Oncogenic RET Receptors Display Different Autophosphorylation Sites and Substrate Binding Specificities

Abstract

The c-ret proto-oncogene encodes a receptor tyrosine kinase which plays an important role in neural crest as well as kidney development. Genetic studies have demonstrated that germ line mutations in the ret oncogene are the direct cause of multiple endocrine neoplasia (MEN) 2A and 2B, familial medullary thyroid carcinoma (FMTC), and Hirschsprung's disease. However, despite the large body of genetic and biological evidence suggesting the importance of RET in development and neoplastic processes, the signal transduction mechanisms of RET remain unknown. To begin to understand the molecular mechanisms of the disease states caused by mutations in RET, the patterns of autophosphorylation of the wild-type RET and the MEN mutants were studied using site-directed mutagenesis and phosphopeptide mapping. Among the 6 autophosphorylation sites found in the wild-type RET receptor, the MEN2B mutant lacked phosphorylation at Tyr-1096, leading to decreased Grb2 binding, while simultaneously creating a new phosphorylation site. These changes in autophosphorylation suggest that the MEN2B mutation may result in the more aggressive MEN2B phenotype by altering the receptor's signaling capabilities.The c-ret proto-oncogene is a member of the receptor tyrosine kinase superfamily and consists of an extracellular ligand binding domain with a cysteine-rich region, a single transmembrane domain, and an intracellular kinase domain (1). Studies involving transgenic mice lacking the RET receptor have demonstrated a critical function for this receptor in kidney and enteric nervous system development (2).Mutations in the ret gene result in several forms of human disease including familial medullary thyroid carcinoma (FMTC), 1 multiple endocrine neoplasia types , and Hirschsprung's disease (3-6). FMTC is a familial cancer syndrome characterized by medullary thyroid carcinoma while MEN2A variably expresses pheochromocytomas, hyperparathyroidism and rarely Hirschsprung's disease in addition to medullary thyroid carcinoma (7,8). These cancerous states arise by substitution of a cysteine residue in the extracellular domain of RET resulting in ligand independent activation of the RET receptor (9, 10). MEN2B displays all the characteristics of MEN2A but is additionally characterized by a more rapid disease progression, skeletal abnormalities, ganglioneuromas of the intestinal tract, and mucosal neuromas (7). In MEN2B, a Met 3 Thr substitution occurs at position 918 in the kinase domain (5). Finally, Hirschsprung's disease is a loss of function mutation characterized by decreased parasympathetic innervation of the lower intestine (10).It is now well established that the phosphotyrosine residues of the receptor tyrosine kinases can serve as docking sites for molecules containing SH2 domains (11) or PTB domains (12). In order to elucidate potential RET signaling pathways, we initiated efforts to map the phosphorylation sites of the wildtype, MEN2A, and MEN2B RET receptors. Both the wild-type RET and the MEN2A mutant were autophosphorylated at identical ...

Expression of RET 3′ splicing variants during human kidney development

“…The RET51 isoform contains two additional tyrosine residues not present in the RET9 or RET43 isoforms (Y1092 and Y1096) (Myers et al, 1995;Takahashi et al, 1988). The Y1096 residue is phosphorylated on activation of wildtype RET (Liu et al, 1996) and direct binding between the Grb2-SH2 interaction domain and pY1096 has been demonstrated (Borrello et al, 1994). The unique amino acids found in RET9 and RET43 do not include tyrosine residues, however, all three isoforms do include the tyrosine residue at 1062 which can be autophosphorylated on RET activation.…”

“…The sequences of the RET 3' alternatively spliced transcripts diverge at codon 1063 which spans the splice boundary and encodes a glycine conserved between RET9 and RET51 and an aspartic acid in RET43 (Myers et al, 1995;Tahira et al, 1990). The ®nal common amino acid for all three isoforms is a tyrosine (Y1062) which has been shown to undergo phosphorylation on RET activation (Arighi et al, 1997;Liu et al, 1996;Lorenzo et al, 1997). Thus, alternative splicing places Y1062 in di erent amino acid contexts in the three RET isoforms conferring it with di erent binding poten-tials.…”

Oncogenic Flt3 receptors display different specificity and kinetics of autophosphorylation

“…The corresponding site in c-MET receptor (D1264N) has been found to be mutated in papillary renal carcinoma, leading not only to activation of the kinase activity of the receptor, but also altering its substrate specificity [20]. Furthermore, the oncogenic M918T mutant of the receptor tyrosine kinase c-RET (found in multiple endocrine neoplasia type 2B) showed both altered substrate specificity towards exogenous substrates as well as altered pattern of autophosphorylation [21]. This prompted us to investigate whether also the oncogenic mutants of FLT3 showed altered pattern of autophosphorylation, compared to wild-type FLT3 and whether this could explain the phenotypic differences of cells expressing the respective mutant.…”

Germline mutation of RET codon 883 in two cases of de novo MEN 2B

“…And indeed the M918T-RET kinase domain does phosphorylate synthetic peptide substrates of cytoplasmic kinases more e ciently than the wild-type kinase domain (Songyang et al, 1995). In addition, changes in M918T-RET autophosphorylation have been observed (Santoro et al, 1995;Liu et al, 1996). The A883F mutation also lies within a region involved in substrate recognition (Hanks et al, 1988), but the F residue is not seen in cytoplasmic kinases, and in fact a F residue at the equivalent position is very uncommon in tyrosine kinases.…”

“…This mutation, which lies in the substrate binding pocket of the kinase domain (Hanks et al, 1988) leads to ligand-independent activation of RET by an uncertain mechanism, and also to altered tyrosine phosphorylation of the receptor and altered catalytic substrate speci®city (Santoro et al, 1995;Songyang et al, 1995;Liu et al, 1996).…”