Tu1216 DCLK1 Regulates Intestinal Epithelial Self-Renewal, Survival Signaling Pathways and DNA Repair Machinery in Response to Genotoxic Injury

The receptor tyrosine kinase Met is known to engage Shc-dependent signaling pathways. Structure/function studies have unveiled critical roles for the p52Shc adaptor protein in Met-regulated biological functions. Although Met can bind to the 3 distinct protein isoforms encoded by the ShcA gene, the role of the longest protein isoform, p66Shc, is still undefined. In the present study, co-immunoprecipitation assays were carried out in HEK293 cells transiently transfected with an oncogenic form of the Met receptor (Tpr-Met) and p66Shc mutants, as such to define the molecular determinants involved in mediating Met/p66Shc interaction. Functional impact of p66Shc overexpression in intestinal epithelial cells transformed by Tpr-Met was investigated. We showed that at steady state, p66Shc interacted constitutively with the Met receptor, and the adaptor protein Grb2. Even though not essential for the recruitment of p66Shc, the aberrant activation of Met enhanced these interactions and promoted tyrosine phosphorylation of p66Shc by mechanisms not reliant on the integrity of the Met multisubstrate binding site. Upon Met activation, p66Shc/Grb2 complex was increased, thereby preventing the recruitment of the adaptor protein Grb2 to activated Met. Also, while the p66Shc PTB and SH2 domains, which are usually known to bind phosphorylated tyrosine motifs, coordinate p66Shc binding to non-activated Met receptor, the SH2 domain was revealed to exert negative constraints. In contrast, p66Shc interaction with activated Met was shown to depend mainly on the integrity of its PTB domain, and to a lesser extent of its SH2 domain. Besides, we demonstrated that p66Shc overexpression in intestinal epithelial cells (IEC-6) transformed by Tpr-Met, an oncogenic form of the Met receptor, evoked contextual-dependent responses, such as apoptosis under adherent conditions, but resistance to cell-death in suspension. In conclusion, we identify for the first time a novel non-canonical dynamic mode of interaction between Met and the p66 protein isoform of Shc, and a duality of p66Shc functions downstream of Met. Hence, our studies provide novel insight into the role of the Shc adaptor proteins in Met-regulated biological functions.