Site-directed mutation of Nm23-H1. Mutations lacking motility suppressive capacity upon transfection are deficient in histidine-dependent protein phosphotransferase pathways in vitro
| Autor: | J M, Freije, P, Blay, N J, MacDonald, R E, Manrow, P S, Steeg |
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| Rok vydání: | 1997 |
| Předmět: |
Dose-Response Relationship
Drug Histidine Kinase NM23 Nucleoside Diphosphate Kinases Transfection Models Biological Recombinant Proteins Nucleoside-Diphosphate Kinase Mutagenesis Site-Directed Serine Humans Histidine Phosphorylation Phosphoenolpyruvate Sugar Phosphotransferase System Protein Kinases Glutathione Transferase Monomeric GTP-Binding Proteins Transcription Factors |
| Zdroj: | The Journal of biological chemistry. 272(9) |
| ISSN: | 0021-9258 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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