Molecular Mechanism of Energy Transduction and regulation of Monovalent cation Pumps

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When pig stomach membrance H,K-ATPase preparations were incubated with ^^<32>ATP,Mg^<2+> and Ca^<2+>, reversible phosphorylation of specific Tyr and Ser residues in the N-terminal alpha-chain of H,K-ATPase occurred without any detectable phosphorylation in other regions of the chain. Amino acid sequence analysis of purified peptides and others showed a sequential phosphorylation of Tyr-10 and Tyr-7 and sub muM Ca^<2+> activated phosphorylation of Ser-27. The apparent molecular weight of a detergent solubilized Tyr-kinase was estimated to be around 50 kDa by a gel filtration column and the kinase activity detected on a gel band after renaturation. The Ser-kinase present was suggested to be a conventional PKC.H,K-ATPase preparations phosphorylated fusion proteins consised of maltose binding protein and a peptide portion of the alpha-chain from Gly-2 to Gln-111. These data and others suggested that N-terminal cytosolic domain is sufficient for phosphorylation by endogenous Tyr-kinase and PKC in the membrane H,K-ATPase preparations.