Abstract
When appended to the epidermal growth factor receptor (EGFR), ubiquitin serves as a sorting signal for lysosomal degradation. Here we demonstrate that the ubiquitin ligase of EGFR, namely c-Cbl, also mediates receptor modification with the ubiquitin-like molecule Nedd8. EGF stimulates receptor neddylation, which enhances subsequent ubiquitylation, as well as sorting of EGFR for degradation. Multiple lysine residues, located within the tyrosine kinase domain of EGFR, serve as attachment sites for Nedd8. A set of clathrin coat-associated binders of ubiquitin also bind Nedd8, but they undergo ubiquitylation, not neddylation. We discuss the emerging versatility of the concerted action of ubiquitylation and neddylation in the process that desensitizes growth factor-activated receptor tyrosine kinases.
Highlights
Growth factors and their transmembrane receptors, harboring intrinsic tyrosine kinase activity, play essential roles in cell fate determination
Neddylation of Cullin1 and Cullin2 activates their E3 ubiquitin ligase activity toward substrates like p27 [18, 19] and HIF␣ [20], which led to the suggestion that Nedd8 assists the positioning of the E2-ubiquitin complex and subsequent ubiquitin transfer [21]
Receptor neddylation strictly depends on ligand stimulation, and in coordination with ubiquitin, it accelerates sorting of epidermal growth factor receptor (EGFR) to degradation
Summary
Materials and Antibodies—Unless indicated, materials were purchased from Sigma, radioactive materials from Amersham Biosciences, and antibodies from Santa Cruz Biotechnology (Santa Cruz, CA). The number of surface-exposed ligand binding sites was determined by incubating cells for 90 min at 4 °C with a radiolabeled EGF. Ligation in an expression plasmid was performed following restriction of DNA encoding EGFR and FLAG with these enzymes. Cbl [4], GST-UIM [26], and ⌬C4R-EGFR [8] expression vectors were previously described. Membranes were blocked for 1 h in TBST buffer (20 mM Tris-HCl (pH 7.6), 0.15 M NaCl, 0.05% Tween 20) supplemented with albumin (3%), followed by blotting for 1 h with a primary antibody, washing with TBST and re-blotting with horseradish peroxidase-conjugated secondary antibodies. Cell lysates were incubated for 1 h at 4 °C with glutathione-agarose beads or with GST fusion proteins precoupled to glutathione-agarose beads. The following sense and the respective antisense sequence were selected for effective knockdown of Nedd in cells (construct C; nucleotides 162–181): 5Ј-GACAGCAGCTGATTACAAG-3Ј
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