FAIMS Enhances the Detection of PTM Crosstalk Sites.

Protein post-translational modifications (PTMs) enable cells to rapidly change in response to biological stimuli. With hundreds of different PTMs, understanding these control mechanisms is complex. To date, efforts have focused on investigating the effect of a single PTM on protein function. Yet, many proteins contain multiple PTMs. Moreover, one PTM can alter the prevalence of another, a phenomenon termed PTM crosstalk. Understanding PTM crosstalk is critical; however, its detection is challenging since PTMs occur substoichiometrically. Here, we develop an enrichment-free, label-free proteomics method that utilizes high-field asymmetric ion mobility spectrometry (FAIMS) to enhance the detection of PTM crosstalk. We show that by searching for multiple combinations of dynamic PTMs on peptide sequences, a 6-fold increase in candidate PTM crosstalk sites is identified compared with that of standard liquid chromatography-tandem mass spectrometry (LC-MS/MS) workflows. Additionally, by cycling through FAIMS compensation voltages within a single LC-FAIMS-MS/MS run, we show that our LC-FAIMS-MS/MS workflow can increase multi-PTM-containing peptide identifications without additional increases in run times. With 159 novel candidate crosstalk sites identified, we envisage LC-FAIMS-MS/MS to play an important role in expanding the repertoire of multi-PTM identifications. Moreover, it is only by detecting PTM crosstalk that we can "see" the full picture of how proteins are regulated.

Differential responses to kinase inhibition in FGFR2-addicted triple negative breast cancer cells: a quantitative phosphoproteomics study.

Fibroblast Growth Factor (FGF) dependent signalling is frequently activated in cancer by a variety of different mechanisms. However, the downstream signal transduction pathways involved are poorly characterised. Here a quantitative differential phosphoproteomics approach, SILAC, is applied to identify FGF-regulated phosphorylation events in two triple- negative breast tumour cell lines, MFM223 and SUM52, that exhibit amplified expression of FGF receptor 2 (FGFR2) and are dependent on continued FGFR2 signalling for cell viability. Comparative Gene Ontology proteome analysis revealed that SUM52 cells were enriched in proteins associated with cell metabolism and MFM223 cells enriched in proteins associated with cell adhesion and migration. FGFR2 inhibition by SU5402 impacts a significant fraction of the observed phosphoproteome of these cells. This study expands the known landscape of FGF signalling and identifies many new targets for functional investigation. FGF signalling pathways are found to be flexible in architecture as both shared, and divergent, responses to inhibition of FGFR2 kinase activity in the canonical RAF/MAPK/ERK/RSK and PI3K/AKT/PDK/mTOR/S6K pathways are identified. Inhibition of phosphorylation-dependent negative-feedback pathways is observed, defining mechanisms of intrinsic resistance to FGFR2 inhibition. These findings have implications for the therapeutic application of FGFR inhibitors as they identify both common and divergent responses in cells harbouring the same genetic lesion and pathways of drug resistance.

Quantitative Phosphoproteomics Reveals a Role for Collapsin Response Mediator Protein 2 in PDGF-Induced Cell Migration.

The Platelet Derived Growth Factor (PDGF) family of ligands have well established functions in the induction of cell proliferation and migration during development, tissue homeostasis and interactions between tumours and stroma. However, the mechanisms by which these actions are executed are incompletely understood. Here we report a differential phosphoproteomics study, using a SILAC approach, of PDGF-stimulated mouse embryonic fibroblasts (MEFs). 116 phospho-sites were identified as up-regulated and 45 down-regulated in response to PDGF stimulation. These encompass proteins involved in cell adhesion, cytoskeleton regulation and vesicle-mediated transport, significantly expanding the range of proteins implicated in PDGF signalling pathways. Included in the down-regulated class was the microtubule bundling protein Collapsin Response Mediator Protein 2 (CRMP2). In response to stimulation with PDGF, CRMP2 was dephosphorylated on Thr514, an event known to increase CRMP2 activity. This was reversed in the presence of micromolar concentrations of the protein phosphatase inhibitor okadaic acid, implicating PDGF-induced activation of protein phosphatase 1 (PP1) in CRMP2 regulation. Depletion of CRMP2 resulted in impairment of PDGF-mediated cell migration in an in vitro wound healing assay. These results show that CRMP2 is required for PDGF-directed cell migration in vitro.

