RAI(ShcC/N-Shc)-dependent recruitment of GAB1 to RET oncoproteins potentiates PI3-K signalling in thyroid tumors Valentina De Falco 1,3 , Valentina Guarino 1,3 , Luca Malorni 1 , Anna Maria Cirafici 1 , Flavia Troglio 2 , Marco Erreni 2 , Giuliana Pelicci 2 , Massimo Santoro 1 and Rosa Marina Melillo* ,1 1 Istituto di Endocrinologia ed Oncologia Sperimentale del CNR ‘G. Salvatore’, c/o Dipartimento di Biologia e Patologia Cellulare e Molecolare, Via S. Pansini 5, 80131 Naples, Italy; 2 Department of Experimental Oncology, European Institute of Oncology, Milan, Italy RAI, also named ShcC/N-Shc, one of the members of the Shc proteins family, is a substrate of the RET receptor tyrosine kinase. Here, we show that RAI forms a protein complex with both RET/MEN2A and RET/PTC onco- proteins. By coimmunoprecipitation, we found that RAI associates with the Grb2-associated binder1 (GAB1) adapter. This association is constitutive, but, in the presence of RET oncoproteins, both RAI and GAB1 are tyrosine-phosphorylated, and the stoichiometry of this interaction remarkably increases. Consequently, the p85 regulatory subunit of phosphatidylinositol-3 kinase (PI- 3K) is recruited to the complex, and its downstream effector Akt is activated. We show that human thyroid cancer cell lines derived from papillary or medullary thyroid carcinoma (PTC or MTC) carrying, respectively, RET/PTC and RET/MEN2A oncoproteins express RAI proteins. We also show that human PTC samples express higher levels of RAI, when compared to normal thyroid tissue. In thyroid cells expressing RET/PTC1, ectopic expression of RAI protects cells from apoptosis; on the other hand, the silencing of endogenous RAI by small inhibitory duplex RNAs in a PTC cell line that expresses endogenous RET/PTC1, increases the rate of sponta- neous apoptosis. These data suggest that RAI is a critical substrate for RET oncoproteins in thyroid carcinomas. Oncogene (2005) 24, 6303–6313. doi:10.1038/sj.onc.1208776; published online 6 June 2005 Keywords: thyroid tumor; RET oncogenes; RAI and GAB1 adaptors Introduction The family of Shc-like proteins includes three members: Shc, SLI (ShcB/ScK) and RAI (ShcC/N-Shc) (Cattaneo and Pelicci, 1998; Luzi et al., 2000). It has been shown that while Shc is ubiquitously expressed, the other two members, RAI and SLI, are predominantly expressed in neural tissues (O’Bryan et al., 1996; Pelicci et al., 1996; Nakamura et al., 1998). In support of their role in the developing brain, it has been shown that null mutations of both RAI and SLI in mice results in a loss of sympathetic neurons. In particular, neurons of the superior cervical ganglia (SCG) are significantly reduced in number in the double-knockout mice, a phenotype that resembles that induced by the knockout of RET tyrosine kinase receptor (Sakai et al., 2000). Accord- ingly, we showed that RAI is a physiological substrate of the RET receptor, and that it potentiates RET- mediated activation of phosphatydilinositol 3-kinase (PI3-K) and RET-dependent survival of neuronal cells. We also defined the molecular determinants of this interaction, which is mediated by tyrosine 1062 of RET and the phosphotyrosine binding (PTB) domain of RAI (Pelicci et al., 2002). The RAI gene codes for two proteins, p64 RAI and p52 RAI, as a consequence of alternative initiation codon usage. These proteins, like the other members of the Shc-like family, share a similar PTB-CH1-SH2 domains modular structure (Luzi et al., 2000; Ravi- chandran, 2001). The PTB and SH2 domains are phospho-tyrosine-recognition modules common to sev- eral different proteins with similar functions. The CH1 region contains tyrosine-phosphorylation residues and several SH3 binding sites. For these features, RAI proteins have been implicated in signal transduction mediated by tyrosine kinase receptors (Nakamura et al., 1996; O’Bryan et al., 1998; Pelicci et al., 2002; Liu and Meakin, 2002; Nakazawa et al., 2002). While RAI and Shc share many common properties, specific features of RAI-mediated signalling have been identified. First, RAI transduces Grb2-SOS-RAS-dependent ERK acti- vation less efficiently than Shc, because it binds less efficiently Grb2. Furthermore, RAI has three novel tyrosine phosphorylation sites that are not present in Shc. These sites represent potential docking sites for signalling adapters, one of which has been identified as the CRK protein. These data implicate that recruitment of RAI may result in specific signalling output and consequent biological activities (Nakamura et al., 2002). The receptor tyrosine kinase RET is specifically activated by members of the GDNF family of ligands, which are involved in the control of neuronal survival Received 21 December 2004; revised 12 April 2005; accepted 15 April 2005; published online 6 June 2005 *Correspondence: RM Melillo; E-mail: rosmelil@unina.it 3 These authors contributed equally to this study Oncogene (2005) 24, 6303–6313 & 2005 Nature Publishing Group All rights reserved 0950-9232/05 $30.00 www.nature.com/onc