title: Site-directed mutation of Nm23-H1. Mutations lacking motility suppressive capacity upon transfection are deficient in histidine-dependent protein phosphotransferase pathways in vitro.
abstract: We previously compared the structure and motility suppressive capacity of nm23-H1 by transfection of wild type and site-directed mutant forms into breast carcinoma cells. Wild type nm23-H1 and an nm23-H1(S44A) (serine 44 to alanine) mutant suppressed motility, whereas the nm23-H1(P96S), nm23-H1(S120G), and to a lesser extent, nm23-H1(S120A) mutant forms failed to do so. In the present study wild type and mutant recombinant Nm23-H1 proteins have been produced, purified, and assayed for phosphorylation and phosphotransfer activities. We report the first association of Nm23-H1 mutations lacking motility suppressive capacity with decreased in vitro activity in histidine-dependent protein phosphotransferase assays. Nm23-H1(P96S), a Drosophila developmental mutation homolog, exhibited normal autophosphorylation and nucleoside-diphosphate kinase (NDPK) characteristics but deficient phosphotransfer activity in three histidine protein kinase assays, using succinic thiokinase, Nm23-H2, and GST-Nm23-H1 as substrates. Nm23-H1(S120G), found in advanced human neuroblastomas, exhibited deficient activity in several histidine-dependent protein phosphotransfer reactions, including histidine autophosphorylation, downstream phosphorylation on serines, and slightly decreased histidine protein kinase activity; significant NDPK activity was observed. The Nm23-H1(S120A) mutant was deficient in only histidine-dependent serine autophosphorylation. Nm23-H1 and Nm23-H1(S44A) exhibited normal activity in all assays conducted. Based on this correlation, we hypothesize that a histidine-dependent protein phosphotransfer activity of Nm23-H1 may be responsible for its biological suppressive effects.
authors.affiliation: Women's Cancers Section, Laboratory of Pathology, Division of Clinical Sciences, NCI, National Institutes of Health, Bethesda, Maryland 20892, USA.
authors.forename: J M
authors.initials: JM
authors.lastname: Freije
authors.forename: P
authors.initials: P
authors.lastname: Blay
authors.forename: N J
authors.initials: NJ
authors.lastname: MacDonald
authors.forename: R E
authors.initials: RE
authors.lastname: Manrow
authors.forename: P S
authors.initials: PS
authors.lastname: Steeg
mesh_terms: D004305:Dose-Response Relationship, Drug; D005982:Glutathione Transferase / Q000378:metabolism; D006639:Histidine; D000071677:Histidine Kinase; D006801:Humans; D008954:Models, Biological; D020559:Monomeric GTP-Binding Proteins; D016297:Mutagenesis, Site-Directed; D054778:NM23 Nucleoside Diphosphate Kinases; D009701:Nucleoside-Diphosphate Kinase / Q000378:metabolism*; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System / Q000378:metabolism*; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D012694:Serine; D014157:Transcription Factors / Q000235:genetics*; D014162:Transfection
chemical_list: D054778:NM23 Nucleoside Diphosphate Kinases; D011994:Recombinant Proteins; D014157:Transcription Factors; D012694:Serine; D006639:Histidine; D005982:Glutathione Transferase; D011494:Protein Kinases; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System; C022409:protein-N-pi-phosphohistidine - sugar phosphotransferase; D000071677:Histidine Kinase; C517938:NME1 protein, human; D009701:Nucleoside-Diphosphate Kinase; D020559:Monomeric GTP-Binding Proteins