Abstract
The RET proto-oncogene encodes a member of the receptor tyrosine kinase family. Multiple endocrine neoplasia type 2B (MEN 2B) is caused by the mutation of a conserved methionine to a threonine in the catalytic domain of the RET kinase. When the MEN 2B point mutation was introduced into the epidermal growth factor (EGF) receptor (M857T EGFR), the intrinsic tyrosine kinase activity of the mutant receptor was similar to that of wild-type EGF receptor and remained ligand-dependent. However, the mutant receptor showed an enhanced transforming capacity compared to the wild-type receptor as judged by its ability to mediate the growth of NIH 3T3 cells in soft agar. Using the oriented peptide library approach to examine substrate specificity, the M857T mutation was found to be associated with a decrease in the selectivity of the receptor for Phe and an increase in the selectivity for acidic residues at the P + 1 position as compared to wild-type EGF receptor. Short-term responses to EGF were similar in cells expressing wild-type and M857T EGF receptors. However, significant differences in receptor down-regulation were observed between the two receptors. These data demonstrate that the MEN 2B point mutation alters the substrate specificity of receptor tyrosine kinases and suggest that the enhanced oncogenesis associated with the MEN 2B mutation may be due in part to alterations in receptor regulation.
Highlights
RET was first identified as a transforming gene by transfection experiments using DNA isolated from a human T-cell lymphoma [1]
Multiple endocrine neoplasia type 2B is the result of a Met 3 Thr mutation in the catalytic domain of the RET tyrosine kinase
Because the RET protein has not been extensively characterized and no ligand has been identified for this receptor, we investigated the effects of this Met 3 Thr mutation within the context of the well studied epidermal growth factor (EGF) receptor
Summary
RET was first identified as a transforming gene by transfection experiments using DNA isolated from a human T-cell lymphoma [1]. Comparison of the sequence of the RET tyrosine kinase domain with the structure of the cyclic AMP-dependent protein kinase with bound inhibitor peptide suggests that Met-918 forms part of the binding site for the substrate residue immediately COOH-terminal to the site of phosphorylation [14, 15]. Based on this information, Carlson et al [4] predicted that the MEN 2B mutation would alter the substrate specificity of the RET tyrosine kinase rather than constitutively activate the enzyme. Songyang et al [16] have shown that wild-type RET and MEN 2B RET exhibit different activity toward three synthetic tyrosine-containing peptides
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