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Two-component kinase-like activity of nm23 correlates with its motility-suppressing activity
P D Wagner1, P S Steeg, N D Vu
1Laboratory of Biochemistry, Division of Basic Sciences, National Cancer Institute, Bethesda, MD 20892, USA. wagnerp@dc37a.nci.nih.gov
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 ATP-citrate 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.
Insights
Nm23-H1 protein suppresses breast cancer metastasis by transferring phosphate to membrane proteins. This phosphate transfer to aspartate/glutamate residues correlates with suppressed cell motility, unlike transfer to histidine residues.
Area of Science:
- Molecular Biology
- Biochemistry
- Cancer Research
Background:
- Nm23 genes encode nucleoside diphosphate kinases, known for suppressing tumor metastasis.
- Wild-type nm23-H1 suppresses human breast carcinoma cell motility, while mutants nm23-H1(S120G) and nm23-H1(P96S) do not.
Purpose of the Study:
- To investigate the mechanism by which nm23-H1 suppresses tumor metastasis.
- To determine the role of phosphate transfer to specific residues in nm23-H1's function.
Main Methods:
- Transfection of human breast carcinoma cells with wild-type and mutant nm23-H1.
- Assaying the phosphorylation of membrane proteins, ATP-citrate lyase, and succinic thiokinase by nm23-H1 and its mutants.
Main Results:
- Wild-type nm23-H1 phosphorylates 43-kDa membrane proteins at aspartate or glutamate residues.
- Mutants nm23-H1(S120G) and nm23-H1(P96S) showed reduced or no phosphorylation of these membrane proteins.
- Nm23-H1 also phosphorylates histidine residues on ATP-citrate lyase and succinic thiokinase, with varying efficiencies for the mutants.
Conclusions:
- Phosphate transfer from nm23-H1 to aspartate/glutamate residues on membrane proteins correlates with suppressed cell motility.
- This mechanism is distinct from phosphate transfer to histidine residues on other enzymes.