Two-component kinase-like activity of nm23 correlates with its motility-suppressing activity

Abstract

Nm23 genes, which encode nucleoside diphosphate kinases, have been implicated in suppressing tumor metastasis. The motility of human breast carcinoma cells can be suppressed by transfection with wild-type nm23-H1, but not by transfections with two nm23-H1 mutants, nm23-H1 S12OG and nm23-H1 P96S . Here we report that nm23-H1 can transfer a phosphate from its catalytic histidine to aspartate or glutamate residues on 43-kDa membrane proteins. One of the 43-kDa membrane proteins was not phosphorylated by either nm23-H1 P96S or nm23-H1 S120G , and another was phosphorylated much more slowly by nm23-H1 P96S and by nm23-H1 S120G than by wild-type nm23-H1. Nm23-H1 also can transfer phosphate from its catalytic histidine to histidines on ATPcitrate lyase and succinic thiokinase. The rates of phosphorylation of ATP-citrate lyase by nm23-H1 S120G and nm23-H1 P96S were similar to that by wild-type nm23-H1. The rate of phosphorylation of succinic thiokinase by nm23-H1 S120 was similar to that by wild-type nm23-H1, and the rate of phosphorylation of succinic thiokinase by nm23-H1 P96S was about half that by wild-type nm23-H1. Thus, the transfer of phosphate from nm23-H1 to aspartates or glutamates on other proteins appears to correlate better with the suppression of motility than does the transfer to histidines.Nm23 genes have been implicated in the control of tumor metastasis (1). For many, but not all, types of tumors, there is an inverse relationship between the level of nm23 expression and metastatic potential (reviewed in ref.2). Overexpression of nm23 in highly metastatic murine melanoma cell lines (3-6), rat mammary adenocarcinoma cells (7), and human breast carcinoma cells (4,8,9) reduces their metastatic potentials, indicating that nm23 genes can suppress tumor metastasis. Increased levels of nm23 expression also have been associated with both cellular proliferation (10) and differentiation (11). In human tissues the two major forms of nm23 are nm23-H1 and nm23-H2. Expression of nm23-H1 has been correlated with suppression of metastasis (2).The abnormal wing disc (awd) gene of Drosophila encodes a protein that is 78% identical to the human nm23 proteins (12). The awd gene is essential for normal Drosophila development (13). The killer of prune mutation in this gene (awd kpn ) causes a dominant lethality but only in flies that do not have a functional prune gene. The prune gene has been cloned, but the biochemical function of the resulting protein is not known (14,15).Nm23 and awd genes encode nucleoside diphosphate kinases (NDPKs) (12). NDPKs catalyze the phosphorylation of nucleoside 5Ј-diphosphates to triphosphates (16). In the first step, a phosphate is transferred from a nucleoside 5Ј-triphosphate to a histidine at the catalytic site of the enzyme. This high-energy phosphate then is transferred to a phosphate group on a nucleoside 5Ј-diphosphate.The synthesis of nucleoside triphosphates does not appear to explain the role of nm23 in suppressing metastasis (2). Similarly, the effect of the awd kpn mutation...

Plakoglobin interacts with and increases the protein levels of metastasis suppressor Nm23-H2 and regulates the expression of Nm23-H1

“…In humans, there are eight members of the Nm23 family (Nm23-H1-8, respectively), and in addition to their nucleoside diphosphate kinase activity, members of this family have both histidine protein kinase and phosphotransferase activities (Wagner et al, 1997;Lacombe et al, 2000;Steeg et al, 2008). Nm23-H1, which has DNase activity (Fan et al, 2003), is primarily cytoplasmic, whereas Nm23-H2 is both nuclear and cytoplasmic, and binds DNA (Postel et al, 2000;Bosnar et al, 2004Bosnar et al, , 2009).…”

Phosphorylation of nm23-H1 by CKI induces its complex formation with h-prune and promotes cell motility

“…However, the NDPK activity alone cannot explain these pleomorphic effects. The nm23 proteins have been reported to possess both a histidine protein kinase activity and a phosphotransferase activity; the latter potentially represents an additional mechanism by which the nm23s can influence the metastasis and differentiation processes (MacDonald et al, 1996;Wagner et al, 1997). To date, several nm23 interactors have been described in mammals, including Rad, Tiam1, Arf6, the nucleosome assembly protein SET, Cdc42, EBNA1 and KSR (Otsuki et al, 2001;Tseng et al, 2001;Palacios et al, 2002), although their roles are not fully understood.…”

Evidence for interaction between human PRUNE and nm23-H1 NDPKinase

“…Extensive studies have shown that these mutants retain: (1) the catalytic activity; (2) the ability to autophosphorylate His118 (see Figure 4b); (3) the phospho-Histidine-dependent Serine autophosphorylation; (4) the ability to transfer phosphate from His118 to ATP-citrate lyase and succinic thiokinase His residues; and (5) the ability to bind DNA (tested only for nm23-H2 P96S) MacDonald et al, 1993;Postel et al, 1996;Wagner et al, 1997). On the contrary, the P96 and S120 residues are essential for the motility suppressing e ect and the`phosphorelay system' from Histidine residue to Aspartate and/or Glutamate residues of nm23-H1 (Hartsough and Steeg, 1998;MacDonald et al, 1996;Wagner et al, 1997). In this regard, our results provide the ®rst evidence that mutations of residues P96 and S120 a ect in di erent ways NDPK protein function.…”

“…Several studies have shown that di erent NDPK oligomer species are involved in a variety of cellular processes where enzymatic activity is required (Engel et al, 1998;Hartsough and Steeg, 1998;Mesnildrey et al, 1998;Wagner et al, 1997). Elucidation of the role of the biological interactions between PRUNE and nm23-H1 would be a major step towards understanding the role of NDPKs in neuroblastoma.…”

“…Consistently, heterohexamers between nm23-H1 and -H2 and a tetramer containing two nm23-H1 and two GAPDH subunits have been found in vivo (Engel et al, 1998;Gilles et al, 1991). This later complex is responsible for the newly described phosphotransferase function of nm23-H1 (Engel et al, 1995(Engel et al, , 1998Wagner et al, 1997). Other studies have shown that the catalytic activity of NDPKs can be dissociated from the antimetastatic function of nm23-H1 and from the indirect enhancement of c-myc expression by nm23-H2 (Berberich and Postel, 1995;Hartsough and Steeg, 1998;Michelotti et al, 1997;Postel et al, 1993).…”