of PLCγ and MAPK is required for a full EMT. We used expression microarrays to assess changes in gene expression downstream of these signalling cascades. COX-2 was transcriptionally upregulated by FGFR1 and caused increased intracellular prostaglandin E(2) levels, which promoted migration. In conclusion, we have demonstrated that FGFR1 activation in UC cells lines promotes EMT via coordinated activation of multiple signalling pathways and by promoting activation of prostaglandin synthesis.

Although activating mutations of fibroblast growth factor receptor 3 (FGFR3) are frequent in bladder tumors, little information is available on their specific effects in urothelial cells or the basis for the observed mutation spectrum. We investigated the phenotypic and signaling consequences of three FGFR3 mutations (S249C, Y375C, and K652E) in immortalized normal human urothelial cells (TERT-NHUC) and mouse fibroblasts (NIH-3T3). In TERT-NHUC, all mutant forms of FGFR3 induced phosphorylation of FRS2alpha and ERK1/2, but not AKT or SRC. PLCgamma1 phosphorylation was only observed in TERT-NHUC expressing the common S249C and Y375C mutations, and not the rare K652E mutation. Cells expressing S249C and Y375C FGFR3 displayed an increased saturation density, related to increased proliferation and viability. This effect was significantly dependent on PLCgamma1 signaling and undetectable in cells expressing K652E FGFR3, which failed to phosphorylate PLCgamma1. In contrast to TERT-NHUC, expression of mutant FGFR3 in NIH-3T3 resulted in phosphorylation of Src and Akt. In addition, all forms of mutant FGFR3 were able to phosphorylate Plcgamma1 and induce morphological transformation, cell proliferation, and anchorage-independent growth. Our results indicate that the effects of mutant FGFR3 are both cell type specific and mutation specific. Mutant FGFR3 may confer a selective advantage in the urothelium by overcoming normal contact inhibition of proliferation.

E2F3 and CDKAL1 are candidate genes from the 6p22 region frequently amplified in bladder cancer. Expression of E2F3 isoforms (E2F3a and b) and CDKAL1 were examined and modulated in 6p22-amplified bladder cell lines. Eight lines with amplification showed overexpression of both E2F3 isoforms and CDKAL1. shRNA-mediated knockdown of CDKAL1 had no effect on proliferation. Knockdown of E2F3a or E2F3b alone induced antiproliferative effects, with the most significant effect on proliferation being observed when both isoforms were knocked down together. As E2Fs interact with the Rb tumour suppressor protein, Rb expression was analysed. There was a striking relationship between 6p22.3 amplification, E2F3 overexpression and lack of Rb expression. This was also examined in primary bladder tumours. Array-CGH detected 6p22.3 amplification in 8/91 invasive tumours. Five were studied in more detail. Four showed 13q14.2 loss (including RB1) and expressed no Rb protein. In the fifth, 13q was unaltered but the CDKN2A locus was deleted. This tumour was negative for p16 and positive for Rb protein. As p16 is a negative regulator of the Rb pathway, its loss represents an alternative mechanism for inactivation. Indeed, a phospho-specific Rb antibody showed much Rb protein in a hyperphosphorylated (inactive) form. We conclude that inactivation of the Rb pathway is required in addition to E2F3 overexpression in this subset of bladder tumours.