title: 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.
abstract: 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.
authors.affiliation: Women's Cancers Section, Laboratory of Pathology, Division of Clinical Sciences, NCI, National Institutes of Health, Bethesda, Maryland 20892, USA.
authors.forename: N J
authors.initials: NJ
authors.lastname: MacDonald
authors.forename: J M
authors.initials: JM
authors.lastname: Freije
authors.forename: M L
authors.initials: ML
authors.lastname: Stracke
authors.forename: R E
authors.initials: RE
authors.lastname: Manrow
authors.forename: P S
authors.initials: PS
authors.lastname: Steeg
mesh_terms: 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
chemical_list: 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