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