Glial cell line-derived neurotrophic factor-stimulated phosphatidylinositol 3-kinase and Akt activities exert opposing effects on the ERK pathway: importance for the rescue of neuroectodermic cells

Glial cell line-derived neurotrophic factor (GDNF) plays a crucial role in rescuing neural crest cells from apoptosis during their migration in the foregut. This survival factor binds to the heterodimer GDNF family receptor alpha1/Ret, inducing the Ret tyrosine kinase activity. ret loss-of-function mutations result in Hirschsprung's disease, a frequent developmental defect of the enteric nervous system. Although critical to enteric nervous system development, the intracellular signaling cascades activated by GDNF and their importance in neuroectodermic cell survival still remain elusive. Using the neuroectodermic SK-N-MC cell line, we found that the Ret tyrosine kinase activity is essential for GDNF to induce phosphatidylinositol 3-kinase (PI3K)/Akt and ERK pathways as well as cell rescue. We demonstrate that activation of PI3K is mandatory for GDNF-induced cell survival. In addition, evidence is provided for a critical up-regulation of the ERK pathway by PI3K at the level of Raf-1. Conversely, Akt inhibits the ERK pathway. Thus, both PI3K and Akt act in concert to finely regulate the level of ERK. We found that Akt activation is indispensable for counteracting the apoptotic signal on mitochondria, whereas ERK is partially involved in precluding procaspase-3 cleavage. Altogether, these findings underscore the importance of the Ret/PI3K/Akt pathway in GDNF-induced neuroectodermic cell survival.     

Biological effects of the dual phenotypic Janus mutation of ret cosegregating with both multiple endocrine neoplasia type 2 and Hirschsprung's disease

Gain-of-function mutations of ret receptor tyrosine kinase, the signaling receptor for glial cell line-derived neurotrophic factor, cause sporadic thyroid and adrenal malignancies as well as endocrine cancer syndromes, such as multiple endocrine neoplasia types 2A and 2B (MEN 2A and MEN 2B) and familial medullary thyroid carcinoma. Loss-of-function mutations of ret cause Hirschsprung's disease (HSCR) or colonic aganglionosis. In 20-30% of families with a mutation at residues 609, 611, 618, or 620 of RET, MEN 2A and familial medullary thyroid carcinoma cosegregate with HSCR. These mutations constitutively activate RET due to aberrant disulfide homodimerization and diminish the level of RET at the plasma membrane. It is not known how these mutations simultaneously lead to both gain- and loss-of-function RET-associated diseases. We provide an explanation for the dual phenotypic Janus mutation at Cys620 of RET. In Madin-Darby canine kidney (MDCK) cells, the Janus mutation impairs the glial cell line-derived neurotrophic factor-induced effects of RET on cell migration, differentiation, and survival but simultaneously promotes rapid cell proliferation.     

Mechanism of Ret activation by a mutation at aspartic acid 631 identified in sporadic pheochromocytoma

Mutations at aspartic acid 631 in Ret were reported in sporadic pheochromocytoma and medullary thyroid carcinoma. We replaced this aspartic acid with four other amino acids including tyrosine, glycine, asparagine, and alanine and investigated the transforming activity of these mutant cDNAs. Among them, RET cDNA with a mutation of aspartic acid to tyrosine (D631Y) that was reported in sporadic pheochromocytoma showed high transforming activity. The D631Y mutation activated Ret by inducing its disulfide-linked dimerization in the transfectant as observed for multiple endocrine neoplasia (MEN) 2A mutations at cysteine 609, 611, 618, 620, 630, or 634. Further mutation analysis suggested that cysteine 630 or 634 could be involved in the disulfide-linked Ret dimerization induced by the D631Y mutation.     

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.     

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.     

