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Tyrosines 1015 and 1062 Are in VivoAutophosphorylation Sites in Ret and Ret-Derived Oncoproteins¹

Centro di Endocrinologia ed Oncologia Sperimentale del Consiglio Nazionale delle Ricerche, Dipartimento di Biologia e Patologia Cellulare e Molecolare (D.S., M.V.B., G.S., R.M., G.V., M.S.); Istituto Nazionale dei Tumori di Napoli, Fondazione Senatore Pascale (G.C., A.M.); and Dipartimento di Endocrinologia ed Oncologia Molecolare e Clinica, Facoltà di Medicina e Chirurgia, Università Federico II (D.S., G.F.), 80131 Naples, Italy; and Dipartimento di Medicina Sperimentale e Clinica, Facoltà di Medicina e Chirurgia, Università Magna Graecia (A.F.), 88100 Catanzaro, Italy

Address all correspondence and requests for reprints to: Dr. Massimo Santoro, Centro di Endocrinologia ed Oncologia Sperimentale del Consiglio Nazionale delle Ricerche, Università di Napoli Federico, Via S. Pansini 5, 80131 Naples, Italy. E-mail: masantor{at}unina.it

Point mutations of the RET receptor tyrosine kinase are responsible for the inheritance of multiple endocrine neoplasia (MEN) type 2 syndromes and are also present in a fraction of sporadic medullary thyroid carcinomas. Somatic rearrangements of the RET gene generating the chimeric RET/papillary thyroid carcinoma (PTC) oncogenes are the predominant molecular lesions associated with papillary carcinoma, the most frequent thyroid malignancy in humans. Oncogenic mutations cause constitutive activation of the kinase function of RET, which, in turn, results in the autophosphorylation of RET tyrosine residues critical for signaling. In vitro kinase assays previously revealed six putative RET autophosphorylation sites. The aim of the present study was to assess the phosphorylation of two such residues, tyrosines 1015 and 1062 (Y1015 and Y1062), in the in vivo signaling of RET and RET-derived oncogenes. Using phosphorylated RET-specific antibodies, we demonstrate that both Y1015 and Y1062 are rapidly phosphorylated upon ligand triggering of RET. Moreover, regardless of the nature of the underlying activating mutation, the concomitant phosphorylation of Y1015 and Y1062 is a common feature of the various oncogenic RET products (MEN2A, MEN2B, and PTC). This study shows that Ab-pY1062 is a useful tool with which to detect activated RET in human tumor cells and surgical samples. Finally, the microinjection of Ab-pY1062 antibodies into living cells demonstrates that Ret/PTC1 signaling is required to maintain the mitogenesis of a human carcinoma cell line expressing the Ret/PTC1 oncoprotein.

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