Signaling of Hepatocyte Growth Factor/Scatter Factor (HGF) to the Small GTPase Rap1 via the Large Docking Protein Gab1 and the Adapter Protein CRKL

Dima Sakkab,Marc Lewitzky,Guido Posern,Ute Schaeper,Martin Sachs,Walter Birchmeier,Stephan M Feller

doi:10.1074/jbc.275.15.10772

Abstract

Hepatocyte growth factor (HGF; scatter factor) is a multipotent protein with mitogenic, motogenic, and developmental functions. Upon activation, the HGF-receptor c-Met binds and phosphorylates the multisite docking protein Gab1. Besides binding motifs for phosphatidylinositol 3-kinase and Grb2, Gab 1 contains multiple Tyr-X-X-Pro (YXXP) motifs which, when phosphorylated, are potential binding sites for the adapter proteins c-Crk and Crk-like (CRKL). Stimulation of human embryonic kidney cells (HEK293) with HGF leads to Gab1 association with CRKL. The Gab1-CRKL interaction requires both, the SH2 domain of CRKL and the region containing the YXXP motifs in Gab1. CRKL binds via its first SH3 domain to several downstream signal transducers, including C3G an activator of the small GTPase Rap1. Indeed, Rap1 was rapidly activated after HGF stimulation of HEK293 cells. Rap1 activation through HGF was suppressed through transfection of a truncated C3G protein which only contains the SH3-binding motifs of C3G. Transfection of nonmutated Gab1 led to a strong increase of Rap1.GTP in the absence of HGF. In contrast, transfection of the GabDeltaYXXP mutant abolished the elevation of Rap1.GTP by HGF. A replating assay indicated that HGF decreases the adhesion of HEK293 cells. The results presented here delineate a novel signaling pathway from HGF to the GTPase Rap1 which depends on the interaction of the adapter protein CRKL with the exchange factor C3G and could be linked to cell migration.

Highlights

Signal transduction events regulating the development and homeostasis of complex multicellular organisms are prominently regulated through the timely activation of different enzymes and through the rapid and selective formation of multiprotein complexes which are often a prerequiste for the appropriate activation of intracellular enzymes
The results presented here delineate a novel signaling pathway from Hepatocyte growth factor (HGF) to the GTPase Rap1 which depends on the interaction of the adapter protein CRKL with the exchange factor C3G and could be linked to cell migration
The SH3(1) domains of c-Crk and CRKL recognize with great preference sequence motifs which conform to the consensus Pro-X-X-Pro-X-Lys [26]

Summary

Introduction

Signal transduction events regulating the development and homeostasis of complex multicellular organisms are prominently regulated through the timely activation of different enzymes and through the rapid and selective formation of multiprotein complexes which are often a prerequiste for the appropriate activation of intracellular enzymes (reviewed in Refs. 1 and 2). The results presented here delineate a novel signaling pathway from HGF to the GTPase Rap1 which depends on the interaction of the adapter protein CRKL with the exchange factor C3G and could be linked to cell migration. CRKL, and their interacting proteins are known to have effects on proliferation, cell differentiation, and migration, cellular responses which all have been reported to occur after HGF stimulation of certain cells.

Results

Conclusion