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.