A novel LKB1 isoform enhances AMPK metabolic activity and displays oncogenic properties.

The LKB1 tumor suppressor gene encodes a master kinase that coordinates the regulation of energetic metabolism and cell polarity. We now report the identification of a novel isoform of LKB1 (named ΔN-LKB1) that is generated through alternative transcription and internal initiation of translation of the LKB1 mRNA. The ΔN-LKB1 protein lacks the N-terminal region and a portion of the kinase domain. Although ΔN-LKB1 is catalytically inactive, it potentiates the stimulating effect of LKB1 on the AMP-activated protein kinase (AMPK) metabolic sensor through a direct interaction with the regulatory autoinhibitory domain of AMPK. In contrast, ΔN-LKB1 negatively interferes with the LKB1 polarizing activity. Finally, combining in vitro and in vivo approaches, we showed that ΔN-LKB1 has an intrinsic oncogenic property. ΔN-LKB1 is expressed solely in the lung cancer cell line, NCI-H460. Silencing of ΔN-LKB1 decreased the survival of NCI-H460 cells and inhibited their tumorigenicity when engrafted in nude mice. In conclusion, we have identified a novel LKB1 isoform that enhances the LKB1-controlled AMPK metabolic activity but inhibits LKB1-induced polarizing activity. Both the LKB1 tumor suppressor gene and the oncogene ΔN-LKB1 are expressed from the same locus and this may account for some of the paradoxical effects of LKB1 during tumorigenesis.

Interaction between nitric oxide signaling and gap junctions: effects on vascular function.

Nitric oxide signaling, through eNOS (or possibly nNOS), and gap junction communication are essential for normal vascular function. While each component controls specific aspects of vascular function, there is substantial evidence for cross-talk between nitric oxide signaling and the gap junction proteins (connexins), and more recently, protein-protein association between eNOS and connexins. This review will examine the evidence for interaction between these pathways in normal and diseased arteries, highlight the questions that remain about the mechanisms of their interaction, and explore the possible interaction between nitric oxide signaling and the newly discovered pannexin channels. This article is part of a Special Issue entitled: The Communicating junctions, composition, structure and characteristics.

Molecular chaperone complexes with antagonizing activities regulate stability and activity of the tumor suppressor LKB1.

LKB1 is a tumor suppressor that is constitutionally mutated in a cancer-prone condition, called Peutz-Jeghers syndrome, as well as somatically inactivated in a sizeable fraction of lung and cervical neoplasms. The LKB1 gene encodes a serine/threonine kinase that associates with the pseudokinase STRAD (STE-20-related pseudokinase) and the scaffolding protein MO25, the formation of this heterotrimeric complex promotes allosteric activation of LKB1. We have previously reported that the molecular chaperone heat shock protein 90 (Hsp90) binds to and stabilizes LKB1. Combining pharmacological studies and RNA interference approaches, we now provide evidence that the co-chaperone Cdc37 participates to the regulation of LKB1 stability. It is known that the Hsp90-Cdc37 complex recognizes a surface within the N-terminal catalytic lobe of client protein kinases. In agreement with this finding, we found that the chaperones Hsp90 and Cdc37 interact with an LKB1 isoform that differs in the C-terminal region, but not with a novel LKB1 variant that lacks a portion of the kinase N-terminal lobe domain. Reconstitution of the two complexes LKB1-STRAD and LKB1-Hsp90-Cdc37 with recombinant proteins revealed that the former is catalytically active whereas the latter is inactive. Furthermore, consistent with a documented repressor function of Hsp90, LKB1 kinase activity was transiently stimulated upon dissociation of Hsp90. Finally, disruption of the LKB1-Hsp90 complex favors the recruitment of both Hsp/Hsc70 and the U-box dependent E3 ubiquitin ligase CHIP (carboxyl terminus of Hsc70-interacting protein) that triggers LKB1 degradation. Taken together, our results establish that the Hsp90-Cdc37 complex controls both the stability and activity of the LKB1 kinase. This study further shows that two chaperone complexes with antagonizing activities, Hsp90-Cdc37 and Hsp/Hsc70-CHIP, finely control the cellular level of LKB1 protein.

