Abstract
Alternative splicing of transcripts encoding the RET kinase receptor leads to isoforms differing in their cytoplasmic tail. Although in vitro studies have demonstrated a higher transforming activity of the long RET isoform (RET51), only the short isoform (RET9) can rescue the effects of a RET null mutation in the enteric nervous system and kidney development. The molecular basis underlying the distinct functions of the two RET isoforms is not understood. Here we demonstrated that activated RET51 associated more strongly with the ubiquitin ligase Cbl than did RET9, leading to increased ubiquitylation and faster turnover of RET51. The association of Cbl with RET was indirect and was mediated through Grb2. A constitutive complex of Grb2 and Cbl could be recruited to both receptor isoforms via docking of Shc to phosphorylated Tyr-1062 in RET. A mutant Shc protein unable to recruit the Grb2.Cbl complex decreased the turnover and prolonged the half-life of RET9, thus ascribing a previously unknown negative role to the Shc adaptor molecule. In addition, phosphorylation of Tyr-1096, which is present in RET51 but absent in RET9, endowed the longer isoform with a second route to recruit the Grb2.Cbl complex. These findings establish a mechanism for the differential down-regulation of RET9 and RET51 signaling that could explain the apparently paradoxical activities of these two RET isoforms. More generally, these results illustrate how alternative splicing can regulate the half-life and function of a growth factor receptor.
Highlights
The RET proto-oncogene encodes a transmembrane tyrosine kinase receptor that plays a crucial role in the development of the neural crest and the excretory system [1, 2]
We demonstrated the association of the ubiquitin ligase Cbl on activated complexes of RET9 and RET51
We found that the levels of RET51 were down-regulated faster in response to ligand stimulation than those of RET9 (Fig. 1A)
Summary
Antibodies and Other Reagents—Antibodies against Cbl, Grb, and isoform-specific antibodies against RET9 and RET51 were from Santa Cruz Biotechnology. Pan-RET polyclonal antibodies were either from R&D Systems or Santa Cruz Biotechnology. Anti-glutathione S-transferase (GST) antibody was purchased from Amersham Biosciences. GST fusion constructs for Cbl were made by subcloning Pfu-amplified fragments in pGEX6P1 vector (Amersham Biosciences). MN1 cells were grown in 7.5% serum in DMEM buffered with 10 mM HEPES. Fusion proteins were affinity captured on glutathione beads (Amersham Biosciences), eluted with reduced glutathione, and dialyzed against phosphate-buffered saline. Immunoprecipitation, Pull-down Assays, and Protein Immunoblots— Cultured cells were lysed in modified radioimmune precipitation assay buffer (50 mM Tris-HCl, pH7.4, 100 mM NaCl, 50 mM NaF, 1 mM EDTA, 10 mM 2-glycerolphosphate, 2 mM Na3VO4, 1% (v/v) IGEPAL CA-630, 0.25% (w/v) sodium deoxycholate, 10% (v/v) glycerol) and supplemented with Complete protease inhibitor mixture (Roche Applied Bioscience). Immune and affinity pull-down complexes were eluted for protein gel analysis by boiling in reducing Laemmli buffer. polyvinylidene difluoride membranes were used for immobilization of gel-fractionated proteins, and Western blot detection was performed by enhanced chemifluorescence method using a Storm PhosphorImager (Amersham Biosciences)
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