Cobalt- and nickel-binding property of cullin-2

Treatment with divalent metal ions such as cobalt (Co(2+)) or nickel (Ni(2+)) result in the stabilization of hypoxia-inducible factor-1alpha (HIF1alpha). Recently, HIF1alpha was shown to be ubiquitinated by an E3-ligase complex and be subsequently targeted for proteasomal degradation. In this study, we demonstrated that Co(2+) and Ni(2+) specifically bind to cullin-2. Mutant analysis revealed that cullin-2 possesses at least three sites for the binding. Furthermore, fluorescence spectroscopy revealed that only Co(2+) and Ni(2+) have the binding activity to cullin-2, but other metal ions, including Cu(2+), Ca(2+), Mg(2+), Mn(2+), and Zn(2+), did not. Finally, we found that Co(2+) and Ni(2+) do not bind to any components of the E3-ligase other than cullin-2, suggesting that cullin-2 is a key target of Co(2+) and Ni(2+). Interestingly, Co(2+) did not affect the complex formation of the ligase, suggesting that the metal binding to cullin-2 affects the function, but not the assembly of the E3-ligase.