Mice expressing a dominant-negative Ret mutation phenocopy human Hirschsprung disease and delineate a direct role of Ret in spermatogenesis

The Ret receptor tyrosine kinase mediates physiological signals of glial cell line-derived neurotrophic factor (GDNF) family ligands (GFLs) and is essential for postnatal survival in mice. It is implicated in a number of human diseases and developmental abnormalities. Here, we describe our analyses of mice expressing a Ret mutant (RetDN) with diminished kinase activity that inhibits wild-type Ret activity, including its activation of AKT. All RetDN/+ mice died by 1 month of age and had distal intestinal aganglionosis reminiscent of Hirschsprung disease (HSCR) in humans. The RetDN/+ proximal small intestine also had severe hypoganglionosis and reduction in nerve fiber density, suggesting a potential mechanism for the continued gastric dysmotility in postsurgical HSCR patients. Unlike Ret-null mice, which have abnormalities in the parasympathetic and sympathetic nervous systems, the RetDN/+ mice only had defects in the parasympathetic nervous system. A small proportion of RetDN/+ mice had renal agenesis, and the remainder had hypoplastic kidneys and developed tubulocystic abnormalities postnatally. Postnatal analyses of the testes revealed a decreased number of germ cells, degenerating seminiferous tubules, maturation arrest and apoptosis, indicating a crucial role for Ret in early spermatogenesis.     

The many faces of RET dysfunction in kidney

Signaling pathways that are activated upon interaction of glial cell-line derived neurotrophic factor (Gdnf), its coreceptor Gfra1, and receptor tyrosine kinase Ret are critical for kidney development and ureter maturation. Outside the kidney, this pathway is implicated in a number of congenital diseases including Hirschsprung disease (intestinal aganglionosis, HSCR) and hereditary cancer syndromes (MEN 2). Total lack of Gdnf, Gfra1 or Ret in mice results in perinatal lethality due to bilateral renal agenesis or aplasia. In humans, RET mutations have been identified in a spectrum of congenital malformations involving the RET axis including isolated HSCR, isolated congenital anomalies of kidney or urinary tract (CAKUT), or CAKUT and HSCR together. The molecular basis for these pleiotropic effects of RET has just begun to be unraveled. In an effort to delineate the pathogenetic mechanisms that underlie these congenital malformations, we and others have characterized Ret''s role in early kidney and urinary system development. Here we present a brief overview of the “many faces” of Ret dysfunction in kidney with particular emphasis on Ret''s signaling specificity and intergenic interactions that confer normal urinary system development.Key words: RET, GDNF, kidney, RTK, CAKUT, branching morphogenesis, ureter     

Human glial cell line-derived neurotrophic factor receptor alpha 4 is the receptor for persephin and is predominantly expressed in normal and malignant thyroid medullary cells

Glial cell line-derived neurotrophic factor (GDNF) family ligands signal through receptor complex consisting of a glycosylphosphatidylinositol-linked GDNF family receptor (GFR) alpha subunit and the transmembrane receptor tyrosine kinase RET. The inherited cancer syndrome multiple endocrine neoplasia type 2 (MEN2), associated with different mutations in RET, is characterized by medullary thyroid carcinoma. GDNF signals via GFRalpha1, neurturin via GFRalpha2, artemin via GFRalpha3, whereas the mammalian GFRalpha receptor for persephin (PSPN) is unknown. Here we characterize the human GFRalpha4 as the ligand-binding subunit required together with RET for PSPN signaling. Human and mouse GFRalpha4 lack the first Cys-rich domain characteristic of other GFRalpha receptors. Unlabeled PSPN displaces (125)I-PSPN from GFRA4-transfected cells, which express endogenous Ret. PSPN can be specifically cross-linked to mammalian GFRalpha4 and Ret, and is able to promote autophosphorylation of Ret in GFRA4-transfected cells. PSPN, but not other GDNF family ligands, promotes the survival of cultured sympathetic neurons microinjected with GFRA4. We identified different splice forms of human GFRA4 mRNA encoding for two glycosylphosphatidylinositol-linked and one putative soluble isoform that were predominantly expressed in the thyroid gland. Overlapping expression of RET and GFRA4 but not other GFRA mRNAs in normal and malignant thyroid medullary cells suggests that GFRalpha4 may restrict the MEN2 syndrome to these cells.     

