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. Author manuscript; available in PMC: 2010 Jun 22.
Published in final edited form as: Sci Signal. 2009 Dec 22;2(102):ra86. doi: 10.1126/scisignal.2000217

Figure 1. Biochemical characterization of Cbl RF tail alanine substitution mutants.

Figure 1

A. The conserved C-terminal flank of the RING finger in human Cbl isoforms. Domains relevant to EGF-R regulation include: the tyrosine kinase-binding domain (TKB); linker region (L); RING finger domain (RF); RING finger C-terminal flank or “tail” domain (T); proline-rich region (PRO); and leucine zipper (LZ). Residue Y371 lies within the linker region. RING finger tail sequences of human Cbl isoforms (c-Cbl, Cbl-b, and Cbl-3) are aligned in the expanded portion of the graphic. Evolutionarily conserved sequences are marked with shaded (sequence identity) or plain (amino acid similarity) boxes. Underlined residues were substituted for this study. The vertical dashed line marks the C-terminal limit of the evolutionarily conserved Cbl sequences sufficient to enhance EGF-R ubiquitination, downregulation, and degradation. B. Cbl RF tail substitution mutants V431A and F434A are compromised for EGF-R downregulation. Surface EGF-R remaining at each stimulation time point was expressed relative to the amount of surface receptor in matched unstimulated cells. Results reflect the mean of three independent experiments ± S.D. C. Cbl RF tail substitution mutants V430A, V431A and F434A effect reduced EGF-R ubiquitination after 10 minutes of cell incubation with EGF. Upper section: 750 μg immunoprecipitates (anti-EGF-R antibody 528). Isotype-matched anti-Syk antibody 4D10 [C] was the specificity control. Immunoprecipitates (I.P.) and 75 μg lysate protein samples were gel-resolved and sequentially immunoblotted (I.B.). Lower section: Quantification of results. Ub signals were normalized to their matching EGF-R signals. Mean values were expressed relative to the signal achieved with GFP-Cbl wild type (1.00). Results represent data from three independent experiments +/− S.D. Using Student’s t-test with α=0.05, only V431A and V430A produced significantly less EGF-R ubiquitination than wt Cbl. D. Cbl mutants F434A and V431A effect reduced and delayed EGF-R ubiquitination and reduced EGF-R degradation. Upper section: 1 mg anti-EGF-R (528) immunoprecipitates. I.P.s (top three panels) and 100 μg of lysate protein from each sample (bottom panel) were sequentially immunoblotted using the antibodies indicated. The double claret marks the position of the predominant species of ubiquitinated EGF-R. Lower section: Quantification of EGF-R degradation effected by wild type and RF tail mutant Cbl proteins (100 μg protein/lane).