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. 2014 Jan 14;289(11):7307–7319. doi: 10.1074/jbc.M113.537878

FIGURE 5.

FIGURE 5.

Cbl-3/c-mediated ubiquitination of Ret51 requires both its ring finger and TKB domains. Cbl-3/c mutants were transfected into NIH3T3 cells along with CD2AP, Ret51, and HA-ubiquitin. These inactivating mutations included three point mutations in the ring finger domain of the E3 ligase (C351A, R390Q, and W378A) as well as a deletion of the entire ring finger (TKB only), a mutation of the phosphotyrosine-binding domain of the TKB region (G276E) and a Y341F mutant that eliminates a phosphorylation site that is conserved among Cbl family members. Wild type Cbl-3/c was also included as a positive control (each variant is indicated above the top panel). A, the extent of Ret51 ubiquitination was evaluated as was done in Fig. 1E and displayed. B, three representative experiments were quantified and graphed as the means ± S.E. Asterisks indicated a statistically significant difference (p < 0.05) between the indicated condition and wild type Cbl-3 (WT). C, identical transfections and inhibitor treatments were performed as in A. The cell extracts were subjected to FLAG immunoprecipitation to isolate the transfected Cbl-3 proteins that were all FLAG-tagged. These immunoprecipitations were then immunoblotted with Ret51 antibodies followed by Cbl-3 antibodies. Although the TKB domain of Cbl-3 has a smaller relative molecular mass, it is displayed in the panel along with the other Cbl-3/c mutants (box). Actin was used as a loading control. D, as a control for the specificity of the immunoprecipitations, cells were transfected with Ret and CD2AP in the presence or absence of FLAG-Cbl-3/c. FLAG immunoprecipitations and subsequent immunoblot analysis were performed as in C. E, primary sympathetic neurons were exposed to PP2, PP3, or vehicle alone (DMSO) for 1 h prior to GDNF stimulation for 10 min (indicated above blots). Ret51 was immunoprecipitated and then analyzed with polyubiquitin immunoblotting, as was done in Fig. 1B. Phospho-Src immunoblotting of the supernatants (third panel) confirmed inhibition of Src with PP2. The experiments shown in this figure were performed two or three times with similar results. IP, immunoprecipitation; W, Western blot.