Authors
Mustafa Nazir OKUR^1,2, 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]

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 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.

Figure Legend : ITSN1 binding to Cbl in the absence of Spry2 binding leads to enhanced Spry2-Cbl interaction and decreased EGFR ubiquitylation. (A) Interaction of Spry2 P304A with Cbl was measured by BiFC in the absence or presence of increasing ITSN1 levels as described in Fig. 5. ITSN1 overexpression results in enhanced binding of Spry2 P304A to Cbl. (B) Quantification of BiFC signal. Interaction of Spry2 P304A and Cbl was quantified as described (36). Results are the average of three independent experiments +/- SEM. Samples marked with asterisk were significantly different from VN-Spyr2 P304A + VC-Cbl sample (p<0.05). (C) Western blot demonstrates the expression of the various proteins. Both ITSN1 and Cbl are HA tagged. The differences in Spry2 P304A-Cbl interaction are not due to changes in the overall expression of these proteins. (D) Overexpression of HA-epitope tagged ITSN1 597 dose-dependently enhances the binding of Spry2 P304A mutant to Cbl. HA-Cbl was immunoprecipitated from cells and the co-precipitation of Spry2 P304A was monitored by Western blot of Cbl precipitates. Top two panels: Western blot of anti-Cbl precipitates with the indicated antibodies. Bottom two panels: Western blot of cell lysates with the indicated antibodies.

This work was a collaboration involving principally Jolene and Chee Wai from the GG lab and the lab of John O'Brien from the University of Illinois in Chicago, USA.