Quantifying receptor trafficking and colocalization with confocal microscopy.

Confocal microscopy is a powerful tool for the study of cellular receptor trafficking and endocytosis. Unbiased and robust image analysis workflows are required for the identification, and study, of aberrant trafficking. After a brief review of related strategies, identifying both good and bad practice, custom workflows for the analysis of live cell 3D time-lapse data are presented. Strategies for data pre-processing, including denoising and background subtraction are considered. We use a 3D level set protocol to accurately segment cells using only the signal from fluorescently labelled receptor. A protocol for the quantification of changes to subcellular receptor distribution over time is then presented. As an example, ligand stimulated trafficking of epidermal growth factor receptor (EGFR) is shown to be significantly reduced in both AG1478 and Dynasore treated cells. Protocols for the quantitative analysis of colocalization between receptor and endosomes are also introduced, including strategies for signal isolation and statistical testing. By calculating the Manders and Pearson coefficients, both co-occurrence and correlation can be assessed. A statistically significant decrease in the level of ligand induced co-occurrence between EGFR and rab5 positive endosomes is demonstrated for both the AG1478 and Dynasore treated cells relative to a control. Finally, a strategy for the visualisation of co-occurrence is presented, which provides an unbiased alternative to colour overlays.

LAR protein tyrosine phosphatase regulates focal adhesions through CDK1.

Focal adhesions are complex multi-molecular structures that link the actin cytoskeleton to the extracellular matrix through integrin adhesion receptors and play a key role in regulation of many cellular functions. LAR (also known as PTPRF) is a receptor protein tyrosine phosphatase that regulates PDGF signalling and localises to focal adhesions. We have observed that loss of LAR phosphatase activity in mouse embryonic fibroblasts results in reduced numbers of focal adhesions and decreased adhesion to fibronectin. To understand how LAR regulates cell adhesion we used phosphoproteomic data, comparing global phosphorylation events in wild-type and LAR phosphatase-deficient cells, to analyse differential kinase activity. Kinase prediction analysis of LAR-regulated phosphosites identified a node of cytoskeleton- and adhesion-related proteins centred on cyclin-dependent kinase-1 (CDK1). We found that loss of LAR activity resulted in reduced activity of CDK1, and that CDK1 activity was required for LAR-mediated focal adhesion complex formation. We also established that LAR regulates CDK1 activity through c-Abl and Akt family proteins. In summary, we have identified a new role for a receptor protein tyrosine phosphatase in regulating CDK1 activity and hence cell adhesion to the extracellular matrix.

Regulation of Platelet Derived Growth Factor Signaling by Leukocyte Common Antigen-related (LAR) Protein Tyrosine Phosphatase: A Quantitative Phosphoproteomics Study.

Intracellular signaling pathways are reliant on protein phosphorylation events that are controlled by a balance of kinase and phosphatase activity. Although kinases have been extensively studied, the role of phosphatases in controlling specific cell signaling pathways has been less so. Leukocyte common antigen-related protein (LAR) is a member of the LAR subfamily of receptor-like protein tyrosine phosphatases (RPTPs). LAR is known to regulate the activity of a number of receptor tyrosine kinases, including platelet-derived growth factor receptor (PDGFR). To gain insight into the signaling pathways regulated by LAR, including those that are PDGF-dependent, we have carried out the first systematic analysis of LAR-regulated signal transduction using SILAC-based quantitative proteomic and phosphoproteomic techniques. We haveanalyzed differential phosphorylation between wild-type mouse embryo fibroblasts (MEFs) and MEFs in which the LAR cytoplasmic phosphatase domains had been deleted (LARΔP), and found a significant change in abundance of phosphorylation on 270 phosphosites from 205 proteins because of the absence of the phosphatase domains of LAR. Further investigation of specific LAR-dependent phosphorylation sites and enriched biological processes reveal that LAR phosphatase activity impacts on a variety of cellular processes, most notably regulation of the actin cytoskeleton. Analysis of putative upstream kinases that may play an intermediary role between LAR and the identified LAR-dependent phosphorylation events has revealed a role for LAR in regulating mTOR and JNK signaling.

