DISCRIMINATION BETWEEN ACID AND ALKALI-LABILE PHOSPHORYLATED RESIDUESON IMMOBILON - PHOSPHORYLATION STUDIES OF NUCLEOSIDE DIPHOSPHATE KINASE

We have critically analyzed current methodologies for distinguishing h istidine and serine phosphorylated residues in proteins and report a s imple technique that assures a reliable discrimination. Electrotransfe r of a phosphorylated enzyme to Immobilon membranes and its treatment at pH 1 and 14 in buffers containing 5% methanol allows unambiguous di stinction between serine/threonine and histidine phosphorylation (O-ph osphomonoesters and phosphoramide, respectively) since under these con ditions only one type of residue is dephosphorylated. The addition of 5% methanol to all buffers was indispensable to deplete phosphate from membranes incubated successively under acid and basic conditions. The technique was applied to the study of nucleoside diphosphate kinase ( NDP kinase) phosphorylation, In this enzyme, autophosphorylation of ac tive site histidine is an accepted intermediate step in the catalytic phosphate transfer activity of nucleoside diphosphate kinase (NDP kina se). Nonetheless, a significant degree of autophosphorylation on other residues has been reported by several laboratories, and the hypothesi s has been advanced that this nonhistidine phosphorylation may play an important role in NDP kinase cellular function, signaling the suppres sion of metastasis in the case of human NDP kinase A. Using this impro ved method, we show that human, Escherichia coli and Candida albicans NDP kinases are only autophosphorylated on histidine residues. In addi tion, we present evidence that the presence of phosphoserine after str ong acid hydrolysis of the histidine autophosphorylated enzyme is in f act a nonenzymatic transphosphorylation from phosphohistidine due to t he harsh acid treatment. This methodology was also applied to in vivo phosphorylation studies of C. albicans NDP kinase. We believe that the technique will be generally useful in histidine phosphorylation scree nings. (C) 1996 Academic Press, Inc.