The RET receptor: function in development and dysfunction in congenital malformation.

Germline mutations in the RET proto-oncogene are responsible for two unrelated neural crest disorders: Hirschsprung disease, a congenital absence of the enteric nervous system in the hindgut, and multiple endocrine neoplasia type 2, a dominantly inherited cancer syndrome. Moreover, somatic rearrangements of RET are causally involved in the genesis of papillary thyroid carcinoma. The receptor tyrosine kinase encoded by the RET gene acts as the subunit of a multimolecular complex that binds four distinct ligands and activates a signalling network crucial for neural and kidney development. Over the past few years, a clearer picture of the mode of RET activation and of its multifaceted role during development has started to emerge. These findings, which provide new clues to the molecular mechanisms underlying RET signalling dysfunction in Hirschsprung disease, are summarized in this review.

Docking protein FRS2 links the protein tyrosine kinase RET and its oncogenic forms with the mitogen-activated protein kinase signaling cascade.

The receptor tyrosine kinase RET functions as the signal transducing receptor for the GDNF (for "glial cell-derived neurotrophic factors") family of ligands. Mutations in the RET gene were implicated in Hirschsprung disease (HSCR), multiple endocrine neoplasia type 2 (MEN 2), and thyroid carcinomas. In this report we demonstrate that the docking protein FRS2 is tyrosine phosphorylated by ligand-stimulated and by constitutively activated oncogenic forms of RET. Complex formation between RET and FRS2 is mediated by binding of the phosphotyrosine-binding domain of FRS2 to pY1062, a residue in RET that also functions as a binding site for Shc. However, overexpression of FRS2 but not Shc potentiates mitogen-activated protein (MAP) kinase activation by RET oncoproteins. We demonstrate that oncogenic RET-PTC proteins are associated with FRS2 constitutively, leading to tyrosine phosphorylation of FRS2, MAP kinase stimulation, and cell proliferation. However, loss-of-function HSCR-associated RET mutants exhibit impaired FRS2 binding and reduced MAP kinase activation. These experiments demonstrate that FRS2 couples both ligand-regulated and oncogenic forms of RET, with the MAP kinase signaling cascade as part of the response of RET under normal biological conditions and pathological conditions, such as MEN 2 and papillary thyroid carcinomas.

The insulin receptor substrate (IRS)-1 recruits phosphatidylinositol 3-kinase to Ret: evidence for a competition between Shc and IRS-1 for the binding to Ret.

Tyrosine 1062 of Ret, which represents an intracytoplasmic docking site for multiple signaling molecules, is essential for Ret-mediated activation of phosphatidylinositol 3-Kinase (PI3-K). PI3-K, in turn, has been implicated in inducing cell survival and neoplastic transformation mediated by Ret. We have examined the mechanisms by which Ret stimulates PI3-K. Here we show that the Insulin Receptor Substrate-1 (IRS-1) is tyrosine phosphorylated and associated with the p85 regulatory subunit of PI3-K in response to Ret activation. IRS-1 coimmunoprecipitates with Ret and co-expression of IRS-1 results in the potentiation of Ret-mediated activation of Akt(PKB), a bona fide effector of PI3-K. The association with the PTB domain of IRS-1 depends on the phosphorylation of tyrosine 1062 of Ret. The deletion of asparagine 1059 (delN1059) and the substitution of leucine 1061 (L1061P), two Ret mutations identified in families affected by congenital megacolon (Hirschsprung's disease), impair the binding of IRS-1 to Ret as well as Ret-mediated Akt(PKB) stimulation. Finally, we show that Shc, which was previously identified as another ligand of Y1062 of Ret, competes with IRS-1 for the binding to Ret pY1062. All together, these findings suggest that IRS-1 is a component of the signaling pathway which leads to Ret-mediated PI3-K activation, a pathway which can be targeted by Hirschsprung-associated Ret mutations. The alternative binding of Shc and IRS-1 to Ret pY1062 can be a system to modulate the activation of different intracellular signaling pathways and to elicit different biological responses following Ret activation.

