Abstract

Ubiquitylation of receptor tyrosine kinases plays a critical role in regulating the trafficking and lysosomal degradation of these important signaling molecules. We identified the multi‐domain scaffolding protein intersectin (ITSN) as an important regulator of this process. ITSN stimulates ubiquitylation of the epidermal growth factor receptor (EGFR) through enhancing the activity of the Cbl E3 ubiquitin ligase. However, the precise mechanism through which ITSN enhanced Cbl activity was unclear. In this study, we find that ITSN enhances Cbl activity through disrupting the interaction of Cbl with the Sprouty2 (Spry2) inhibitory protein. We demonstrate that ITSN binds Pro‐rich regions in both Cbl and Spry2 and that interaction of ITSN1 with Spry2 disrupts Spry2‐Cbl interaction resulting in enhanced ubiquitylation of the EGFR. Disruption of ITSN1 binding to Spry2, ITSN1 binding site in Spry2 results in enhanced Cbl‐Spry2 interaction and inhibition of receptor ubiquitylation. This study demonstrates that ITSN enhances Cbl activity by modulating the interaction of Cbl with Spry2. In addition, our results reveal a new level of complexity in the regulation of Cbl through the interaction with ITSN and Spry2.

Highlights

  • Receptor tyrosine kinases (RTKs) play critical roles in the regulation of multiple aspects of metazoan life

  • Our results demonstrate that intersectin 1 (ITSN1) binds both Cbl and Spry2 and that ITSN1 releases Cbl from Spry2 inhibition leading to enhanced epidermal growth factor receptor (EGFR) ubiquitylation

  • Using a biotinylated peptide derived from the Spry2 sequence encompassing this site (TVCCKVPTVPPRNFEKPT ), we identified the SH3 domains of both ITSN1 and ITSN2 as potential binding partners for Spry2

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Summary

Introduction

Receptor tyrosine kinases (RTKs) play critical roles in the regulation of multiple aspects of metazoan life. Binding of ligand stimulates the intrinsic kinase activity of the receptor leading to the recruitment and activation of numerous intracellular signaling pathways. A number of mechanisms exist to regulate the extent and duration of RTK signaling. One such mechanism involves the covalent attachment of ubiquitin to activated receptors. This post-translational modification targets the activated receptors for lysosomal degradation [19]. Regulation of RTK ubiquitylation represents a critical step in cellular signaling

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