Site-directed Mutation of Nm23-H1
Journal of Biological Chemistry
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
... ion 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. The nm23 family of genes has been characterized on the basis of its reduced expression in certain highly metastatic cell lines (1) and tumors (reviewed in Ref. 2). In human tissues, three members have been identified, nm23-H1 (3), nm23-H2 (4), and nm23-DR (5). Transfection of nm23 cDNAs into murine K-1735 TK (6, 7), B16F10 (8), or B16FE7 (9) melanoma cells, rat MTLn3 mammary adenocarcinoma cells (10), or human 1 The abbreviations used are: awd, abnormal wing discs; ATX, autotaxin; awd k-pn , awd killer of prune; GST, glutathione S-transferase; HPLC, high performance liquid chromatography; NDP, nucleoside diphosphate; NDPK, NDP kinase; PAGE, polyacrylamide gel electrophoresis; PVDF, polyvinylidene fluoride; r, recombinant; STK, succinic thiokinase.