SITE-DIRECTED MUTATION OF NM23-H1 - MUTATIONS LACKING MOTILITY SUPPRESSIVE CAPACITY UPON TRANSFECTION ARE DEFICIENT IN HISTIDINE-DEPENDENT PROTEIN PHOSPHOTRANSFERASE PATHWAYS IN-VITRO

Citation
Jmp. Freije et al., SITE-DIRECTED MUTATION OF NM23-H1 - MUTATIONS LACKING MOTILITY SUPPRESSIVE CAPACITY UPON TRANSFECTION ARE DEFICIENT IN HISTIDINE-DEPENDENT PROTEIN PHOSPHOTRANSFERASE PATHWAYS IN-VITRO, The Journal of biological chemistry, 272(9), 1997, pp. 5525-5532
Citations number
61
Categorie Soggetti
Biology
ISSN journal
00219258
Volume
272
Issue
9
Year of publication
1997
Pages
5525 - 5532
Database
ISI
SICI code
0021-9258(1997)272:9<5525:SMON-M>2.0.ZU;2-O
Abstract
We previously compared the structure and motility suppressive capacity of nm23-H1 by transfection of wild type and site-directed mutant form s 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 recom binant Nm23-H1 proteins have been produced, purified, and assayed for phosphorylation and phosphotransfer activities, We report the first as sociation of Nm23-H1 mutations lacking motility suppressive capacity w ith decreased in vitro activity in histidine-dependent protein phospho transferase assays. Nm23-H1(P96S), a Drosophila developmental mutation homolog, exhibited normal autophosphorylation and nucleoside-diphosph ate kinase (NDPK) characteristics but deficient phosphotransfer activi ty in three histidine protein kinase assays, using succinic thiokinase , Nm23-H2, and GST-Nm23-H1 as substrates. Nm23-H1(S120G), found in adv anced human neuroblastomas, exhibited deficient activity in several hi stidine-dependent protein phosphotransfer reactions, including histidi ne autophosphorylation, downstream phosphorylation on serines, and sli ghtly decreased histidine protein kinase activity; significant NDPK ac tivity was observed. The Nm23-H1(S120A) mutant was deficient in only h istidine-dependent serine autophosphorylation. Nm23-H1 and Nm23-H1(S44 A) exhibited normal activity in all assays conducted. Based on this co rrelation, we hypothesize that a histidine-dependent protein phosphotr ansfer activity of Nm23-H1 may be responsible for its biological suppr essive effects.