Metastasis suppressor function of NM23-H1 requires its 3'-5' exonuclease activity

Abstract

The metastasis suppressor NM23-H1 possesses 3 enzymatic activities in vitro, a nucleoside diphosphate kinase (NDPK), a protein histidine kinase and a more recently characterized 3'-5' exonuclease. Although the histidine kinase has been implicated in suppression of motility in breast carcinoma cell lines, potential relevance of the NDPK and 3'-5' exonuclease to metastasis suppressor function has not been addressed in detail. To this end, site-directed mutagenesis and biochemical analyses of bacterially expressed mutant NM23-H1 proteins have identified mutations that disrupt the 3'-5' exonuclease alone (Glu(5) to Ala, or E(5) A), the NDPK and histidine kinase activities tandemly (Y(52) A, H(118) F) or all 3 activities simultaneously (K(12) Q). Although forced expression of NM23-H1 potently suppressed spontaneous lung metastasis of subcutaneous tumor explants derived from the human melanoma cell line 1205LU, no significant metastasis suppressor activity was obtained with the exonuclease-deficient variants E(5) A and K(12) Q. The H(118) F mutant, which lacked both the NDPK and histidine kinase while retaining the 3'-5' exonuclease, also exhibited compromised suppressor activity. In contrast, each mutant retained the ability to suppress motility and invasive characteristics of 1205LU cells in culture, indicating that the NM23-H1 molecule possesses an additional activity(s) mediating these suppressor functions. These studies provide the first demonstration that the 3'-5' exonuclease activity of NM23-H1 is necessary for metastasis suppressor function and further indicate cooperativity of the 3 enzymatic activities of the molecule on suppression of the metastatic process.

FIGURE 1

Location of amino acid residues in NM23-H1 targeted by site-directed mutagenesis. (a) Shown is a three-dimensional surface model of the NM23-H1 hexamer, with one subunit represented in a ribbon format. Amino acid residues targeted by mutagenesis in this study are highlighted with a ball-and-stick format. Negative charges are identified in red and positive charges in blue . (b) The amino acid sequence of NM23-H1 is displayed in a linear alignment with secondary structure elements. Residues targeted by site-directed mutagenesis are underlined and highlighted in bold font.

FIGURE 2

Analysis of secondary structure for NM23-H1 mutant variants by circular dichroism spectrometry. Wild-type (WT) and mutant variants E₅A, Q₁₇N, Y₅₂A and P₉₆S were expressed in E. coli, purified to near homogeneity, and subjected to circular dichroism spectrometry, as described previously.

FIGURE 3

Determination of 3’–5’ exonuclease and protein histidine kinase activity for selected NM23-H1 mutant variants. (a) Purified preparations of wild-type or mutant NM23-H1 proteins (500 ng) were incubated with 10 fmol of a single-stranded, 5’-radiolabeled DNA substrate for the indicated times at room temperature, as described in Materials and Methods. Extent of DNA cleavage was analyzed by electrophoresis through denaturing 10% polyacrylamide gels and visualization by phosphorimaging. P, radiolabeled probe. (b) 3’–5’ exonuclease assay of E₅A mutant is shown. C, Quantitation of results in panels A and B are shown, expressed as total nucleotides (nt) cleaved. (c) Protein histidine kinase activity was analyzed for wild-type and mutant variants of NM23-H1. NM23-H1 proteins were auto-radiolabeled with [³²P-γ] ATP, followed by incubation with unlabeled NM23-H2 substrate for the indicated times.

FIGURE 4

Forced expression of NM23-H1 variants has minimal effects on the transformed phenotype of 1205LU melanoma cells in culture and as tumor explants in athymic nude mice. (a) Cell proliferation assays were conducted on 1205LU cells using the MTS procedure, as described in Materials and Methods. (b) Data represents the mean tumor volume obtained over the indicated time courses following subcutaneous injection into athymic nude mice of the 1205LU parent and transfected derivatives expressing wild-type NM23-H1 (H1-WT), P₉₆S and H₁₁₈F variants (left panel), and in a separately conducted series of experiments, H1-WT, E₅A, and K₁₂Q mutants (right panel). No significant differences between growth curves were detected between any of the cell lines in panels a and b, as determined by two-way ANOVA.

FIGURE 5

NM23-H1 inhibits motility and invasive capacities of 1205LU melanoma cells in a manner independent of its 3’–5’ exonuclease, NDPK and histidine kinase activities. (a) The 1205LU panel of cell lines were evaluated for motility and (b) invasive characteristics in Boyden chamber assays. Asterisks denote means that are significantly different (p < 0.05) from all other treatment means within a panel, as determined by one-way ANOVA and Student’s t-Test.

FIGURE 6

Number and diameter of pulmonary metastatic lesions in spontaneous metastasis assay. Closed circles represent diameters for individual nodules, horizontal bars represent the average number of visible metastases per lung, and values listed above each column of data indicate the total number of nodules observed within a given lung. No significant differences between the cell lines were found in either the number of lesions per lung or the average lesion size, as determined by Kruskal-Wallis one-way ANOVA (p > 0.05).