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.