Interactions of Phosphorylation and Dimerizing Domains of the α-Subunits of Na+/K+-ATPase

Abstract

Chemical cross-linking studies are among a number of experimental approaches that have suggested the functional significance of higher association states of α,β-protomers of Na+/K+-ATPase. Formation of the phosphointermediate of the enzyme on Asp369 of the α-subunit is known to induce oxidative cross-linking of the α-subunits catalyzed by Cu2+-phenanthroline. To localize the phosphorylation-induced α,α-interface, we cleaved α at Arg438-Ala439 by controlled proteolysis and exposed the partially cleaved enzyme to the cross-linking reagent. In addition to the α,α-dimer, two other phosphorylation-induced cross-linked products were obtained. Using gel electrophoretic resolution of the cross-linked 32P-labeled enzyme, N-terminal analyses of the products, and their reactivities with sequence-specific antibodies, the two products were identified as a homodimer of the C-terminal 64-kDa fragment of α and a heterodimer of α and the 64-kDa peptide. The latter dimer was also obtained when the cross-linked α,α-dimer was formed first and then subjected to proteolysis. The findings localize the dimerizing domain to the C-terminal side of Ala439 and indicate that intersubunit proximities of dimerizing domains are regulated by phosphorylation-dephosphorylation of Asp369 during the reaction cycle of the enzyme. Chemical cross-linking studies are among a number of experimental approaches that have suggested the functional significance of higher association states of α,β-protomers of Na+/K+-ATPase. Formation of the phosphointermediate of the enzyme on Asp369 of the α-subunit is known to induce oxidative cross-linking of the α-subunits catalyzed by Cu2+-phenanthroline. To localize the phosphorylation-induced α,α-interface, we cleaved α at Arg438-Ala439 by controlled proteolysis and exposed the partially cleaved enzyme to the cross-linking reagent. In addition to the α,α-dimer, two other phosphorylation-induced cross-linked products were obtained. Using gel electrophoretic resolution of the cross-linked 32P-labeled enzyme, N-terminal analyses of the products, and their reactivities with sequence-specific antibodies, the two products were identified as a homodimer of the C-terminal 64-kDa fragment of α and a heterodimer of α and the 64-kDa peptide. The latter dimer was also obtained when the cross-linked α,α-dimer was formed first and then subjected to proteolysis. The findings localize the dimerizing domain to the C-terminal side of Ala439 and indicate that intersubunit proximities of dimerizing domains are regulated by phosphorylation-dephosphorylation of Asp369 during the reaction cycle of the enzyme.