Regulation of EGF Receptor Signaling by a Cbl Dimerization Interface
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Date
2011-08
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The Ohio State University
Abstract
Epidermal growth factor receptor (EGFR) overexpression is associated with many tumors, including those of the breast, lung and brain. Understanding how endogenous suppressors of EGFR signaling work could reveal new therapeutic interventions for cancer patients. The ubiquitin ligase Cbl targets EGFR for destruction by ubiquitinating Sprouty2, EGFR, and Hrs at distinct endocytosis checkpoints. These events increase receptor delivery to lysosomes. The Lill laboratory has reported that Cbl’s RING finger (RF) tail domain is essential for degradative targeting of EGFR. My project goal is to identify the mechanism through which RF tail amino acids (aa) act. Based on available crystal structure data, our lab hypothesized that the RF tail mediates the formation of Cbl homodimers with enhanced ubiquitin ligase activity, and that the dimer interface comprises the RF tail of one Cbl molecule and aa 286‐354 of a second. In this model, Cbl homodimers are critical for negotiating the Sprouty2 and Hrs trafficking checkpoints of the endocytic pathway. For my project, I analyzed mammalian cells expressing Cbl mutants that were substituted in aa 286‐354 (one side of the putative dimer interface). The experiments included assays of EGFR downregulation, in which intact cells were immunostained for fluorescent detection of surface EGFR molecules (red) through flow cytometry. By comparing green fluorescent protein (GFP)‐tagged wild‐type and mutant Cbl proteins, I determined their relative abilities to program EGFR for removal from the cell surface. The data revealed that specific amino acids within the putative 286‐354 interface region critically control EGFR surface levels, as their mutation compromised EGFR downregulation. These results support the hypothesis that the RF tail and aa 286‐354 regions of Cbl jointly regulate EGFR fate through their interaction at a Cbl dimer interface. Therefore, it may be possible to develop molecular tools that modulate Cbl dimer formation by mimicking one or the other surface at the interface,thereby altering EGFR signaling in EGFR‐positive tumors.
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Cbl, endocytic trafficking, EGF receptor, dimerization, downregulation, signaling