Investigating the relationship between the RET receptor, Cbl and ARHGEF7 in downregulation of RET

Abstract

The RET proto-oncogene encodes a receptor tyrosine kinase (RTK), with two major isoforms, RET9 and RET51, which differ in their C-termini, and therefore recruit different signaling complexes. RET plays an important role in cell growth, differentiation, migration and survival. Regulation of RET is critical for normal cellular functioning, however, the biochemical mechanisms underlying the downregulation of RET isoforms, are still not clear. Cbl (Casitas B-lineage Lymphoma) is an E3-ubiquitin ligase that plays an essential role in mediating the degradation of RET. Recently, a negative regulator of Cbl, ARHGEF7 (β-pix/ Cool-1) was found to prevent Cbl-catalyzed deregulation of the Epidermal Growth Factor Receptor (EGFR). In the current study, we characterized and further examined the association between RET and Cbl. We showed that RET can associate with the c-Cbl and Cbl-b homologues, in co-immunoprecipitations. Using far western assays and GST-pulldowns, with the purified tyrosine kinase binding (TKB) domain of c-Cbl, we detected a potential novel direct interaction between RET and c-Cbl. Previously, an indirect association between RET and Cbl had been established, indicating that a bimodal interaction may occur. Furthermore, we proposed that ARHGEF7 may interfere with RET-Cbl interactions, either by sequestring Cbl, so that it is unable to bind to RET, or by forming a complex with both RET and Cbl, thereby blocking Cbl activation. Here, we investigated the possibility of a complex formation using co-immunoprecipitations. We showed that ARHGEF7 and c-Cbl can co-immunoprecipitate, but we could not detect either of the RET isoforms in this complex. Further examination of a possible relationship between RET isoforms, and ARHGEF7, showed that ARHGEF7 phosphorylation was dependent on RET activation. However, in an in vitro kinase assay, we showed that this phosphorylation did not occur directly, but may occur indirectly through a pathway yet unknown. Our data predicts that ARHGEF7 may modulate Cbl-binding to RET, and subsequently inhibit its degradation, in a manner similar to that seen for EGFR.