FAIMS and Phosphoproteomics of Fibroblast Growth Factor Signaling: Enhanced Identification of Multiply Phosphorylated Peptides.

We have applied liquid chromatography high-field asymmetric waveform ion mobility spectrometry tandem mass spectrometry (LC-FAIMS-MS/MS) and liquid chromatography tandem mass spectrometry (LC-MS/MS) to the investigation of site-specific phosphorylation in fibroblast growth factor (FGF) signaling. We have combined a SILAC approach with chemical inhibition by SU5402 (an FGF receptor tyrosine kinase inhibitor) and dasatinib (a Src family kinase inhibitor). The results show that incorporation of FAIMS within the workflow results in (a) an increase in the relative proportion of phosphothreonine and phosphotyrosine sites identified, (b) an increase in phosphopeptide identifications from precursors with charge states ≥ +3 (with an associated increase in peptide length), and (c) an increase in the identification of multiply phosphorylated peptides. Approximately 20% of the phosphorylation sites identified via the FAIMS workflow had not been reported previously, and over 80% of those were from multiply phosphorylated peptides. Moreover, FAIMS provided access to a distinct set of phosphorylation sites regulated in response to SU5402 and dasatinib. The enhanced identification of multiply phosphorylated peptides was particularly striking in the case of sites regulated by SU5402. In addition to providing a compelling example of the complementarity of FAIMS in phosphoproteomics, the results provide a valuable resource of phosphorylation sites for further investigation of FGF signaling and trafficking.

Inference of Low and High-Grade Glioma Gene Regulatory Networks Delineates the Role of Rnd3 in Establishing Multiple Hallmarks of Cancer.

Gliomas are a highly heterogeneous group of brain tumours that are refractory to treatment, highly invasive and pro-angiogenic. Glioblastoma patients have an average survival time of less than 15 months. Understanding the molecular basis of different grades of glioma, from well differentiated, low-grade tumours to high-grade tumours, is a key step in defining new therapeutic targets. Here we use a data-driven approach to learn the structure of gene regulatory networks from observational data and use the resulting models to formulate hypothesis on the molecular determinants of glioma stage. Remarkably, integration of available knowledge with functional genomics datasets representing clinical and pre-clinical studies reveals important properties within the regulatory circuits controlling low and high-grade glioma. Our analyses first show that low and high-grade gliomas are characterised by a switch in activity of two subsets of Rho GTPases. The first one is involved in maintaining normal glial cell function, while the second is linked to the establishment of multiple hallmarks of cancer. Next, the development and application of a novel data integration methodology reveals novel functions of RND3 in controlling glioma cell migration, invasion, proliferation, angiogenesis and clinical outcome.

Eps8 controls Src- and FAK-dependent phenotypes in squamous carcinoma cells.

Eps8 is an actin regulatory scaffold protein whose expression is increased in squamous cell carcinoma (SCC) cells. It forms a complex with both focal adhesion kinase (FAK, also known as PTK2) and Src in SCC cells derived from skin carcinomas induced by administration of the chemical DMBA followed by TPA (the DMBA/TPA model). Here, we describe two new roles for Eps8. Firstly, it controls the spatial distribution of active Src in a FAK-dependent manner. Specifically, Eps8 participates in, and regulates, a biochemical complex with Src and drives trafficking of Src to autophagic structures that SCC cells use to cope with high levels of active Src when FAK is absent. Secondly, when FAK is expressed in SCC cells, thereby meaning active Src becomes tethered at focal adhesion complexes, Eps8 is also recruited to focal adhesions and is required for FAK-dependent polarization and invasion. Therefore, Eps8 is a crucial mediator of Src- and FAK-regulated processes; it participates in specific biochemical complexes and promotes actin re-arrangements that determine the spatial localization of Src, and modulates the functions of Src and FAK during invasive migration.

