Abstract
The Cbl proteins (Cbl, Cbl-b, and Cbl-c) are a highly conserved family of RING finger ubiquitin ligases (E3s) that function as negative regulators of tyrosine kinases in a wide variety of signal transduction pathways. In this study, we identify a new Cbl-c interacting protein, Enigma (PDLIM7). This interaction is specific to Cbl-c as Enigma fails to bind either of its closely related homologues, Cbl and Cbl-b. The binding between Enigma and Cbl-c is mediated through the LIM domains of Enigma as removal of all three LIM domains abrogates this interaction, while only LIM1 is sufficient for binding. Here we show that Cbl-c binds wild-type and MEN2A isoforms of the receptor tyrosine kinase, RET, and that Cbl-c enhances ubiquitination and degradation of activated RET. Enigma blocks Cbl-c-mediated RETMEN2A ubiquitination and degradation. Cbl-c decreased downstream ERK activation by RETMEN2A and co-expression of Enigma blocked the Cbl-c-mediated decrease in ERK activation. Enigma showed no detectable effect on Cbl-c-mediated ubiquitination of activated EGFR suggesting that this effect is specific to RET. Through mapping studies, we show that Cbl-c and Enigma bind RETMEN2A at different residues. However, binding of Enigma to RETMENA prevents Cbl-c recruitment to RETMEN2A. Consistent with these biochemical data, exploratory analyses of breast cancer patients with high expression of RET suggest that high expression of Cbl-c correlates with a good outcome, and high expression of Enigma correlates with a poor outcome. Together, these data demonstrate that Cbl-c can ubiquitinate and downregulate RETMEN2A and implicate Enigma as a positive regulator of RETMEN2A through blocking of Cbl-mediated ubiquitination and degradation.
Highlights
Receptor Tyrosine Kinase (RTK) signaling is essential for normal biological processes and disruption of this regulation can lead to tumor initiation and progression
Cbl proteins are a family of RING finger ubiquitin ligases (E3) that negatively regulate a variety of RTKs, tyrosine kinase dependent receptors such as the T-cell receptor, and nonreceptor tyrosine kinases such as Src
Cbl proteins have a highly conserved N-terminus consisting of a tyrosine kinase binding (TKB) domain that binds to specific phosphorylated tyrosines on substrates, a catalytic RING Finger (RF) domain, and an alpha helical linker region separating the TKB and RF domains [1,2]
Summary
Receptor Tyrosine Kinase (RTK) signaling is essential for normal biological processes and disruption of this regulation can lead to tumor initiation and progression. Cbl proteins have a highly conserved N-terminus consisting of a tyrosine kinase binding (TKB) domain that binds to specific phosphorylated tyrosines on substrates, a catalytic RING Finger (RF) domain, and an alpha helical linker region separating the TKB and RF domains [1,2]. The C-termini of the Cbl proteins are the least conserved; each is comprised of a proline rich (PR) region which mediates interactions with SH3-domain containing proteins. The C-termini of Cbl and Cbl-b share tyrosines that, upon phosphorylation, serve as sites of SH2 protein interaction [3] as well as a ubiquitin associated (UBA) domain which has been shown to mediate homodimerization and ubiquitin binding [4,5]
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