The RET proto-oncogene induces apoptosis: a novel mechanism for Hirschsprung disease.

The RET (rearranged during transfection) proto-oncogene encodes a tyrosine kinase receptor involved in both multiple endocrine neoplasia type 2 (MEN 2), an inherited cancer syndrome, and Hirschsprung disease (HSCR), a developmental defect of enteric neurons. We report here that the expression of RET receptor induces apoptosis. This pro-apoptotic effect of RET is inhibited in the presence of its ligand glial cell line-derived neurotrophic factor (GDNF). Furthermore, we present evidence that RET induces apoptosis via its own cleavage by caspases, a phenomenon allowing the liberation/exposure of a pro-apoptotic domain of RET. In addition, we report that Hirschsprung-associated RET mutations impair GDNF control of RET pro-apoptotic activity. These results indicate that HSCR may result from apoptosis of RET-expressing enteric neuroblasts.

Tyrosine 1062 of RET-MEN2A mediates activation of Akt (protein kinase B) and mitogen-activated protein kinase pathways leading to PC12 cell survival.

The RET tyrosine kinase is a functional receptor for neurotrophic ligands of the glial cell line-derived neurotrophic factor (GDNF) family. Loss of function of RET is associated with congenital megacolon or Hirschsprung's disease, whereas germ-line point mutations causing RET activation are responsible for multiple endocrine neoplasia type 2 (MEN2A, MEN2B, and familial medullary thyroid carcinoma) syndromes. Here we show that the expression of a constitutively active RET-MEN2A oncogene promotes survival of rat pheochromocytoma PC12 cells upon growth factor withdrawal. Moreover, we show that the RET-MEN2A-mediated survival depends on signals transduced by the phosphoinositide 3-kinase (PI3K) and mitogen-activated protein kinase (MAPK) cascades. Thus, in PC12 cells, RET-MEN2A associates with the PI3K regulatory subunit p85 and promotes activation of Akt (also referred to as protein kinase B) in a PI3K-dependent fashion; in addition, RET-MEN2A promotes MAPK activation. PI3K recruitment and Akt activation as well as MAPK activation depend on RET-MEN2A tyrosine residue 1062. As a result, tyrosine 1062 of RET-MEN2A is essential for RET-MEN2A-mediated survival of PC12 cells cultured in growth factor-depleted media.

Transforming ability of MEN2A-RET requires activation of the phosphatidylinositol 3-kinase/AKT signaling pathway.

The RET gene codes for a receptor tyrosine kinase that plays a crucial role during the development of both the enteric nervous system and the kidney. Germ line missense mutations at one of six codons specifying extracytoplasmic cysteines are responsible for two related cancer disorders as follows: multiple endocrine neoplasia type2A (MEN2A) and familial medullary thyroid carcinoma (FMTC). MEN2A and FMTC mutations result in a constitutive catalytic activity and as a consequence convert RET into a dominantly acting transforming gene. Although it has been shown that RET-MEN2 mutants activate several transduction pathways, their respective contribution to the neoplastic phenotype remains poorly understood. Over the past few years, it has become increasingly clear that the transforming ability of several viral and cellular oncoproteins depends on their capacity to activate phosphatidylinositol 3-kinase (PI3K). We now report that RET carrying a representative MEN2A mutation at Cys-634 (termed RET-MEN2A) activates PI3K and its downstream effector, the serine/threonine kinase AKT/protein kinase B. Previous studies have demonstrated that mutation of Tyr-1062, which is the intracellular docking site for Shc and Enigma on RET, abolishes the RET-MEN2A transforming activity. We provide evidence that mutation of Tyr-1062 abrogates the binding of the p85 regulatory subunit of PI3K to RET-MEN2A and the subsequent stimulation of the PI3K/AKT pathway. Furthermore, infection of rat fibroblasts with a retrovirus expressing a dominant-interfering form of PI3K suppresses RET-MEN2A-dependent transformation, whereas overexpression of AKT enhances the RET-MEN2A oncogenic potential. In summary, these data are consistent with the notion that RET-mediated cell-transforming effect is critically dependent on the activation of the PI3K/AKT pathway.