The tyrosine phosphatase Shp2 acts downstream of GDNF/Ret in branching morphogenesis of the developing mouse kidney

The tyrosine phosphatase Shp2 acts downstream of various growth factors, hormones or cytokine receptors. Mutations of the Shp2 gene are associated with several human diseases. Here we have ablated Shp2 in the developing kidneys of mice, using the ureteric bud epithelium-specific Hoxb7/Cre. Mutant mice produced a phenotype that is similar to mutations of the genes of the GDNF/Ret receptor system, that is: strongly reduced ureteric bud branching and downregulation of the Ret target genes Etv4 and Etv5. Shp2 mutant embryonic kidneys also displayed reduced cell proliferation at the branch tips and branching defects, which could not be overcome by GDNF in organ culture. We also examined compound mutants of Shp2 and Sprouty1, which is an inhibitor of receptor tyrosine kinase signaling in the kidney. Sprouty1 single mutants produce supernumerary ureteric buds, which branch excessively. Sprouty1 mutants rescued branching deficits in Ret^−/− and GDNF^−/− kidneys. Sprouty1; Shp2 double mutants showed no rescue of kidney branching. Our data thus indicate an intricate interplay of Shp2 and Sprouty1 in signaling downstream of receptor tyrosine kinases during kidney development. Apparently, Shp2 mediates not only GDNF/Ret but also signaling by other receptor tyrosine kinases in branching morphogenesis of the embryonic kidney.     

Identification of the key amino acids of glial cell line-derived neurotrophic factor family receptor alpha1 involved in its biological function

Glial cell line-derived neurotrophic factor (GDNF) plays a critical role in neurodevelopment and survival of midbrain dopaminergic and spinal motor neurons in vitro and in vivo. The biological actions of GDNF are mediated by a two-receptor complex consisting of a glycosylphosphatidylinositol-linked cell surface molecule, the GDNF family receptor alpha1 (GFRalpha1), and receptor protein tyrosine kinase Ret. Although structural analysis of GDNF has been extensively examined, less is known about the structural basis of GFRalpha1 function. In this study, based on evolutionary trace method and relative solvent accessibility prediction of residues, a set of trace residues that are solvent-accessible was selected for site-directed mutagenesis. A series of GFRalpha1 mutations was made, and PC12 cell lines stably expressing different GFRalpha1 mutants were generated. According to the survival and differentiation responses of these stable PC12 cells upon GDNF stimulation and the GDNF-GFRalpha1-Ret interaction assay, residues 152NN153, Arg259, and 316SNS318 in the GFRalpha1 central region were found to be critical for GFRalpha1 binding to GDNF and eliciting downstream signal transduction. The single mutation R259A in the GFRalpha1 molecule simultaneously lost its binding ability to GDNF and Ret. However N152A/N153A or S316A/N317A/S318A mutation in the GFRalpha1 molecule still retained the ability to bind with Ret. These findings suggest that distinct structural elements in GFRalpha1 may be involved in binding to GDNF and Ret.     

Molecular genetics of Hirschsprung disease: a model of multigenic neurocristopathy

Hirschsprung's disease (HSCR, aganglionic megacolon) is a frequent congenital malformation regarded as a multigenic neurocristopathy. Three susceptibility genes have been recently identified in HSCR, namely the RET proto-oncogene, the endothelin B receptor (EDNRB) gene, and the endothelin 3 (EDN3) gene. RET gene mutations were found in significant proportions of familial (50%) and sporadic (15-20%) HSCR, while homozygosity for EDNRB or EDN3 mutations accounted for the rare HSCR-Waardenburg syndrome (WS) association. More recently, heterozygous EDNRB an EDN3 missense mutations have been reported in isolated HSCR patients. Some of these results were obtained after the identification of mouse genes whose natural or site-directed mutations resulted in megacolon and coat color spotting. There is also conclusive evidence for the involvement of other independent loci in HSCR. In particular, the recent identification of neurotrophic factors acting as RET ligands (GDNF and Neurturin) provide additional candidate genes for HSCR. The dissection of the genetic etiology of HSCR disease may then provide a unique opportunity to distinguish between a polygenic and a genetically heterogeneous disease, thereby helping to understand other complex disorders and congenital malformations hitherto considered as multifactorial in origin. Finally, the study of the molecular bases of HSCR is also a step towards the understanding of developmental genetics of the enteric nervous system giving support to the role of the tyrosine kinase and endothelin-signaling pathways in the development of neural crest-derived enteric neurons in human.     

GDNF - a stranger in the TGF-beta superfamily?

Glial cell line-derived neurotrophic factor (GDNF) family, consisting of GDNF, neurturin, artemin and persephin are distant members of the transforming growth factor-beta (TGF-beta) superfamily. Unlike other members of the TGF-beta superfamily, which signal through the receptor serine-threonine kinases, GDNF family ligands activate intracellular signalling cascades via the receptor tyrosine kinase Ret. GDNF family ligands first bind to the glycosylphosphatidylinositol (GPI)-anchored GDNF family receptor alpha (GFRalpha) and then the GDNF family ligand-GFRalpha complex binds to and stimulates autophosphorylation of Ret. Alternatively, a preassociated complex between GFRalpha and Ret could form the binding site for the GDNF family ligand. GFRalpha1, GFRalpha2, GFRalpha3 and GFRalpha4 are the physiological coreceptors for GDNF, neurturin, artemin and persephin, respectively. Although all GDNF family ligands signal via activated Ret, GDNF can signal also via GFRalpha1 in the absence of Ret. GPI-anchored GFRalpha receptors are localized in plasma membrane to lipid rafts. GDNF binding to GFRalpha1 also recruits Ret to the lipid rafts and triggers association with Src, which is required for effective downstream signalling, leading to differentiation and neuronal survival. GDNF family ligands are potent survival factors for midbrain dopamine neurons, motoneurons, noradrenergic neurons, as well as for sympathetic, parasympathetic and sensory neurons. However, for most neuronal populations, except for motoneurons, TGF-beta is required as a cofactor for GDNF family ligand signalling. Because GDNF and neurturin can rescue dopamine neurons in the animal models of Parkinson disease, as well as motoneurons in vivo, hopes have been raised that GDNF family ligands may be new drugs for the treatment of neurodegenerative diseases. GDNF also has distinct functions outside the nervous system, promoting ureteric branching in kidney development and regulating spermatogenesis.     

The Shp-1 and Shp-2, tyrosine phosphatases, are recruited on cell membrane in two distinct molecular complexes including Ret oncogenes

The Shp-2 and Shp-1 non-transmembrane tyrosine phosphatases display different and even opposing effects on downstream signaling events initiated by Ret activation. By using rat pheochromocytoma-derived PC12 cells, here we studied the interactions of Shp-2 and Shp-1 with two activated mutants of Ret receptor, Ret(C634Y) and Ret(M918T). Each of these mutated receptors causes inheritance of distinct cancer syndromes, multiple endocrine neoplasia (MEN) type 2A and type 2B, respectively. We show that: (i) both Shp-1 and Shp-2 are associated to a multiprotein complex that includes Ret mutants; (ii) the Shp-1-Ret complexes are distinct from Shp-2-Ret complexes, and these complexes are differently distributed inside and outside lipid rafts; (iii) constitutively activated Ret proteins neither directly bind to nor are substrates of these phosphatases. Our results well support the evidence that Ret complexes within and outside rafts mediate distinct biological functions, and indicate that the presence of either Shps participates to determine such functions.     

The many faces of RET dysfunction in kidney

Signaling pathways that are activated upon interaction of glial cell-line derived neurotrophic factor (Gdnf), its coreceptor Gfrα1, and receptor tyrosine kinase Ret are critical for kidney development and ureter maturation. Outside the kidney, this pathway is implicated in a number of congenital diseases including Hirschsprung disease (intestinal aganglionosis, HSCR) and hereditary cancer syndromes (MEN 2). Total lack of Gdnf, Gfrα1 or Ret in mice results in perinatal lethality due to bilateral renal agenesis or aplasia. In humans, RET mutations have been identified in a spectrum of congenital malformations involving the RET axis including isolated HSCR, isolated congenital anomalies of kidney or urinary tract (CAKUT), or CAKUT and HSCR together. The molecular basis for these pleiotropic effects of RET has just begun to be unraveled. In an effort to delineate the pathogenetic mechanisms that underlie these congenital malformations, we and others have characterized Ret’s role in early kidney and urinary system development. Here we present a brief overview of the “many faces” of Ret dysfunction in kidney with particular emphasis on Ret’s signaling specificity and intergenic interactions that confer normal urinary system development.     

Molecular heterogeneity of RET loss of function in Hirschsprung's disease.

The RET proto-oncogene encodes a receptor with tyrosine kinase activity (RET) that is involved in several neoplastic and non-neoplastic diseases. Oncogenic activation of RET, achieved by different mechanisms, is detected in a sizeable fraction of human thyroid tumors, as well as in multiple endocrine neoplasia types 2A and 2B (MEN2A and MEN2B) and familial medullary thyroid carcinoma tumoral syndromes. Germline mutations of RET have also been associated with a non-neoplastic disease, the congenital colonic aganglionosis, i.e. Hirschsprung's disease (HSCR). To analyse the impact of HSCR mutations on RET function, we have introduced into wild-type RET and activated RET(MEN2A) and RET(MEN2B) alleles three missense mutations associated with HSCR. Here we show that the three mutations caused a loss of function of RET when assayed in two model cell systems, NIH 3T3 and PC12 cells. The effect of different HSCR mutations was due to different molecular mechanisms. The HSCR972 (Arg972-->Gly) mutation, mapping in the intracytoplasmic region of RET, impaired its tyrosine kinase activity, while two extracellular mutations, HSCR32 (Ser32-->Leu) and HSCR393 (Phe393-->Leu), inhibited the biological activity of RET by impairing the correct maturation of the RET protein and its transport to the cell surface.     

GDNF promotes tubulogenesis of GFRalpha1-expressing MDCK cells by Src-mediated phosphorylation of Met receptor tyrosine kinase

Glial cell line-derived neurotrophic factor (GDNF) and hepatocyte growth factor (HGF) are multifunctional signaling molecules in embryogenesis. HGF binds to and activates Met receptor tyrosine kinase. The signaling receptor complex for GDNF typically includes both GDNF family receptor alpha1 (GFRalpha1) and Ret receptor tyrosine kinase. GDNF can also signal independently of Ret via GFRalpha1, although the mechanism has remained unclear. We now show that GDNF partially restores ureteric branching morphogenesis in ret-deficient mice with severe renal hypodysplasia. The mechanism of Ret-independent effect of GDNF was therefore studied by the MDCK cell model. In MDCK cells expressing GFRalpha1 but no Ret, GDNF stimulates branching but not chemotactic migration, whereas both branching and chemotaxis are promoted by GDNF in the cells coexpressing Ret and GFRalpha1, mimicking HGF/Met responses in wild-type MDCK cells. Indeed, GDNF induces Met phosphorylation in several ret-deficient/GFRalpha1-positive and GFRalpha1/Ret-coexpressing cell lines. However, GDNF does not immunoprecipite Met, making a direct interaction between GDNF and Met highly improbable. Met activation is mediated by Src family kinases. The GDNF-induced branching of MDCK cells requires Src activation, whereas the HGF-induced branching does not. Our data show a mechanism for the GDNF-induced branching morphogenesis in non-Ret signaling.     

Signaling through Ras is essential for ret oncogene-induced cell differentiation in PC12 cells

Specific germline mutations of the receptor tyrosine kinase, Ret, predispose to multiple endocrine neoplasia types 2A and 2B and familial medullary thyroid carcinoma. The mechanisms by which different Ret isoforms (Ret-2A and Ret-2B) cause distinct neoplastic diseases remain largely unknown. On the other hand, forced expression of these mutated versions of Ret induces the rat pheochromocytoma cell line, PC12, to differentiate. Here we used an inducible vector encoding a dominant-negative Ras (Ras p21(N17)) to investigate the contributions of the Ras pathway to the phenotype induced in PC12 cells by the expression of either Ret-2A or Ret-2B mutants. We show that the Ret-induced molecular and morphological changes are both mediated by Ras-dependent pathways. However, even though inhibition of Ras activity was sufficient to revert Ret-induced differentiation, the kinetics of morphological reversion of the Ret-2B- was more rapid than the Ret-2A-transfected cells. Further, we show that in Ret-transfected cells the suc1-associated neurotrophic factor-induced tyrosine phosphorylation target, SNT, is chronically phosphorylated in tyrosine residues, and associates with the Sos substrate. These results indicate the activation of the Ras cascade as an essential pathway triggered by the chronic active Ret mutants in PC12 cells. Moreover, our data indicate SNT as a substrate for both Ret mutants, which might mediate the activation of this cascade.     

A rapid method for the purification of wild-type and V804M mutant ret catalytic domain: A tool to study thyroid cancer

RET (rearranged during transfection) is a transmembrane tyrosine kinase and acts as co-receptor of glial-derived neurotrophic factor (GDNF) family neurothrofic factors in complex with GFRalpha family proteins; RET is important for development of enteric nervous system and renal organogenesis during embryonal life. Alterations in Ret gene are related to several neoplasias: point mutations are identified in medullary thyroid carcinoma (MTC) and multiple endocrine neoplasias 2A and B (MEN2A and B), while translocations and chromosomal inversions cause papillary thyroid carcinoma (PTC). We expressed recombinant RET kinase domain (rRET) containing the active site, the ATP binding pocket, and the activation loop with regulatory activity, with the Baculovirus expression system. RET was purified by a two-step procedure consisting of an anion exchange chromatography followed by nickel affinity chromatography. Moreover a biochemical characterization of the recombinant product was performed in order to verify its activity (by ELISA) and physical state (dynamic light scattering). We used rRET to validate an ELISA-based kinase assay, by testing inhibitors reported in literature such as PP1 and PP2. This method represents an easy system to screen potential inhibitors found by computational methods. We also produced V804M mutants to identify inhibitors that can overcome resistance to PP1 and ZD6474. The catalytic domain of RET can be used also for X-ray diffraction to obtain information about the three-dimensional structure, necessary for a rational design of selective inhibitors: it represents an important tool to understand the molecular mechanisms causing thyroid cancer and to care it.     

Ultraviolet light induces redox reaction-mediated dimerization and superactivation of oncogenic Ret tyrosine kinases

The c-RET proto-oncogene encodes a receptor-type tyrosine kinase, and its mutations in the germ line are responsible for the inheritance of multiple endocrine neoplasia type 2A (MEN2A) and 2B (MEN2B). Ret kinases are constitutively activated as a result of MEN2A mutations (Ret-MEN2A) or MEN2B mutations (Ret-MEN2B). Here we demonstrate that UV light (UV) irradiation induces superactivation of the constitutively activated Ret-MEN2A and Ret-MEN2B as well as activation of c-Ret. Before UV irradiation, small percentages of c-Ret (3–4%) and Ret-MEN2B (1–2%) and large percentages of Ret-MEN2A (30–40%) were dimerized through disulfide bonds. These dimerized Ret proteins were preferentially autophosphorylated, suggesting a close relation between up-regulated kinase activity and disulfide bond–mediated dimerization of Ret proteins. We found that UV irradiation promotes the disulfide bond–mediated dimerization of the Ret proteins, in close association with activation and superactivation of Ret kinases. UV irradiation also induced dimerization and activation of the extracellular domain–deleted mutant Ret (Ret-PTC-1). Interestingly, the levels of basic kinase activity and dimerization of Ret-PTC-1–C376A, in which cysteine 376 in the tyrosine kinase domain of Ret-PTC-1 was replaced by alanine, were low and were not increased by UV irradiation. These results suggest that Ret-PTC-1 cysteine 376 is one of possibly multiple critical target amino acids of UV for Ret kinase activation. Overexpression of Cu/Zn superoxide dismutase in cells as a result of gene transfection prevented both the UV-mediated promotion of dimerization and the superactivation of Ret-MEN2A kinase. These results suggest that the UV-induced free radicals in cells attack intracellular domains of Ret to dimerize the kinase proteins for superactivation.     

Tyrosine 981, a novel ret autophosphorylation site, binds c-Src to mediate neuronal survival

The glial cell line-derived neurotrophic factor (GDNF) family ligands (GFLs) are neurotrophic factors that influence several aspects of the developing and injured nervous system. GFLs signal through a common receptor tyrosine kinase (Ret) and one of the four ligand-binding co-receptors (GFRalpha1 to 4). Ligand-induced translocation of Ret to lipid rafts, where it interacts with the nonreceptor tyrosine kinase Src, is a prerequisite for full biological activity of these neurotrophic factors. This interaction and subsequent activation of Src are required for GFL-mediated neuronal survival, neurite outgrowth, or cell proliferation. Here we show by multiple approaches that Ret tyrosine 981 constitutes the major binding site of the Src homology 2 domain of Src and therefore the primary residue responsible for Src activation upon Ret engagement. Other tyrosines such as 1015 and 1029 may contribute to the overall interaction between Ret and Src, as judged by overexpression experiments. By generating a phosphospecific antibody, we demonstrate that tyrosine 981 is a novel autophosphorylation site in Ret. Importantly, we also show that this tyrosine becomes phosphorylated in dissociated sympathetic neurons after ligand stimulation. Mutation of tyrosine 981 to phenylalanine reduces GDNF-mediated survival in a transfected cerebellar granule neuron paradigm.