Abstract
Cell migration is a physiological process that requires endocytic trafficking and polarization of adhesion molecules and receptor tyrosine kinases (RTKs) to the leading edge. Many growth factors are able to induce motility by binding to specific RTK on target cells. Among them, keratinocyte growth factor (KGF or FGF7) and fibroblast growth factor 10 (FGF10), members of the FGF family, are motogenic for keratinocytes, and exert their action by binding to the keratinocyte growth factor receptor (KGFR), a splicing variant of FGFR2, exclusively expressed on epithelial cells. Here we analyzed the possible role of cortactin, an F-actin binding protein which is tyrosine phosphorylated by Src and is involved in KGFR-mediated cell migration, in the KGFR endocytosis and polarization to the leading edge of migrating cells upon ligand-induced stimulation. Biochemical phosphorylation study revealed that both KGF and FGF10 were able to induce tyrosine phosphorylation of Src and in turn of cortactin, as demonstrated by using the specific pharmacological Src-inhibitor SU6656, although FGF10 effect was delayed with respect to that promoted by KGF. Immunofluorescence analysis demonstrated the polarized localization of KGFR upon ligand stimulation to the leading edge of migrating keratinocytes, process that was regulated by Src. Moreover, we showed that the colocalization of cortactin with KGFR at the plasma membrane protrusions and on early endosomes after KGF and FGF10 treatment was Src-dependent. Further, by using a RNA interference approach through microinjection, we showed that cortactin is required for KGFR endocytosis and that the clathrin-dependent internalization of the receptor is a critical event for its polarization. Finally, KGFR expression and polarization enhanced cell migration in a scratch assay. Our results indicate that both Src and cortactin play a key role in the KGFR endocytosis and polarization at the leading edge of migrating keratinocytes, supporting the crucial involvement of RTK trafficking in cell motility.
Highlights
Cell migration is a physiological process that involves actin cytoskeleton remodeling in lamellipodia and membrane ruffles at the leading edge of the cell, and the assembly and disassembly of adhesion contacts at the rear part of the cell [1]
Src activity controls keratinocyte growth factor receptor (KGFR) polarization at the leading edge of migrating cells Since cortactin is an important component of the receptormediated endocytosis machinery [12,13,15], and its function is dependent on tyrosine phosphorylation mediated by Src [8], to evaluate the role of cortactin and Src in KGFR internalization, we first determined whether Src would be tyrosine phosphorylated by KGFR activation
Since cortactin has been described to associate with endosomes [9,10,11] and here we demonstrate that KGFR polarization is the result of the receptor endocytosis and recycling (Fig. 3), we wondered if the KGFR and cortactin could colocalize in the early endosomal compartment when keratinocyte migration was triggered by KGFR ligand stimulation
Summary
Cell migration is a physiological process that involves actin cytoskeleton remodeling in lamellipodia and membrane ruffles at the leading edge of the cell, and the assembly and disassembly of adhesion contacts at the rear part of the cell [1]. A direct correlation between RTK endocytosis and cell motility has not yet been clarified, studies conducted by different groups suggest a key role of different actin-binding proteins in the regulation of RTKs internalization and consequent polarization following the ligand-dependent motogenic stimulus. A recent study demonstrated the essential role of Src-dependent tyrosine phosphorylation of cortactin and dynamin 2 in transferrin endocytosis [17]. All of these findings suggest that cortactin is an important component of the receptor-mediated endocytic machinery, regulating together with actin and dynamin the scission of clathrin pits from the plasma membrane
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