Tyrosines 1015 and 1062 are in vivo autophosphorylation sites in RET and RET-derived oncoproteins

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. Somat ic rearrangements of the RET gene generating the chimeric RET/papillary thy roid carcinoma (PTC) oncogenes are the predominant molecular lesions associ ated with papillary carcinoma, the most frequent thyroid malignancy in huma ns. Oncogenic mutations cause constitutive activation of the kinase functio n of RET, which, in turn, results in the autophosphorylation of RET tyrosin e residues critical for signaling. In vitro kinase assays previously reveal ed 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 PET-derived on cogenes. Using phosphorylated RET-specific antibodies, we demonstrate that both Y1015 and Y1062 are rapidly phosphorylated upon ligand triggering of R ET. Moreover, regardless of the nature of the underlying activating mutatio n, the concomitant phosphorylation of Y1015 and Y1062 is a common feature o f the various oncogenic RET products (MEN2A, MEN2B, and PTC). This study sh ows that Ab-pY1062 is a useful tool with which to detect activated RET in h uman tumor cells and surgical samples. Finally, the microinjection of Ab-pY 1062 antibodies into living cells demonstrates that Ret/PTC1 signaling is r equired to maintain the mitogenesis of a human carcinoma cell line expressi ng the Ret/PTC1 oncoprotein.