Frank-ter Haar Syndrome Protein Tks4 Regulates Epidermal Growth Factor-dependent Cell Migration

Julian Downward,Gábor Bögel,Árpád Lányi,Szabolcs Sipeki,Miklós Geiszt,László Buday,Anna Fekete,Annamária Gujdár

doi:10.1074/jbc.m111.324897

Abstract

Mutations in the SH3PXD2B gene coding for the Tks4 protein are responsible for the autosomal recessive Frank-ter Haar syndrome. Tks4, a substrate of Src tyrosine kinase, is implicated in the regulation of podosome formation. Here, we report a novel role for Tks4 in the EGF signaling pathway. In EGF-treated cells, Tks4 is tyrosine-phosphorylated and associated with the activated EGF receptor. This association is not direct but requires the presence of Src tyrosine kinase. In addition, treatment of cells with LY294002, an inhibitor of PI 3-kinase, or mutations of the PX domain reduces tyrosine phosphorylation and membrane translocation of Tks4. Furthermore, a PX domain mutant (R43W) Tks4 carrying a reported point mutation in a Frank-ter Haar syndrome patient showed aberrant intracellular expression and reduced phosphoinositide binding. Finally, silencing of Tks4 was shown to markedly inhibit HeLa cell migration in a Boyden chamber assay in response to EGF or serum. Our results therefore reveal a new function for Tks4 in the regulation of growth factor-dependent cell migration.

Highlights

The mechanism by which Tks4 regulates actin cytoskeleton is largely unknown
EGF Induces Tyrosine Phosphorylation of Tks4 and Its Interaction with the Epidermal growth factor receptor (EGFR)—We have investigated whether Tks4 is involved directly in the EGF signaling pathway
A 150-kDa phosphotyrosine protein was consistently observed in the immunoprecipitates, the association of this unknown protein seemed to be independent of growth factor treatment

Summary

Background

The mechanism by which Tks regulates actin cytoskeleton is largely unknown. Results: In response to EGF treatment, Tks is tyrosine-phosphorylated and associated with the EGF receptor. In EGF-treated cells, Tks is tyrosine-phosphorylated and associated with the activated EGF receptor. This association is not direct but requires the presence of Src tyrosine kinase. Treatment of cells with LY294002, an inhibitor of PI 3-kinase, or mutations of the PX domain reduces tyrosine phosphorylation and membrane translocation of Tks. We demonstrate that Tks is tyrosine-phosphorylated and associated with the activated EGF receptor upon EGF stimulation of cells. Tks Regulates Cell Migration not direct but requires the presence of Src tyrosine kinase. A PX domain mutant (R43W) Tks carrying a reported point mutation in a Frank-ter Haar syndrome patient showed aberrant intracellular expression and reduced phosphoinositide binding. Silencing of Tks was shown to inhibit HeLa cell migration markedly in response to EGF or serum. Our results reveal a new function for Tks in the regulation of growth factor-dependent cell migration

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION