Ultraviolet Light Induces Redox Reaction–mediated Dimerization and Superactivation of Oncogenic Ret Tyrosine Kinases

Abstract

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 UVmediated 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.

The mouse soluble GFRα4 receptor activates RET independently of its ligand persephin

“…In order to assay if the soluble GFRa4 also has the capacity to activate RET, we added the sGFRa4-concentrate to naive Neuro 2a cells and immunoprecipitated RET from the lysates of these samples. The myelin basic protein has previously been reported to function as an exogenous substrate in in vitro kinase assays with kinase activated RET (Asai et al, 1995;Kato et al, 2000). The result of our in vitro kinase assay (Figure 2d) is in line with our co-immunoprecipitation results, and shows that soluble GFRa4 activates RET both in the absence and presence of PSPN.…”

RET tyrosine kinase enhances hair growth in association with promotion of melanogenesis

“…After the membranes had been reacted with the anti-Ret (reactive to both c-Ret and RFP-RET; Immuno-Biological laboratories, Gunma, Japan) antibody, the reaction was examined using Western Blot Chemiluminescence Reagent (DuPont NEN, Boston, MA, USA). Immunoprecipitation and immunohistochemistry with antiRet antibody (Kato et al, 1999(Kato et al, , 2000 or anti-S-100 protein (Iwamoto et al, 1991) were performed as described previously.…”

FLT3-driven redox-modulation of Ezrin regulates leukaemic cell migration

“…Pourmahram et al (2008) provides an example of such an opportunity, as the authors detected a shift in sub-cellular localisation in PKCα in intrapulmonary arteries upon exposure to low concentrations of H 2 O 2 . Similarly, Kato (2000) and colleagues" discovery of redox-sensitive cysteine residues in c-Ret which are conserved across a range of kinases should prompt investigation of the redox-sensitivity of those kinases.…”

“…Close investigation of the redox-regulation of proto-oncogenic RTK c-Ret by Kato et al(2000) has revealed that oxidative activation of the kinase occurs via disulfide bond formation between cysteine residues on two different c-Ret molecules, resulting in dimerisation. The cysteine residue in question, Cys376, is necessary for the basal activity of the kinase, as well as its up-regulation in response to UV irradiation.…”