Preliminary screening data showed that the ShcD adaptor protein associates with the proto-oncogene RET receptor tyrosine kinase. In the present study, we aimed to investigate the molecular interaction between ShcD and RET in human neuroblastoma cells and study the functional impact of this interaction. We were able to show that ShcD immunoprecipitated with RET from SK-N-AS neuroblastoma cell lysates upon GDNF treatment. This result was validated by ShcD-RET co-localization, which was visualized using a fluorescence microscope. ShcD-RET coexpression promoted ShcD and RET endosomal localization, resulting in unexpected inhibition of the downstream ERK and AKT pathways. Interestingly, ShcD-RET association reduced the viability and migration of SK-N-AS cells. Although ShcD was previously shown to trigger melanoma cell migration and tumorigenesis, our data showed an opposite role for ShcD in neuroblastoma SK-N-AS cells via its association with RET in GDNF-treated cells. In conclusion, ShcD acts as a switch molecule that promotes contrasting biological responses depending on the stimulus ad cell type.
Keywords: ALK,, Anaplastic Lymphoma Kinase; Akt,, Protein kinase B;; CMV,, Cytomegalovirus; DMEM,, Dulbecoo Modified Eagle's Medium;; DNA,, Deoxyribonucleic Acid; ECL,, Enhanced Chemiluminescence;; EGF,, Epidermal Growth Factor;; EGFR,, Epidermal Growth Factor Receptor;; ERK,, Extracellular Signal–Regulated Kinases;; Endosomes; FBS,, Fetal Bovine Serum; FGFR,, fibroblast growth factor receptors; GDNF; GDNF,, Glial Cell Line-Derived Neurotropic Factor;; GFLs,, GDNF Family Ligands;; GFP,, Green Fluorescent Protein; GPCR,, G-Protein Coupled Receptor; GRB2,, Growth Factor Receptor-Bound Protein 2;; HGFR,, hepatocyte growth factor receptor;; HRP,, Horseradish Peroxidase; IGF,, Insulin Growth Factor;; LB,, Luria-Bertani; MAP,, Mitogen-Activated Protein;; MAPK,, Mitogen-Activated Protein Kinases; MuSK,, Muscle Specific Kinase; NFDM,, Non-Fat Dry Milk; Neuroblastoma; PBS,, Phosphate-Buffered Saline; PBST,, Phosphate-Buffered Saline Tween; PDGF,, Platelet-Derived Growth Factor;; PI3K,, Phosphoinositide 3-Kinase; PMSF,, Phenylmethylsulfonyl Fluoride; PVDF,, Polyvinylidene Fluoride; RET; RET,, Rearranged During Transfection; RT,, Room Temperature;; RTKs,, Receptor Tyrosine Kinase; SDS-PAGE,, Sodium Dodecylsulphate Polyacrylamide Gel Electrophoresis; ShcD; ShcD,, Src Homology And Collagen D; Src,, Proto-Oncogene Tyrosine-Protein Kinase Src; TKRs,, Tyrosine Kinase Receptor;; TrkA/B/C,, Tropomyosin-Related Kinase Receptor A/B/C; hrs,, Hours; mAb,, Monoclonal Antibody; min,, Minute; pAb,, Polyclonal Antibody; pTyr,, Phospho-Tyrosine; rpm,, revolution per minute;.