Intersectin Can Regulate the Ras/MAP Kinase Pathway Independent of Its Role in Endocytosis

Xin-Kang Tong,Natasha K Hussain,Anthony G Adams,John P O'Bryan,Peter S Mcpherson

doi:10.1074/jbc.m004096200

Abstract

We previously identified intersectin, a multiple EH and SH3 domain-containing protein, as a component of the endocytic machinery. Overexpression of the SH3 domains of intersectin blocks transferrin receptor endocytosis, possibly by disrupting targeting of accessory proteins of clathrin-coated pit formation. More recently, we identified mammalian Sos, a guanine-nucleotide exchange factor for Ras, as an intersectin SH3 domain-binding partner. We now demonstrate that overexpression of intersectin's SH3 domains blocks activation of Ras and MAP kinase in various cell lines. Several studies suggest that activation of MAP kinase downstream of multiple receptor types is dependent on endocytosis. Thus, the dominant-negative effect of the SH3 domains on Ras/MAP kinase activation may be indirectly mediated through a block in endocytosis. Consistent with this idea, incubating cells at 4 degrees C or with phenylarsine oxide, treatments previously established to inhibit EGF receptor endocytosis, blocks EGF-dependent activation of MAP kinase. However, under these conditions, Ras activity is unaffected and overexpression of the SH3 domains of intersectin is still able to block Ras activation. Thus, intersectin SH3 domain overexpression can effect EGF-mediated MAP kinase activation directly through a block in Ras, consistent with a functional role for intersectin in Ras activation.

Highlights

Src homology 3 (SH3)1 domains are 50- to 70-amino acid protein modules that mediate protein-protein interactions through binding to specific proline-rich peptide sequences
Through its SH3 domains, Grb2 interacts with proline-rich sequences in mammalian son-of-sevenless (mSos), and through its SH2 domain, Grb2 interacts with SHC or activated growth factor receptors, thereby recruiting mSos to the membrane where it can activate Ras [7, 8]
In non-stimulated (0 EGF), serum-starved HEK-293 cells, infected with the green fluorescence protein (GFP), with GFP fused to the five tandem SH3 domains of intersectin (GFPSH3), or with a virus encoding the 170-kDa isoform of synaptojanin [28], little binding of Ras to GSTRaf-1 was observed (Fig. 1A)

Summary

Introduction

Src homology 3 (SH3)1 domains are 50- to 70-amino acid protein modules that mediate protein-protein interactions through binding to specific proline-rich peptide sequences. We demonstrate that overexpression of intersectin’s five SH3 domains blocks EGF-dependent activation of Ras and MAP kinase in HEK-293 cells and COS-7 cells. We examined the activation of this pathway in cells in which EGF receptor endocytosis was blocked and found that, MAP kinase activation was attenuated, Ras activation was not affected.

Results

Conclusion