GDNF triggers a novel ret-independent Src kinase family-coupled signaling via a GPI-linked GDNF receptor alpha1.

Glial cell line-derived neurotrophic factor (GDNF) has potentially great clinical importance in the treatment of Parkinson's disease and several other neurodegenerative diseases, however its intracellular signaling mechanisms are poorly understood. Here we show that upon GDNF binding glycosyl-phosphatidylinositol (GPI)-linked GDNF receptor alpha1 (GFRalpha1) activates cytoplasmic Src family tyrosine kinase(s) in Ret tyrosine kinase-deficient cultured mouse dorsal root ganglion neurons and in two Ret-negative cell lines. GFRalpha1-mediated Src-type kinase activation subsequently triggers phosphorylation of mitogen-activated protein kinase, cAMP response element binding protein and phospholipase Cgamma. We therefore conclude that GDNF can activate intracellular signaling pathways Ret-independently via GPI-linked GFRalpha1.

Two distinct mutations of the RET receptor causing Hirschsprung's disease impair the binding of signalling effectors to a multifunctional docking site.

The RET gene codes for a transmembrane tyrosine kinase which is a subunit of a multimeric complex that acts as a receptor for four structurally related molecules: the glial cell line-derived neurotrophic factor (GDNF), neurturin, artemin and persephin. Germline mutations of RET cause a dominantly inherited dysgenesis of the enteric nervous system known as Hirschsprung's disease (HSCR; aganglionosis megacolon). The majority of HSCR mutations results either in a reduction of dosage of the RET protein or in the loss of RET function. Two novel distinct mutations of RET that led either to the deletion of codon 1059 (denoted Delta1059) or to the substitution of a Pro for Leu1061 have been identified in five HSCR families. In one large pedigree, two children born from asymptomatic consanguineous parents presented a severe form of HSCR and were found to carry the mutation at codon 1061 in the homozygous state. A tyrosine residue at position 1062 is an intracytoplasmic docking site that enables RET to recruit several signalling molecules, including the Shc adaptor protein. We now report that both HSCR mutations impair the fixation of Shc to RET and consequently prevent its phosphorylation. In addition, quantitative analysis in PC12 cells reveals that mutation Delta1059 inactivates the ability of RET to transduce a downstream signal whereas mutation L1061P only partially inhibits the signalling of RET. Finally, we provide evidence that these effects are partly mediated via the disruption of the RET/Shc interaction. Collectively, these results demonstrate that HSCR can be ascribed to mutations of RET which interfere with the binding of transduction effectors, such as Shc, and further provide a biochemical explanation for the phenotype of patients carrying a homozygous mutation at codon 1061. Finally, these data indicate that Y1062 is a multifunctional docking site that confers to RET the capacity to engage downstream signalling pathways which exert a crucial role during enteric neurogenesis.

Co-segregation of MEN2 and Hirschsprung's disease: the same mutation of RET with both gain and loss-of-function?

