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

Abstract

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 EGFmediated 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.

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

“…Interestingly, T693 on EGFR was found phosphorylated only in the proximal phosphoproteome (Fig. 5e), consistent with its role in regulating FGFR2b recycling at the REs 22 . Therefore, the SRP approach selectively enriches for phosphorylation proteins localized at the REs which could not be detected in the global phosphoproteome.…”

“…As shown for FGFR1 36 , also FGFR2b co-localized with the marker of EEs, EEA1, and with DnRAB11 in cells expressing DnRAB11 and was not found at the PM after longer stimulation with FGF10 (Fig. 1a-b) 22 , suggesting that FGFR2b is trapped in EEA1/DnRAB11-positive vescicles under this experimental condition. Therefore, expressing DnDNM2 and DnRAB11 impair FGFR2b trafficking and will be used here to study recycling-dependent changes in FGFR2b signalling in response to FGF10.…”

“…However, this analysis did not affect the overall quality of the proximal phosphoproteome data, as we found that >89% (6224 and 11726 for global and proximal, respectively) of the phosphorylated sites identified were Class I (≥ 0.75 localisation probability 39 ) and had the expected proportions of single or multiple phosphorylated sites or serine, threonine, or tyrosine residues (Supplementary Fig. 4i) 22 . Finally, the APEX2 tag did not affect the quantification of the phosphoproteome (Supplementary Fig.…”

“…6c-d). The phosphorylation of ULK1 downstream of FGFR2b recycling is a specific event, as other FGFR2b downstream pathways were only marginally affected in cells expressing either DnRAB11 or DnDNM2, treated with the primaquine and dynasore compounds, or stimulated with FGF7, all conditions that impaired FGFR2b trafficking 13,22,50,51 (Fig. 1, Fig.…”

“…This novel method has allowed us to uncover FGFR2b signalling partners localized at the REs and to study their impact on FGFR2b responses. To dissect the spatially restricted signalling modules regulated by FGF10/FGFR2b during recycling, we combined the SRP approach with traditional quantitative phosphoproteomics of epithelial cells where FGFR2b recycling was blocked 22 . We found novel FGFR2b signalling partners localized at the REs during recycling that regulate mTOR signalling with functional consequences for autophagy and cell survival.…”