CD2-associated Protein (CD2AP) Enhances Casitas B-lineage Lymphoma-3/c (Cbl-3/c)-mediated Ret Isoform-specific Ubiquitination and Degradation via its Amino-terminal SRC Homology 3 (SH3) Domains [Neurobiology]

January 14th, 2014 by Calco, G. N., Stephens, O. R., Donahue, L. M., Tsui, C. C., Pierchala, B. A.

Ret is the receptor tyrosine kinase for the glial cell line-derived neurotrophic factor (GDNF) family of neuronal growth factors. Upon activation by GDNF, Ret is rapidly polyubiquitinated and degraded. This degradation process is isoform selective, with the longer Ret51 isoform exhibiting different degradation kinetics than the shorter isoform, Ret9. In sympathetic neurons Ret degradation is induced, at least in part, by a complex consisting of the adaptor protein CD2AP and the E3-ligase Cbl-3/c. Knock down of Cbl-3/c using siRNA reduced the GDNF-induced ubiquitination and degradation of Ret51 in neurons and podocytes, suggesting that Cbl-3/c was a predominant E3 ligase for Ret. Co-expression of CD2AP with Cbl-3/c augmented the ubiquitination of Ret51 as compared to the expression of Cbl-3/c alone. Ret51 ubiquitination by the CD2AP/Cbl-3/c complex required a functional ring finger and TKB domain in Cbl-3/c. The SH3 domains of CD2AP were sufficient to drive the Cbl-3/c-dependent ubiquitination of Ret51, while the C-terminal coiled coil domain of CD2AP was dispensable. Interestingly, activated Ret induced the degradation of CD2AP, but not Cbl-3/c, suggesting a potential inhibitory feedback mechanism. There were only two major ubiquitination sites in Ret51, K1060 and K1107, and the combined mutation of these lysines almost completely eliminated both the ubiquitination and degradation of Ret51. Ret9 was not ubiquitinated by the CD2AP/Cbl-3/c complex, suggesting that Ret9 was downregulated by a fundamentally different mechanism. Taken together, these results suggest that only the SH3 domains of CD2AP were necessary to enhance the E3 ligase activity of Cbl-3/c towards Ret51.
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