Arighi E, Alberti L, Torriti F, Ghizzoni S, Rizzetti M G, Pelicci G, Pasini B, Bongarzone I, Piutti C, Pierotti M A, Borrello M G
Division of Experimental Oncology A, Istituto Nazionale Tumori, Milan, Italy.
Oncogene. 1997 Feb 20;14(7):773-82. doi: 10.1038/sj.onc.1200896.
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 findings, 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 identified 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 identified as Tyr586 of Ret/ptc2 (Tyr1062 on proto-Ret). In keeping with this finding, 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.
RET原癌基因编码一种受体型酪氨酸激酶的两种亚型，该激酶在神经嵴和肾脏发育中起作用。RET的不同种系突变与遗传性癌症综合征MEN2A、MEN2B和FMTC以及先天性疾病先天性巨结肠（HSCR）相关，而体细胞重排（RET/PTCs）在甲状腺乳头状癌中经常被检测到。尽管有这些发现，表明RET产物在发育和肿瘤形成过程中具有相关作用，但关于该受体触发的信号传导知之甚少。在本研究中，我们已经证明转导衔接分子Shc被两种Ret亚型、重排的细胞质Ret/ptc2癌蛋白以及由MEN2A或MEN2B相关突变激活的膜结合受体募集并激活。此外，我们的分析确定了参与相互作用的Ret酪氨酸残基和Shc结构域。事实上，我们在此表明，Shc的两个磷酸酪氨酸结合结构域PTB和SH2在体外均与Ret/ptc2相互作用。然而，PTB结构域结合的Ret/ptc2量比SH2高20倍。已确定SH2和PTB结构域的假定结合位点为Ret/ptc2的Tyr586（原Ret上的Tyr1062）。与这一发现一致，通过使用RET/PTC2 - Y586F突变体，我们已经证明这个酪氨酸残基，即两种Ret亚型分歧前的倒数第二个氨基酸，是Shc的对接位点。