Multiple endocrine neoplasia type 2 (MEN2) and Hirschsprung's disease (HSCR) are two dominantly inherited neurocristopathies ascribed to mutations in the RET gene [Chakravarti, 1996; Pasini et al., 1996; Eng and Mulligan, 1997]. MEN2 is a cancer syndrome comprising three related clinical subtypes: (1) MEN type 2A (MEN2A; MIM# 171400) characterized by the association of medullary thyroid carcinoma (MTC), pheochromocytoma (Pheo), and hyperparathyroidism; (2) MEN type 2B (MEN2B; MIM# 162300), which includes MTC, Pheo, mucosal neuromas, ganglioneuromatosis of the digestive tract, and skeletal abnormalities; and (3) familial MTC (FMTC; MIM# 155240), defined by the sole occurrence of MTC. HSCR (MIM# 142623) is a congenital malformation caused by the absence of enteric plexuses in the hindgut, leading to bowel obstruction in neonates. The RET gene (MIM# 164761) codes for a transmembrane tyrosine kinase, a component of a multimeric complex that also comprises one of four members of a novel family of glycosylphosphatidylinositol (GPI)-anchored receptor, GFRalpha((1-4) (e.g., GFRA1, MIM# 601496; references are detailed in Baloh et al. [1998]. Four structurally related soluble factors-glial cell line-derived neurotrophic factor (GDNF), neurturin, persephin, and artemin-are the ligands of these multimolecular receptors in which the nature of the GFRalpha determines the ligand specificity of the complex [see Baloh et al., 1998, for references]. It is well documented that RET/GFRalpha-1/GDNF delivers a signal critical for the survival of the early neural crest-derived precursors that colonize the intestine below the rostral foregut and give rise to the enteric nervous plexuses [Gershon, 1997; Cacalano et al., 1998; Enomoto et al., 1998].

Regulation of dauer larva development in Caenorhabditis elegans by daf-18, a homologue of the tumour suppressor PTEN.

The tumour suppressor gene PTEN (also called MMAC1 or TEP1) is somatically mutated in a variety of cancer types [1] [2] [3] [4]. In addition, germline mutation of PTEN is responsible for two dominantly inherited, related cancer syndromes called Cowden disease and Bannayan-Ruvalcaba-Riley syndrome [4]. PTEN encodes a dual-specificity phosphatase that inhibits cell spreading and migration partly by inhibiting integrin-mediated signalling [5] [6] [7]. Furthermore, PTEN regulates the levels of phosphatidylinositol 3,4,5-trisphosphate (PIP3) by specifically dephosphorylating position 3 on the inositol ring [8]. We report here that the dauer formation gene daf-18 is the Caenorhabditis elegans homologue of PTEN. DAF-18 is a component of the insulin-like signalling pathway controlling entry into diapause and adult longevity that is regulated by the DAF-2 receptor tyrosine kinase and the AGE-1 PI 3-kinase [9]. Others have shown that mutation of daf-18 suppresses the life extension and constitutive dauer formation associated with daf-2 or age-1 mutants. Similarly, we show that inactivation of daf-18 by RNA-mediated interference mimics this suppression, and that a wild-type daf-18 transgene rescues the dauer defect. These results indicate that PTEN/daf-18 antagonizes the DAF-2-AGE-1 pathway, perhaps by catalyzing dephosphorylation of the PIP3 generated by AGE-1. These data further support the notion that mutations of PTEN contribute to the development of human neoplasia through an aberrant activation of the PI 3-kinase signalling cascade.

[The RET gene in thyroid pathology]. / Le gène RET en pathologie thyroïdienne.

The RET proto-oncogene encodes a receptor tyrosine kinase which plays a crucial role during the embryonic development of the enteric nervous system and of the kidney. Cytogenetic analyses of papillary thyroid carcinoma (PTC), a neoplasm which originates from thyrocytes, have revealed that somatic rearrangements of the RET gene are involved in the etiology of a significant proportion of this tumour. Medullary thyroid carcinoma (MTC) which arises from neural-crest derived C-cells is the cardinal disease feature of multiple endocrine neoplasia type 2 (MEN 2), a dominantly inherited cancer syndrome. Recent studies have provided evidence that germline mutations of the RET gene are the underlying genetic events responsible for MEN 2. This review focuses on the role of RET mutations in the pathogenesis of PTC and MTC and summarizes our present knowledge on the consequences of these alterations on the RET tyrosine kinase function. We further describe a transgenic mouse model for hereditary MTC. Mice carrying a MEN 2A allele of RET under the control of the CGRP/calcitonin promoter develop bilateral and multifocal MTC, morphologically and biologically similar to human MTC.

