Site-directed mutagenesis of nm23-H1. Mutation of proline 96 or serine 120 abrogates its motility inhibitory activity upon transfection into human breast carcinoma cells.
We report the first correlation of Nm23 sequence and its tumor metastasis-suppressive capacity using site-directed mutagenesis and an in vitro tumor cell motility assay. MDA-MB-435 human breast carcinoma cells were transfected with a control expression vector (pCMVBamneo), the vector containing the wild type nm23-H1, or the nm23-H1 vector encoding mutations at the following amino acids: serine 44, a phosphorylation site; proline 96, the k-pn mutation in the Drosophila nm23 homolog that causes developmental defects; histidine 118, involved in Nm23's nucleoside diphosphate kinase activity; and serine 120, a site of mutation in human neuroblastomas and phosphorylation. The wild type nm23-H1 transfectants were 44-98% less motile to serum and 86-99% less motile to autotaxin than control vector transfectants. The proline 96 k-pn, serine 120 to glycine, and to a lesser extent serine 120 to alanine mutant nm23-H1-transfected cell lines exhibited motility levels at or above the control transfectants, indicating that these mutations can abrogate the motility-suppressive phenotype of nm23-H1. No effect was observed on cellular proliferation, nor were the serine 44 to alanine nm23-H1 mutant transfectants motile, demonstrating the specificity of the data. The data identify the first structural motifs of nm23-H1 that influence its metastasis suppressive effect and suggest complex biochemical associations or activities in the Nm23 suppressive pathway.
MacDonald
N J
NJ
Women's Cancers Section, Laboratory of Pathology, Division of Clinical Sciences, NCI, National Institutes of Health, Bethesda, Maryland 20892, USA.
Freije
J M
JM
Stracke
M L
ML
Manrow
R E
RE
Steeg
P S
PS
D000409:Alanine; D000595:Amino Acid Sequence; D000818:Animals; D001943:Breast Neoplasms; D002455:Cell Division; D002633:Chemotaxis; D004330:Drosophila; D005260:Female; D005998:Glycine; D006639:Histidine; D006801:Humans; D008969:Molecular Sequence Data; D020559:Monomeric GTP-Binding Proteins; D016297:Mutagenesis, Site-Directed; D054778:NM23 Nucleoside Diphosphate Kinases; D009447:Neuroblastoma / Q000235:genetics; D009701:Nucleoside-Diphosphate Kinase / Q000096:biosynthesis / Q000378:metabolism*; D010766:Phosphorylation; D016133:Polymerase Chain Reaction; D011392:Proline; D011994:Recombinant Proteins / Q000378:metabolism; D012694:Serine; D014157:Transcription Factors / Q000096:biosynthesis / Q000502:physiology*; D014162:Transfection; D014407:Tumor Cells, Cultured
D054778:NM23 Nucleoside Diphosphate Kinases; D011994:Recombinant Proteins; D014157:Transcription Factors; D012694:Serine; D006639:Histidine; D011392:Proline; C517938:NME1 protein, human; D009701:Nucleoside-Diphosphate Kinase; D020559:Monomeric GTP-Binding Proteins; D000409:Alanine; D005998:Glycine