RhoJ interacts with the GIT-PIX complex and regulates focal adhesion disassembly.

RhoJ is a Rho GTPase expressed in endothelial cells and tumour cells, which regulates cell motility, invasion, endothelial tube formation and focal adhesion numbers. This study aimed to further delineate the molecular function of RhoJ. Using timelapse microscopy RhoJ was found to regulate focal adhesion disassembly; small interfering RNA (siRNA)-mediated knockdown of RhoJ increased focal adhesion disassembly time, whereas expression of an active mutant (daRhoJ) decreased it. Furthermore, daRhoJ co-precipitated with the GIT-PIX complex, a regulator of focal adhesion disassembly. An interaction between daRhoJ and GIT1 was confirmed using yeast two-hybrid experiments, and this depended on the Spa homology domain of GIT1. GIT1, GIT2, ß-PIX (also known as ARHGEF7) and RhoJ all colocalised in focal adhesions and depended on each other for their recruitment to focal adhesions. Functionally, the GIT-PIX complex regulated endothelial tube formation, with knockdown of both GIT1 and GIT2, or ß-PIX phenocopying RhoJ knockdown. RhoJ-knockout mice showed reduced tumour growth and diminished tumour vessel density, identifying a role for RhoJ in mediating tumour angiogenesis. These studies give new insight into the molecular function of RhoJ in regulating cell motility and tumour vessel formation.

DiME: a scalable disease module identification algorithm with application to glioma progression.

Disease module is a group of molecular components that interact intensively in the disease specific biological network. Since the connectivity and activity of disease modules may shed light on the molecular mechanisms of pathogenesis and disease progression, their identification becomes one of the most important challenges in network medicine, an emerging paradigm to study complex human disease. This paper proposes a novel algorithm, DiME (Disease Module Extraction), to identify putative disease modules from biological networks. We have developed novel heuristics to optimise Community Extraction, a module criterion originally proposed for social network analysis, to extract topological core modules from biological networks as putative disease modules. In addition, we have incorporated a statistical significance measure, B-score, to evaluate the quality of extracted modules. As an application to complex diseases, we have employed DiME to investigate the molecular mechanisms that underpin the progression of glioma, the most common type of brain tumour. We have built low (grade II)--and high (GBM)--grade glioma co-expression networks from three independent datasets and then applied DiME to extract potential disease modules from both networks for comparison. Examination of the interconnectivity of the identified modules have revealed changes in topology and module activity (expression) between low- and high- grade tumours, which are characteristic of the major shifts in the constitution and physiology of tumour cells during glioma progression. Our results suggest that transcription factors E2F4, AR and ETS1 are potential key regulators in tumour progression. Our DiME compiled software, R/C++ source code, sample data and a tutorial are available at http://www.cs.bham.ac.uk/~szh/DiME.

The non-receptor tyrosine kinase Ack1 regulates the fate of activated EGFR by inducing trafficking to the p62/NBR1 pre-autophagosome.

Growth factor signalling regulates multiple cellular functions and its misregulation has been linked to the development and progression of cancer. Ack1 (activated Cdc42-associated kinase 1, also known as TNK2) is a non-receptor tyrosine kinase that has been implicated in trafficking and degradation of epidermal growth factor receptor (EGFR), yet its precise functions remain elusive. In this report, we investigate the role of Ack1 in EGFR trafficking and show that Ack1 partially colocalises to Atg16L-positive structures upon stimulation with EGF. These structures are proposed to be the isolation membranes that arise during formation of autophagosomes. In addition, we find that Ack1 colocalises and interacts with sequestosome 1 (p62/SQSTM1), a receptor for selective autophagy, through a ubiquitin-associated domain, and this interaction decreases upon treatment with EGF, thus suggesting that Ack1 moves away from p62/SQSTM1 compartments. Furthermore, Ack1 interacts and colocalises with NBR1, another autophagic receptor, and this colocalisation is enhanced in the presence of ectopically expressed p62/SQSTM1. Finally, knockdown of Ack1 results in accelerated localisation of EGFR to lysosomes upon treatment with EGF. Structure-function analyses of a panel of Ack1 deletion mutants revealed key mechanistic aspects of these relationships. The Mig6-homology domain and clathrin-binding domain both contribute to colocalisation with EGFR, whereas the UBA domain is essential for colocalisation with p62/SQSTM1, but not NBR1. Taken together, our studies demonstrate a novel role for Ack1 in diverting activated EGFR into a non-canonical degradative pathway, marked by association with p62/SQSTM1, NBR1 and Atg16L.

