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 metasta sis-suppressive capacity using site-directed mutagenesis and an in vit ro tumor cell motility assay. MDA-MB-435 human breast carcinoma cells were transfected with a control expression vector (pCMVB-amneo), the v ector 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 tha t causes developmental defects; histidine 118, involved in Nm23's nucl eoside diphosphate kinase activity; and serine 120, a site of mutation in human neuroblastomas and phosphorylation. The wild type nm23-H1 tr ansfectants were 44-98% less motile to serum and 86-99% less motile to autotaxin than control vector transfectants. The proline 96 k-pn, ser ine 120 to glycine, and to a lesser extent serine 120 to alanine mutan t nm23-H1-transfected cell lines exhibited motility levels at or above the control transfectants, indicating that these mutations can abroga te the motility-suppressive phenotype of nm23-H1. No effect was observ ed on cellular proliferation, nor were the serine 44 to alanine nm23-H 1 mutant transfectants motile, demonstrating the specificity of the da ta. The data identify the first structural motifs of nm23-H1 that infl uence its metastasis suppressive effect and suggest complex biochemica l associations or activities in the Nm23 suppressive pathway.