Engineering the Recruitment of Phosphotyrosine Binding Domain-containing Adaptor Proteins Reveals Distinct Roles for RET Receptor-mediated Cell Survival

T Kalle Lundgren,Rizaldy P Scott,Matthew Smith,Tony Pawson,Patrik Ernfors

doi:10.1074/jbc.m600473200

Abstract

The RET receptor tyrosine kinase is important for several different biological functions during development. The recruitment at the phosphorylated Tyr(1062) site in RET of a number of different phosphotyrosine binding (PTB) domain-containing adaptor proteins, including Shc and Frs2, plays a dominant role for the multiple different biological functions of the RET receptor during development, including stimulation of cell survival. Here, we demonstrate that a competitive recruitment of Shc as opposed to Frs2 mediates the survival signaling arising from RET activation. Based on results from a peptide array, we have genetically engineered the PTB domain binding site of RET to rewire its recruitment of the PTB proteins Shc and Frs2. An engineered RET that has a competitive interaction with Shc at the expense of Frs2, but not a RET receptor that only recruits Frs2, activates cell survival signaling pathways and is protective from cell death in neuronal SK-N-MC cells. Thus, cell type-specific functions involve a competitive recruitment of different PTB adaptor molecules by RET that activate selective signaling pathways.

Highlights

Sites that selectively recruit the SH2 domains of downstream targets in a fashion that can be critical for cellular responses to receptor tyrosine kinases (RTKs) [1,2,3,4]
Phosphorylation of Tyr1062 appears functionally important, since substituting this tyrosine with phenylalanine impairs the transforming activity of the oncogenic MEN2A and MEN2B RET receptors [28], and targeted mutation of Tyr1062 in mice causes a decrease in enteric neurons and induces renal hypoplasia [29], both of which resemble the phenotype of RET null mutant mice
Activated RET can potentially recruit a number of different docking proteins through the autophosphorylated Tyr1062 site, including Shc (28, 30 –31), Enigma [32], SNT/ Frs2 [33, 34], Dok [35, 36], and IRS-1 [37], accounting for the ability of the phosphorylated Tyr1062 site to stimulate the Ras/ ERK, phosphatidylinositol-3 kinase (PI3K)/Akt, p38 mitogenactivated protein kinase (MAPK), c-Jun amino-terminal kinase (JNK), and ERK5 pathways

Summary

EXPERIMENTAL PROCEDURES

DNA Constructs and Mutagenesis—The human cDNA for ret was originally obtained in the PJ7⍀ vector as a gift from Marc Billaud (Universite Claude Bernard). After repeated washings, captured protein was eluted by the addition of buffer containing 12 mM reduced gluthatione (Sigma), 50 mM Tris, pH 7.4, and protease inhibitors Membranes were blocked overnight in 10% skimmed milk, incubated with 1 ␮M purified GST fusion proteins in 0.1% Tween in Tris-buffered saline for 2 h at 4 °C, washed three times, and probed with rabbit polyclonal antiGST antibody. Immunoprecipitations were carried out overnight at 4 °C using the appropriate antibodies immobilized on protein G-Sepharose. Medium was replaced with anisomycin-free growth medium after 6 h, and cells were incubated for an additional 10 h. Apoptosis Assay—SK-N-MC cells grown on glass coverslips were transfected and stimulated with GDNF the following day. After washings in PBS, cells were incubated with Cy2conjugated secondary antibodies for 1 h at room temperature. Images were captured with a Jenoptik ProgRes-C14 camera using OpenLab image acquisition software

RESULTS

Wild type

DISCUSSION