Novel binding partners and differentially regulated phosphorylation sites clarify Eps8 as a multi-functional adaptor.

Eps8 is involved in both cell signalling and receptor trafficking. It is a known phosphorylation substrate for two proteins involved in the fibroblast growth factor receptor (FGFR) signalling pathway: the receptor itself and Src. Here we report a differential proteomic analysis of Eps8 aimed to identify specific FGFR and Src family kinase dependent phosphosites and co-associated phosphodependent binding partners. This study reveals a total of 22 Eps8 pTyr and pSer/Thr phosphorylation sites, including those that are dependent on Src family and FGFR kinase activity. Peptide affinity purification of proteins that bind to a selection of the pTyr phosphosites has identified a range of novel Eps8 binding partners including members of the intracellular vesicle trafficking machinery (clathrin and AP-2), proteins which have been shown to regulate activated receptor trafficking (NBR1 and Vav2), and proteins involved in receptor signalling (IRS4 and Shp2). Collectively this study significantly extends the understanding of Eps8 post-translational modification by regulated phosphorylation, identifies novel Eps8 binding partners implicated in receptor trafficking and signalling, and confirms the functions of Eps8 at the nexus of receptor signalling and vesicular trafficking.

Probing the complementarity of FAIMS and strong cation exchange chromatography in shotgun proteomics.

High field asymmetric waveform ion mobility spectrometry (FAIMS), also known as differential ion mobility spectrometry, coupled with liquid chromatography tandem mass spectrometry (LC-MS/MS) offers benefits for the analysis of complex proteomics samples. Advantages include increased dynamic range, increased signal-to-noise, and reduced interference from ions of similar m/z. FAIMS also separates isomers and positional variants. An alternative, and more established, method of reducing sample complexity is prefractionation by use of strong cation exchange chromatography. Here, we have compared SCX-LC-MS/MS with LC-FAIMS-MS/MS for the identification of peptides and proteins from whole cell lysates from the breast carcinoma SUM52 cell line. Two FAIMS approaches are considered: (1) multiple compensation voltages within a single LC-MS/MS analysis (internal stepping) and (2) repeat LC-MS/MS analyses at different and fixed compensation voltages (external stepping). We also consider the consequence of the fragmentation method (electron transfer dissociation or collision-induced dissociation) on the workflow performance. The external stepping approach resulted in a greater number of protein and peptide identifications than the internal stepping approach for both ETD and CID MS/MS, suggesting that this should be the method of choice for FAIMS proteomics experiments. The overlap in protein identifications from the SCX method and the external FAIMS method was ~25% for both ETD and CID, and for peptides was less than 20%. The lack of overlap between FAIMS and SCX highlights the complementarity of the two techniques. Charge state analysis of the peptide assignments showed that the FAIMS approach identified a much greater proportion of triply-charged ions.

Regulation of fibroblast growth factor receptor signalling and trafficking by Src and Eps8.

