Reciprocal priming between receptor tyrosine kinases at recycling endosomes orchestrates cellular signalling outputs.

Integration of signalling downstream of individual receptor tyrosine kinases (RTKs) is crucial to fine-tune cellular homeostasis during development and in pathological conditions, including breast cancer. However, how signalling integration is regulated and whether the endocytic fate of single receptors controls such signalling integration remains poorly elucidated. Combining quantitative phosphoproteomics and targeted assays, we generated a detailed picture of recycling-dependent fibroblast growth factor (FGF) signalling in breast cancer cells, with a focus on distinct FGF receptors (FGFRs). We discovered reciprocal priming between FGFRs and epidermal growth factor (EGF) receptor (EGFR) that is coordinated at recycling endosomes. FGFR recycling ligands induce EGFR phosphorylation on threonine 693. This phosphorylation event alters both FGFR and EGFR trafficking and primes FGFR-mediated proliferation but not cell invasion. In turn, FGFR signalling primes EGF-mediated outputs via EGFR threonine 693 phosphorylation. This reciprocal priming between distinct families of RTKs from recycling endosomes exemplifies a novel signalling integration hub where recycling endosomes orchestrate cellular behaviour. Therefore, targeting reciprocal priming over individual receptors may improve personalized therapies in breast and other cancers.

Discovery of a Gatekeeper Residue in the C-Terminal Tail of the Extracellular Signal-Regulated Protein Kinase 5 (ERK5).

The extracellular signal-regulated protein kinase 5 (ERK5) is a non-redundant mitogen-activated protein kinase (MAPK) that exhibits a unique C-terminal extension which comprises distinct structural and functional properties. Here, we sought to elucidate the significance of phosphoacceptor sites in the C-terminal transactivation domain of ERK5. We have found that Thr732 acted as a functional gatekeeper residue controlling C-terminal-mediated nuclear translocation and transcriptional enhancement. Consistently, using a non-bias quantitative mass spectrometry approach, we demonstrated that phosphorylation at Thr732 conferred selectivity for binding interactions of ERK5 with proteins related to chromatin and RNA biology, whereas a number of metabolic regulators were associated with full-length wild type ERK5. Additionally, our proteomic analysis revealed that phosphorylation of the Ser730-Glu-Thr732-Pro motif could occur independently of dual phosphorylation at Thr218-Glu-Tyr220 in the activation loop. Collectively, our results firmly establish the significance of C-terminal phosphorylation in regulating ERK5 function. The post-translational modification of ERK5 on its C-terminal tail might be of particular relevance in cancer cells where ERK5 has be found to be hyperphosphoryated.

Oncogenic ERRB2 signals through the AP-1 transcription factor to control mesenchymal-like properties of oesophageal adenocarcinoma.

Oesophageal adenocarcinoma (OAC) is a deadly disease with poor survival statistics and few targeted therapies available. One of the most common molecular aberrations in OAC is amplification or activation of the gene encoding the receptor tyrosine kinase ERBB2, and ERBB2 is targeted in the clinic for this subset of patients. However, the downstream consequences of these ERBB2 activating events are not well understood. Here we used a combination of phosphoproteomics, open chromatin profiling and transcriptome analysis on cell line models and patient-derived datasets to interrogate the molecular pathways operating downstream from ERBB2. Integrated analysis of these data sets converge on a model where dysregulated ERBB2 signalling is mediated at the transcriptional level by the transcription factor AP-1. AP-1 in turn controls cell behaviour by acting on cohorts of genes that regulate cell migration and adhesion, features often associated with EMT. Our study therefore provides a valuable resource for the cancer cell signalling community and reveals novel molecular determinants underlying the dysregulated behaviour of OAC cells.

Spatially resolved phosphoproteomics reveals fibroblast growth factor receptor recycling-driven regulation of autophagy and survival.

Receptor Tyrosine Kinase (RTK) endocytosis-dependent signalling drives cell proliferation and motility during development and adult homeostasis, but is dysregulated in diseases, including cancer. The recruitment of RTK signalling partners during endocytosis, specifically during recycling to the plasma membrane, is still unknown. Focusing on Fibroblast Growth Factor Receptor 2b (FGFR2b) recycling, we reveal FGFR signalling partners proximal to recycling endosomes by developing a Spatially Resolved Phosphoproteomics (SRP) approach based on APEX2-driven biotinylation followed by phosphorylated peptides enrichment. Combining this with traditional phosphoproteomics, bioinformatics, and targeted assays, we uncover that FGFR2b stimulated by its recycling ligand FGF10 activates mTOR-dependent signalling and ULK1 at the recycling endosomes, leading to autophagy suppression and cell survival. This adds to the growing importance of RTK recycling in orchestrating cell fate and suggests a therapeutically targetable vulnerability in ligand-responsive cancer cells. Integrating SRP with other systems biology approaches provides a powerful tool to spatially resolve cellular signalling.

Oxidative stress from DGAT1 oncoprotein inhibition in melanoma suppresses tumor growth when ROS defenses are also breached.

Dysregulated cellular metabolism is a cancer hallmark for which few druggable oncoprotein targets have been identified. Increased fatty acid (FA) acquisition allows cancer cells to meet their heightened membrane biogenesis, bioenergy, and signaling needs. Excess FAs are toxic to non-transformed cells but surprisingly not to cancer cells. Molecules underlying this cancer adaptation may provide alternative drug targets. Here, we demonstrate that diacylglycerol O-acyltransferase 1 (DGAT1), an enzyme integral to triacylglyceride synthesis and lipid droplet formation, is frequently up-regulated in melanoma, allowing melanoma cells to tolerate excess FA. DGAT1 over-expression alone transforms p53-mutant zebrafish melanocytes and co-operates with oncogenic BRAF or NRAS for more rapid melanoma formation. Antagonism of DGAT1 induces oxidative stress in melanoma cells, which adapt by up-regulating cellular reactive oxygen species defenses. We show that inhibiting both DGAT1 and superoxide dismutase 1 profoundly suppress tumor growth through eliciting intolerable oxidative stress.