Molecular Mechanism of Activation and Superactivation of Ret Tyrosine Kinases by Ultraviolet Light Irradiation

The catalytic activities of Ret tyrosine kinases as the products of oncogene RET with multiple endocrine neoplasia type 2A (Ret-MEN2A) or 2B (Ret-MEN2B) mutations and the hybrid gene from c-RET and RFP (Rfp-Ret) were higher than those of c-Ret. We demonstrated that ultraviolet light (UV) irradiation induced activation of c-Ret and superactivation of genetically mutated, and thereby constitutively activated, Ret-MEN2A, Ret-MEN2B, and Rfp-Ret. We found that small proportions of c-Ret and Ret-MEN2B and a large proportion of MEN2A were dimerized due to disulfide bonds and that high kinase activity resided in these fractions. The UV-induced activation of c-Ret and superactivation of Ret-MEN2A and Ret-MEN2B were then shown to be closely associated with promotion of the disulfide bond-mediated dimerization of the Ret proteins. Furthermore, we showed that a large proportion of Rfp-Ret was dimerized or polymerized and that almost all kinase activities resided in the highly polymerized but not dimerized fraction. The UV-induced superactivation of Rfp-Ret was also found to be closely associated with promotion of polymerization but not with dimerization of Rfp-Ret. Further experiments revealed that UV induced intracellular dimerization and activation of the extracellular domain-deleted mutant Ret (Ret-PTC-1). Most importantly, the levels of basal kinase activity and dimerization of Ret-TPC-1-C376A, in which cysteine 376 in the tyrosine kinase domain of Ret-TPC-1 was replaced with alanine, were low and were not increased by UV irradiation. These results suggest that the cysteine at this position works as the primary target of dimerization of Ret proteins inside the cell for both the maintenance of the basal kinase activity and its promotion by UV, possibly in co-operation with the cysteine(s) in the extracellular domain of Ret-MEN2A and Rfp-Ret, which is the target of dimerization and polymerization outside the cell. The potential biological significance of the UV-mediated superactivation of mutant Ret through the newly proposed mechanism in oncogenesis is discussed.