A serine phosphorylation of Nm23, and not its nucleoside diphosphate kinase activity, correlates with suppression of tumor metastatic potential.
We describe a serine phosphorylation of the putative metastasis suppressor protein Nm23, and present evidence of its relevance to the signal transduction and tumor metastatic processes. Nm23 was previously demonstrated to exhibit nucleoside diphosphate kinase (NDPK) activity, which transfers a phosphate among nucleoside tri- and diphosphates via an Nm23-phospho-histidine intermediate. Recent data have dissociated the NDPK activity of Nm23 from its phenotypic effects; therefore we have asked whether Nm23 possesses additional biochemical functions. An acid-stable (nonhistidine) phosphorylation was identified on autophosphorylated purified recombinant Nm23 proteins and [32P]orthophosphate-labeled human breast carcinoma and murine melanoma Nm23. Phosphoamino acid analysis identified serine as the acid-stable phosphorylation and serine 44 as the major site of phosphorylation. The acid stable phosphorylation (serine) of Nm23 was inhibited by cAMP in vitro and forskolin in vivo, suggesting that this phosphorylation pathway is regulated in signal transduction. No effect of cAMP was observed on Nm23 NDPK activity. Once phosphorylated, Nm23-phosphoserine can release free phosphate in vitro. The biological relevance of the novel phosphorylation identified herein is suggested by the direct correlation of in vivo Nm23 acid-stable phosphorylation levels, but not Nm23 NDPK activity, with suppression of tumor metastatic potential among control and nm23-1 transfected murine melanoma cells.
MacDonald
N J
NJ
Women's Cancer Section, National Cancer Institute, Bethesda, Maryland 20892.
De la Rosa
A
A
Benedict
M A
MA
Freije
J M
JM
Krutsch
H
H
Steeg
P S
PS
D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D016147:Genes, Tumor Suppressor; D006801:Humans; D008546:Melanoma, Experimental / Q000473:pathology*; D051379:Mice; D008969:Molecular Sequence Data; D020559:Monomeric GTP-Binding Proteins; D054778:NM23 Nucleoside Diphosphate Kinases; D009362:Neoplasm Metastasis / Q000235:genetics; D009701:Nucleoside-Diphosphate Kinase / Q000378:metabolism*; D010710:Phosphates / Q000378:metabolism; D010766:Phosphorylation; D010768:Phosphoserine / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping; D012694:Serine / Q000378:metabolism*; D014157:Transcription Factors / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D014162:Transfection; D014407:Tumor Cells, Cultured
D017931:DNA Primers; D054778:NM23 Nucleoside Diphosphate Kinases; D010710:Phosphates; D011994:Recombinant Proteins; D014157:Transcription Factors; D010768:Phosphoserine; D012694:Serine; D000255:Adenosine Triphosphate; C517938:NME1 protein, human; C517939:Nme1 protein, mouse; D009701:Nucleoside-Diphosphate Kinase; D020559:Monomeric GTP-Binding Proteins