Mutation of the RET ligand, neurturin, supports multigenic inheritance in Hirschsprung disease.

Hirschsprung disease (HSCR) is a frequent neurocristopathy characterized by the absence of submucosal and myenteric plexuses in a variable length of the gastrointestinal tract. Pedigrees and segregation analyses suggested the involvement of one or several dominant genes with low penetrance in HSCR. Considering that RET and glial cell line-derived neurotrophic factor (GDNF) mutations have been reported in the disease, we regarded the other RET ligand, neurturin (NTN), as an attractive candidate gene, especially as it shares large homologies with GDNF. Here, we report on the finding of a heterozygous missense NTN mutation in a large non-consanguineous family including four children affected with a severe aganglionosis phenotype extending up to the small intestine. Interestingly, it appears that the NTN mutation reported here is not sufficient to cause HSCR, and this multiplex family also segregates a RET mutation. This cascade of independent and additive genetic events fits well with the multigenic pattern of inheritance expected in HSCR, and further support the role of RET ligands in development of the enteric nervous system.

Interaction of BTG1 and p53-regulated BTG2 gene products with mCaf1, the murine homolog of a component of the yeast CCR4 transcriptional regulatory complex.

Both BTG1 and BTG2 are involved in cell-growth control. BTG2 expression is regulated by p53, and its inactivation in embryonic stem cells leads to the disruption of DNA damage-induced G2/M cell-cycle arrest. In order to investigate the mechanism underlying Btg-mediated functions, we looked for possible functional partners of Btg1 and Btg2. Using yeast two-hybrid screening, protein-binding assays, and transient transfection assays in HeLa cells, we demonstrated the physical in vitro and in vivo interaction of both Btg1 and Btg2 with the mouse protein mCaf1 (i.e. mouse CCR4-associated factor 1). mCaf1 was identified through its interaction with the CCR4 protein, a component of a general transcription multisubunit complex, which, in yeast, regulates the expression of different genes involved in cell-cycle regulation and progression. These data suggest that Btg proteins, through their association with mCaf1, may participate, either directly or indirectly, in the transcriptional regulation of the genes involved in the control of the cell cycle. Finally, we found that box B, one of two conserved domains which define the Btg family, plays a functional role, namely that it is essential to the Btg-mCaf1 interaction.

Various mechanisms cause RET-mediated signaling defects in Hirschsprung's disease.

Hirschsprung's disease (HSCR) is a common congenital malformation characterized by the absence of intramural ganglion cells of the hindgut. Recently, mutations of the RET tyrosine kinase receptor have been identified in 50 and 15-20% of familial and sporadic HSCR, respectively. These mutations include deletion, insertion, frameshift, nonsense, and missense mutations dispersed throughout the RET coding sequence. To investigate their effects on RET function, seven HSCR missense mutations were introduced into either a 1114-amino acid wild-type RET isoform (RET51) or a constitutively activated form of RET51 (RET-MEN 2A). Here, we report that one mutation affecting the extracytoplasmic cadherin domain (R231H) and two mutations located in the tyrosine kinase domain (K907E, E921K) impaired the biological activity of RET-MEN 2A when tested in Rat1 fibroblasts and pheochromocytoma PC12 cells. However, the mechanisms resulting in RET inactivation differed since the receptor bearing R231H extracellular mutation resulted in an absent RET protein at the cell surface while the E921K mutation located within the catalytic domain abolished its enzymatic activity. In contrast, three mutations mapping into the intracytoplasmic domain neither modified the transforming capacity of RET-MEN 2A nor stimulated the catalytic activity of RET in our ligand-independent system (S767R, P1039L, M1064T). Finally, the C609W HSCR mutation exerts a dual effect on RET since it leads to a decrease of the receptor at the cell surface and converted RET51 into a constitutively activated kinase due to the formation of disulfide-linked homodimers. Taken together, our data show that allelic heterogeneity at the RET locus in HSCR is associated with various molecular mechanisms responsible for RET dysfunction.

