The Multiple Endocrine Neoplasia Type 2B Point Mutation Alters Long-term Regulation and Enhances the Transforming Capacity of the Epidermal Growth Factor Receptor

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 wildtype 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.RET was first identified as a transforming gene by transfection experiments using DNA isolated from a human T-cell lymphoma (1). Subsequently, physical and genetic mapping data have implicated the RET gene in several dominantly inherited human neoplasias, including multiple endocrine neoplasia type 2A (MEN 2A), 1 multiple endocrine neoplasia type 2B (MEN 2B), and familial medullary thyroid carcinoma (FMTC) (2-6). MEN 2A and FMTC are both characterized by the development of medullary thyroid carcinoma between the ages of 20 and 50. Approximately one-third to one-half of MEN 2A patients also develop pheochromocytomas and parathyroid hyperplasia. MEN 2B is a more severe form of the disease with onset of symptoms during the first decade of life. Patients exhibit ganglioneuromas of the intestinal tract, mucosal neuromas, and opthalmalogic and skeletal abnormalities, in addition to a particularly aggressive form of medullary thyroid carcinoma.The RET proto-oncogene encodes a protein that is a member of the family of transmembrane tyrosine kinase growth factor receptors. The RET protein is comprised of a 635-amino acid extracellular domain, a single transmembrane spanning region, and an intracellular tyrosine kinase domain that contains a short, 26-amino acid kinase insert domain. Residues 515 to 634 of the extracellular domain constitute a region of high cysteine content similar to those found in the EGF receptor and the insulin receptor (7, 8). Immediately amino-terminal to this high cysteine region is a domain of ϳ110 amino acids that is 20 -30% homologous to cadherins (9) The presence of this cadherin-like domain is unique among ...

Point mutations in the tyrosine kinase domain release the oncogenic and metastatic potential of the ron receptor

“…Sarcomas compress the normal alveolar tissue (d), are highly vascularized in appearance (e) and contain typical cell whorl structures (f). d and e,f were photographed at 610 and 633 respectively substrate speci®city (Pandit et al, 1996). Our data suggest that the D1232V and M1254T mutations alter substrate speci®city also in the Ron receptor.…”

“…These proteins were not phosphorylated in lysates of cells expressing wildtype Tpr ± Ron and Ron proteins (Figure 4a). This set of phosphorylated proteins is reminiscent of two phosphoproteins of similar molecular mass observed in ®broblasts expressing the EGF receptor carrying the MEN2B, Ret-type mutation (Pandit et al, 1996). A panel of antibodies speci®c for known proteins in this size range, namely GAP, GAB-1, Cbl, p85, failed to react with these phosphorylated putative substrates (data not shown).…”

The multiple endocrine neoplasia type 2B point mutation switches the specificity of the Ret tyrosine kinase towards cellular substrates that are susceptible to interact with Crk and Nck

“…Accordingly, it has been proposed that the MEN 2B mutation changes the Ret TK substrate speci®city by substituting the Met 918 , a residue highly conserved in the transmembrane RTK to a Thr residue that is characteristic of cytosolic nonreceptor TK family (Hanks et al, 1988). Consistently, when the MEN 2B mutation was introduced into the RET or EGFR sequences, their speci®city, as measured on synthetic substrates, switched to that characteristic of cytosolic Src-like kinases (Pandit et al, 1996;Songyang et al, 1995). Our data support the idea that the MEN 2B mutation is sucient to orientate the kinase activity towards substrates that selectively interact with Nck and Crk.…”

Identification of Shc docking site on Ret tyrosine kinase

“…Given that Ret/ptc oncoproteins localize in the cytoplasm, whereas Ret-C634R and Ret-M918T maintain the transmembrane localization as proto-Ret, these ®ndings indicate that, irrespectively of the subcellular localizations, the cytoplasmic domain of Ret is suited to recruit Shc. In addition, although the M?T mutation associated with MEN2B, as well as the M?T mutation at the corresponding amino acid in EGFR, was demonstrated responsible for changing the substrate speci®city of the kinase (Songyang et al, 1995;Pandit et al, 1996), Shc appears a common substrate for wild type and MEN2B Ret kinase. This ®nding is in agreement with the evidence that both the receptor-type tyrosine kinases, harboring a methionine, and the Src-like tyrosine kinases, harboring a threonine in the corresponding position (Hanks et al, 1994), are able to activate Shc (e.g.…”