Poster abstracts

Poster number 27 submitted by Nurettin Ilter Sever

Structure-Function Studies of a Putative Cbl Dimer Interface

Nurettin Ilter Sever (Department of Pathology, The Ohio State University), Rajeev Sachdeva (Medical Physiology Graduate Program, Case Western Reserve University), Nancy L.Lill, Ph.D (Department of Pathology, The Ohio State University)

Abstract:

Cbl (Casitas B-lineage) is a RING-type E3 ubiquitin ligase that ubiquitinates its substrates, thereby marking them for degradation. Epidermal growth factor receptor (EGFR) is a prominent substrate for Cbl. EGFR plays roles in epithelial development by activating distinct signaling pathways. Deregulated EGF receptor signaling has been implicated in a variety of cancers, such as breast, lung and glioblastoma. To maintain the desired amplitude and duration of signaling for normal cell proliferation and survival, cellular EGFR levels must be tightly controlled. When a cell is activated by epidermal growth factor (EGF), an EGFR ligand, Cbl reduces cellular EGFR levels by ubiquitinating receptors and directing them for degradation.
An early crystal structure of unmodified Cbl suggested that Cbl might function as a dimer, whose interface consists of different residues from each Cbl subunit. We hypothesize that hydrogen bonds or hydrophobic interactions between residues in this putative dimer interface play a very important role in Cbl function. In this study, alanine and charge or polarity substitutions of selected interface residues were generated. One set of the mutant Cbl proteins were tested in terms of EGFR downregulation from the cell surface and EGFR degradation following EGF stimulation of HEK 293 cells.
Alanine substitution for H286 and R333 (potential hydrogen-bonds) and Q345 (hydrophobic) impaired EGFR degradation in HEK 293 cells following EGF stimulation. Alanine substitution for H286 and D348 (potential hydrogen-bonding residues) impaired EGFR downregulation in HEK 293 cells following EGF stimulation. Direct correlation of downregulation and degradation was observed for the H286A Cbl mutant but not for R333A, Q345A, or D348A.
These and additional Cbl mutant proteins have been tested in terms of: EGFR binding and ubiquitination; EGFR downregulation from the cell surface; EGFR trafficking through the degradative pathway and receptor degradation and also the effect of the mutant Cbl proteins on EGFR-regulated signaling pathways following EGF stimulation.
Based on our results, we conclude that a subset of dimerization interface residues is critical for Cbl’s function in regulating EGFR.

References:
Visser Smit GD et al. (2009) Cbl controls EGFR fate by regulating early endosome fusion. Sci Signal. 2(102):ra86.

Zheng N et al. (2000) Structure of a c-Cbl-UbcH7 complex: RING domain function in ubiquitin-protein ligases. Cell 102(4):533-9

Keywords: Cbl, EGFR, downregulation