Mustafa Nazir OKUR1,2, Jolene Yu Zhu OOI3, Chee-Wai FONG3, Natalia MARTINEZ4, Carlota Garcia-DOMINGUEZ4, Jose M. ROJAS4, Graeme GUY3, John P. O’BRYAN1
1 - Departments of Pharmacology, University of Illinois at Chicago
2 - Biochemistry and Molecular Genetics, University of Illinois at Chicago
3 - Institute of Molecular and Cell Biology, Singapore
4 - Unidad de Biologia Celular, Área de Biología Celular y del Desarrollo,
Centro Nacional de Microbiología, Instituto de Salud Carlos III (ISCIII), 28220 Majadahonda, Madrid, Spain.
Published in Mol Cell Biol. 2011 Dec 12. [Epub ahead of print]
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 1 (ITSN1) as an important regulator of this process. ITSN1 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 ITSN1 enhanced Cbl activity was unclear. In this study, we find that ITSN1 enhances Cbl activity through disrupting the interaction of Cbl with the Sprouty2 (Spry2) inhibitory protein. We demonstrate that ITSN1 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 through point mutation of the Pro-rich, ITSN1 binding site in Spry2 results in enhanced Cbl-Spry2 interaction and inhibition of receptor ubiquitylation. This study demonstrates that ITSN1 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 ITSN1 and Spry2.