Fibroblast growth factor receptors (FGFRs) mediate a wide spectrum of cellular responses that are crucial for development and wound healing. However, aberrant FGFR activity leads to cancer. Activated growth factor receptors undergo stimulated endocytosis, but can continue to signal along the endocytic pathway. Endocytic trafficking controls the duration and intensity of signalling, and growth factor receptor signalling can lead to modifications of trafficking pathways. We have developed live-cell imaging methods for studying FGFR dynamics to investigate mechanisms that coordinate the interplay between receptor trafficking and signal transduction. Activated FGFR enters the cell following recruitment to pre-formed clathrin-coated pits (CCPs). However, FGFR activation stimulates clathrin-mediated endocytosis; FGF treatment increases the number of CCPs, including those undergoing endocytosis, and this effect is mediated by Src and its phosphorylation target Eps8. Eps8 interacts with the clathrin-mediated endocytosis machinery and depletion of Eps8 inhibits FGFR trafficking and immediate Erk signalling. Once internalized, FGFR passes through peripheral early endosomes en route to recycling and degredative compartments, through an Src- and Eps8-dependent mechanism. Thus Eps8 functions as a key coordinator in the interplay between FGFR signalling and trafficking. This work provides the first detailed mechanistic analysis of growth factor receptor clustering at the cell surface through signal transduction and endocytic trafficking. As we have characterised the Src target Eps8 as a key regulator of FGFR signalling and trafficking, and identified the early endocytic system as the site of Eps8-mediated effects, this work provides novel mechanistic insight into the reciprocal regulation of growth factor receptor signalling and trafficking.

Plakoglobin-dependent regulation of keratinocyte apoptosis by Rnd3.

The human epidermis is a self-renewing, stratified epithelial tissue that provides the protective function of the skin. The principal cell type within the epidermis is the keratinocyte, and normal function of the epidermis requires that keratinocyte proliferation, differentiation and cell death be carefully controlled. There is clear evidence that signalling through adhesion receptors such as integrins and cadherins plays a key role in regulating epidermal function. Previous work has shown that Rho family GTPases regulate cadherin- and integrin-based adhesion structures and hence epidermal function. In this study, we show that a member of this family, Rnd3, regulates desmosomal cell-cell adhesion in that loss of Rnd3 expression leads to an increase in desmosomes at sites of cell-cell adhesion and altered colony morphology. Loss of Rnd3 expression is also associated with resistance to cisplatin-mediated apoptosis in keratinocytes and this resistance is mediated through the desmosomal protein plakoglobin. We propose a novel plakoglobin-dependent role for Rnd3 in the regulation of keratinocyte cell death.

Identification and characterization of an inhibitory fibroblast growth factor receptor 2 (FGFR2) molecule, up-regulated in an Apert Syndrome mouse model.

AS (Apert syndrome) is a congenital disease composed of skeletal, visceral and neural abnormalities, caused by dominant-acting mutations in FGFR2 [FGF (fibroblast growth factor) receptor 2]. Multiple FGFR2 splice variants are generated through alternative splicing, including PTC (premature termination codon)-containing transcripts that are normally eliminated via the NMD (nonsense-mediated decay) pathway. We have discovered that a soluble truncated FGFR2 molecule encoded by a PTC-containing transcript is up-regulated and persists in tissues of an AS mouse model. We have termed this IIIa-TM as it arises from aberrant splicing of FGFR2 exon 7 (IIIa) into exon 10 [TM (transmembrane domain)]. IIIa-TM is glycosylated and can modulate the binding of FGF1 to FGFR2 molecules in BIAcore-binding assays. We also show that IIIa-TM can negatively regulate FGF signalling in vitro and in vivo. AS phenotypes are thought to result from gain-of-FGFR2 signalling, but our findings suggest that IIIa-TM can contribute to these through a loss-of-FGFR2 function mechanism. Moreover, our findings raise the interesting possibility that FGFR2 signalling may be a regulator of the NMD pathway.

Computational modeling of biological pathways by executable biology.

"In silico" experiments (i.e., computer simulation) constitute an aid to traditional biological research, by allowing biologists to execute efficient simulations taking into consideration the data obtained in wet experiments and to generate new hypotheses, which can be later verified in additional wet experiments. In addition to being much cheaper and faster than wet experiments, computer simulation has other advantages: it allows us to run experiments in which several species can be monitored at the same time, to explore quickly various conditions by varying species and parameters in different runs, and in some cases to observe the behavior of the system at a greater level of detail than the one permitted by experimental techniques. In the past few years there has been a considerable effort in the computer science community to develop computational languages and software tools for modeling and analysing biochemical systems. Among the challenges which must be addressed in this context, there are: the definition of languages powerful enough to express all the relevant features of biochemical systems, the development of efficient algorithms to analyze models and interpret the results, and the implementation of modeling platforms which are usable by nonprogrammers. In this chapter, we focus on the use of computational modeling to the analysis of biochemical systems. Computational modeling, in conjunction with the use of formal intuitive modeling languages, enables biologists to define models using a notation very similar to the informal descriptions they commonly use, but formal and, hence, automatically executable. We describe the main features of the existing textual computational languages and the tool support available for model development and analysis.

