Spatially Resolved Phosphoproteomics Reveals Fibroblast Growth Factor Receptor Recycling-driven Regulation of Autophagy and Survival

SummaryReceptor 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 revealed FGFR signalling partners proximal to recycling endosomes (REs) by developing a Spatially Resolved Phosphoproteomics (SRP) approach based on APEX2-driven biotinylation followed by phosphopeptide enrichment. Combining this with traditional phosphoproteomics, bioinformatics, and targeted assays, we uncovered that FGFR2b stimulated by its recycling ligand FGF10 activates mTOR-dependent signalling and ULK1 at the REs, 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 celllar signalling.

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A role for the fibroblast growth factor receptor in cell fate decisions in the developing vertebrate retina

Development ◽

10.1242/dev.125.20.3967 ◽

1998 ◽

Vol 125 (20) ◽

pp. 3967-3975 ◽

Cited By ~ 2

Author(s):

S. McFarlane ◽

M.E. Zuber ◽

C.E. Holt

Keyword(s):

Growth Factor ◽

Fibroblast Growth Factor ◽

Cell Fate ◽

Fibroblast Growth Factor Receptor ◽

Growth Factor Receptor ◽

Retinal Cell ◽

Fibroblast Growth ◽

Cell Fate Decisions ◽

Mutant Receptor ◽

Vertebrate Retina

The mature vertebrate retina contains seven major cell types that develop from an apparently homogenous population of precursor cells. Clonal analyses have suggested that environmental influences play a major role in specifying retinal cell identity. Fibroblast growth factor-2 is present in the developing retina and regulates the survival, proliferation and differentiation of developing retinal cells in culture. Here we have tested whether fibroblast growth factor receptor signaling biases retinal cell fate decisions in vivo. Fibroblast growth factor receptors were inhibited in retinal precursors in Xenopus embryos by expressing a dominant negative form of the receptor, XFD. Dorsal animal blastomeres that give rise to the retina were injected with cDNA expression constructs for XFD and a control non-functional mutant receptor, D48, and the cell fates of transgene-expressing cells in the mature retina determined. Fibroblast growth factor receptor blockade results in almost a 50% loss of photoreceptors and amacrine cells, and a concurrent 3.5-fold increase in Muller glia, suggesting a shift towards a Muller cell fate in the absence of a fibroblast growth factor receptor signal. Inhibition of non-fibroblast-growth-factor-mediated receptor signaling with a third mutant receptor, HAVO, alters cell fate in an opposite manner. These results suggest that it is the balance of fibroblast growth factor and non-fibroblast growth factor ligand signals that influences retinal cell genesis.

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Ubiquitination of Fibroblast Growth Factor Receptor 1 Is Required for Its Intracellular Sorting but Not for Its Endocytosis

Molecular Biology of the Cell ◽

10.1091/mbc.e07-12-1219 ◽

2008 ◽

Vol 19 (8) ◽

pp. 3390-3403 ◽

Cited By ~ 77

Author(s):

Ellen Margrethe Haugsten ◽

Jędrzej Małecki ◽

Sunniva Maria Stordal Bjørklund ◽

Sjur Olsnes ◽

Jørgen Wesche

Keyword(s):

Growth Factor ◽

Tyrosine Kinase ◽

Fibroblast Growth Factor ◽

Fibroblast Growth Factor Receptor ◽

Growth Factor Receptor ◽

Kinase Domain ◽

Tyrosine Kinase Domain ◽

Fibroblast Growth ◽

Recycling Endosomes ◽

Lysine Residues

Endocytosis and targeting of growth factor receptors for lysosomal degradation have been associated with ubiquitination of the intracellular part of the receptors. To elucidate the role of receptor ubiquitination in internalization and sorting of fibroblast growth factor receptor (FGFR), we constructed several mutants of FGFR1 in which lysines, potential ubiquitination sites, were substituted for arginines. Substitution of all lysine residues in the intracellular part of FGFR1 resulted in inactivation of the tyrosine kinase domain of the receptor. However, several multilysine FGFR1 mutants, where up to 26 of 29 lysines in the intracellular part of the receptor were mutated, retained tyrosine kinase activity. The active multilysine mutants were poorly ubiquitinated, but internalized normally, indicating that ubiquitination of the receptor is not required for endocytosis. In contrast, degradation of the multilysine mutants was dramatically reduced as the mutants were inefficiently transported to lysosomes but rather sorted to recycling endosomes. The altered sorting resulted in sustained signaling. The duration of FGFR1 signaling seems to be tightly regulated by receptor ubiquitination and subsequent sorting to the lysosomes for degradation.

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