Dual effect on the RET receptor of MEN 2 mutations affecting specific extracytoplasmic cysteines.

The RET gene encodes a receptor tyrosine kinase whose function is essential during the development of kidney and the intestinal nervous system. Germline mutations affecting one of five cysteines (Cys609, 611, 618, 620 and 634) located in the juxtamembrane domain of the RET receptor are responsible for the vast majority of two cancer-prone disorders, multiple endocrine neoplasia type 2A (MEN 2A) and familial medullary thyroid carcinoma (FMTC). These mutations lead to the replacement of a cysteine by an alternate amino acid. Mutations of the RET gene are also the underlying genetic cause of Hirschsprung disease (HSCR), a congenital aganglionosis of the hindgut. In a fraction of kindreds, MEN 2A cosegregate with HSCR and affected individuals carry a single mutation at codons 609, 618 or 620. To examine the consequences of cysteine substitution on RET function, we have introduced a Cys to Arg mutation into the wild-type RET at either codons 609, 618, 620, 630 or 634. We now report that each mutation induces a constitutive catalytic activity due to the aberrant disulfide homodimerization of RET. However, mutations 630 and 634 activate RET more strongly than mutations 609, 618 or 620 as demonstrated by quantitative assays in rodent fibroblasts and pheochromocytoma PC12 cells. Biochemical analysis revealed that mutations 618 and 620, and to a lesser extent mutation 609, result in a marked reduction of the level of RET at the cell surface and as a consequence decrease the amount of RET covalent dimer. These findings provide a molecular basis explaining the range of phenotype engendered by alterations of RET cysteines and suggest a novel mechanism whereby mutations of cysteines 609, 618 and 620 exert both activating and inactivating effects.

Oncogenic activation of RET by two distinct FMTC mutations affecting the tyrosine kinase domain.

Multiple endocrine neoplasia type 2A (MEN 2A) and familial medullary thyroid carcinoma (FMTC) are two dominantly inherited disorders caused by germline mutations of the RET proto-oncogene. The RET gene codes for a receptor tyrosine kinase. The majority of MEN2A and FMTC mutations are clustered in the extra-cellular cysteine-rich domain and result in constitutive activation of the tyrosine kinase through the formation of disulfide-bonded RET homodimers. Recently, two novel point mutations have been identified in the germline of five distinct FMTC families. Both mutations occur within the catalytic domain of the RET kinase and lead to the substitution of either glutamic acid 768 or valine 804 by an aspartic acid and a leucine respectively. We have introduced each FMTC mutation in two RET isoforms: RET51 the long isoform (1114 aa) and RET9 the short isoform (1072 aa) which differ in the C-terminal region of the protein. The RET51 isoform carrying either E768D or V804L mutation was autophosphorylated, displayed a transforming activity upon expression in Rat1 fibroblasts and induced neuronal differentiation of PC12 cells. However, the transforming capacity of these RET51-FMTC mutants was found to be severalfold less potent compared to the same isoform carrying either the MEN2A mutation (C634R) or the MEN2B mutation (M918T). In contrast, RET9 containing mutations E768D or V804L was not autophosphorylated, exhibited a poor oncogenic potential in fibroblasts and did not promote neuritic outgrowth upon expression in PC12 cells. Overall, these findings demonstrate that mutations E768D and V804L are gain-of-function mutations that confer to the long RET isoform the capacity to exert a biological effect, although these mutations are more weakly activating than the MEN2A and MEN2B mutations. These results may provide a biochemical basis as to why the phenotypic consequences of these mutations are restricted to thyroid C-cells.