Nbr1 is a novel inhibitor of ligand-mediated receptor tyrosine kinase degradation.

Neighbor of BRCA1 (Nbr1) is a highly conserved multidomain scaffold protein with proposed roles in endocytic trafficking and selective autophagy. However, the exact function of Nbr1 in these contexts has not been studied in detail. Here we investigated the role of Nbr1 in the trafficking of receptor tyrosine kinases (RTKs). We report that ectopic Nbr1 expression inhibits the ligand-mediated lysosomal degradation of RTKs, and this is probably done via the inhibition of receptor internalization. Conversely, the depletion of endogenous NBR1 enhances RTK degradation. Analyses of truncation mutations demonstrated that the C terminus of Nbr1 is essential but not sufficient for this activity. Moreover, the C terminus of Nbr1 is essential but not sufficient for the localization of the protein to late endosomes. We demonstrate that the C terminus of Nbr1 contains a novel membrane-interacting amphipathic α-helix, which is essential for the late endocytic localization of the protein but not for its effect on RTK degradation. Finally, autophagic and late endocytic localizations of Nbr1 are independent of one another, suggesting that the roles of Nbr1 in each context might be distinct. Our results define Nbr1 as a negative regulator of ligand-mediated RTK degradation and reveal the interplay between its various regions for protein localization and function.

Critical role of FLRT1 phosphorylation in the interdependent regulation of FLRT1 function and FGF receptor signalling.

BACKGROUND: Fibronectin leucine rich transmembrane (FLRT) proteins have dual properties as regulators of cell adhesion and potentiators of fibroblast growth factor (FGF) mediated signalling. The mechanism by which the latter is achieved is still unknown and is the subject of this investigation. PRINCIPAL FINDINGS: Here we show that FLRT1 is a target for tyrosine phosphorylation mediated by FGFR1 and implicate a non-receptor Src family kinase (SFK). We identify the target tyrosine residues in the cytoplasmic domain of FLRT1 and show that these are not direct substrates for Src kinase suggesting that the SFK may exert effects via potentiation of FGFR1 kinase activity. We show that whilst FLRT1 expression results in a ligand-dependent elevation of MAP kinase activity, a mutant version of FLRT1, defective as an FGFR1 kinase substrate (Y3F-FLRT1), has the property of eliciting ligand-independent chronic activation of the MAP kinase pathway which is suppressed by pharmacological inhibition of either FGFR1 or Src kinase. Functional investigation of FGFR1 and FLRT1 signalling in SH-SY5Y neuroblastoma cells reveals that FLRT1 alone acts to induce a multi-polar phenotype whereas the combination of FLRT1 and FGFR activation, or expression of Y3F-FLRT1, acts to induce neurite outgrowth via MAPK activation. Similar results were obtained in a dendrite outgrowth assay in primary hippocampal neurons. We also show that FGFR1, FLRT1 and activated Src are co-localized and this complex is trafficked toward the soma of the cell. The presence of Y3F-FLRT1 rather than FLRT1 resulted in prolonged localization of this complex within the neuritic arbour. CONCLUSIONS: This study shows that the phosphorylation state of FLRT1, which is itself FGFR1 dependent, may play a critical role in the potentiation of FGFR1 signalling and may also depend on a SFK-dependent phosphorylation mechanism acting via the FGFR. This is consistent with an 'in vivo' role for FLRT1 regulation of FGF signalling via SFKs. Furthermore, the phosphorylation-dependent futile cycle mechanism controlling FGFR1 signalling is concurrently crucial for regulation of FLRT1-mediated neurite outgrowth.