Identification of Shc docking site on Ret tyrosine kinase

Abstract

The RET proto-oncogene encodes two isoforms of a receptor type tyrosine kinase which plays a role in neural crest and kidney development. Distinct germ-line mutations of RET have been associated with the inherited cancer syndromes MEN2A, MEN2B and FMTC as well as with the congenital disorder Hirschsprung disease (HSCR), whereas somatic rearrangements (RET/PTCs) have been frequently detected in the papillary thyroid carcinoma. Despite these ®ndings, suggesting a relevant role for RET product in development and neoplastic processes, little is known about the signalling triggered by this receptor. In this study, we have demonstrated that the transducing adaptor molecule Shc is recruited and activated by both Ret isoforms and by the rearranged cytoplasmatic Ret/ptc2 oncoproteins as well as by the membrane bound receptor activated by MEN2A or by MEN2B associated mutations. Moreover, our analysis has identi®ed the Ret tyrosine residue and the Shc domains involved in the interaction. In fact, here we show that both the phosphotyrosine binding domains of Shc, PTB and SH2, interact with Ret/ptc2 in vitro. However, PTB domain binds 20 folds higher amount of Ret/ptc2 than SH2. The putative binding site for either SH2 and PTB domains has been identi®ed as Tyr586 of Ret/ptc2 (Tyr1062 on proto-Ret). In keeping with this ®nding, by using RET/PTC2-Y586F mutant, we have demonstrated that this tyrosine residue, the last amino acid but one before the divergence of the two Ret isoforms, is the docking site for Shc.

Signalling of the Ret receptor tyrosine kinase through the c-Jun NH2-terminal protein kinases (JNKs): evidence for a divergence of the ERKs and JNKs pathways induced by Ret

“…We constructed a Y1062F Ret/MEN2A protein mutant (RET/MEN2A Y1062F ) and we con®rmed that, when expressed in COS-1 cells, it was correctly synthesized (Figure 7a, lower). Moreover, an immunoprecipitation with anti-Ret followed by blotting with anti-phosphotyrosine antibodies, indicated that, as reported Lorenzo et al, 1997;Arighi et al, 1997), Y1062F mutation did not alter the intrinsic kinase activity of Ret/MEN2A (see for an example Figure 7b). As expected, Y1062F mutation abrogated in vitro binding of Ret/MEN2A to the PTB domain of Shc (Figure 7a, upper).…”

“…Conversely, both isoforms are able to bind and to induce the tyrosine phosphorylation of Shc (Borrello et al, 1994;Lorenzo et al, 1997). In particular, Ret tyrosine residue 1062, which is common to both Ret 1072 and Ret 1114 , is part of a docking site for the Shc phosphotyrosine binding (PTB) domain Lorenzo et al, 1997;Arighi et al, 1997). Our preliminary results indicate that Y1062F mutation impairs Ret activation of endogenous Ras (Visconti et al, unpublished).…”

“…Mutation of tyrosine 1062 abrogates both Shc binding to Ret and Shc tyrosine phosphorylation induced by Ret Lorenzo et al, 1997;Arighi et al, 1997;Melillo et al, in preparation). We constructed a Y1062F Ret/MEN2A protein mutant (RET/MEN2A Y1062F ) and we con®rmed that, when expressed in COS-1 cells, it was correctly synthesized (Figure 7a, lower).…”

“…Ret has been found to activate the g isoenzyme of phospholypase C (Santoro et al, 1994;Borrello et al, 1996), Ras (Santoro et al, 1994), and ERKs (van Weering et al, 1995). Borrello and coworkers have reported that Ret protein binds to and phosphorylates Shc (Borrello et al, 1994;Asai et al, 1996) and other groups (Lorenzo et al, 1997;Arighi et al, 1997) have demonstrated that tyrosine residue 1062 of Ret is a binding site for the Shc-PTB domain and that its mutation abrogates Shc binding and reduces Ret transforming ability.…”

Dependence receptors: a new paradigm in cell signaling and cancer therapy

“…The receptor complex also includes (GPI)-anchored proteins GFRa1, 2, 3 and 4 that are required for RET dimerization and dictate ligand selectivity (Baloh et al, 2000;Scott and Ibanez, 2001). After interaction with its ligands, RET undergoes autophosphorylation and then interacts with multiple effectors such as phospholipase C, Shc, enigma, Grb2, Grb7/ Grb10, Src kinase and Ras-GAP (Santoro et al, 1994;Arighi et al, 1997;Lorenzo et al, 1997). Gainof-function mutations of the RET gene have been associated with multiple endocrine neoplasia type 2 (MEN 2), an autosomal dominant inherited cancer syndrome (Mulligan et al, 1993), whereas loss-offunction mutations of RET have been associated with Hirschsprung disease (aganglionosis, HSCR), a frequent congenital intestinal malformation (1 in 5000 live births) characterized by the absence of neural crest-derived parasympathetic neurons of the hindgut (Edery et al, 1994;Romeo et al, 1994).…”