Point mutations affecting the oligomeric structure of Nm23-H1 abrogates its inhibitory activity on colonization and invasion of prostate cancer cells.

In order to identify Nm23-H1's structural motifs influencing its metastasis-inhibitory activity, we transfected DU 145 human prostate carcinoma cells with the expression vector encoding the Nm23-H1 protein with mutations at the following amino acids: serine-44, a phosphorylation site; proline-96, a site corresponding to the k-pn mutation that causes developmental defects in Drosophila; and serine-120, a site of mutation in human neuroblastoma and phosphorylation. Significant decrease in colonization in soft agar and invasiveness of DU 145 cells was observed in the wild type nm23-H1 transfectants, and also in the serine-44 and serine-120 to alanine mutant nm23-H1-transfected cell lines. However, the k-pn type proline-96 to serine (P96S) and neuroblastoma type serine-120 to glycine (S120G) mutations of Nm23-H1 abrogated its inhibitory activity on colonization and invasion. Meanwhile, all of the recombinant mutant Nm23-H1 proteins produced in Escherichia coli exhibited NDP kinase activity levels at the wild type protein, although the P96S and S120G mutant proteins exhibited decreased histidine protein kinase activity and autophosphorylation level, respectively. Interestingly, only two of the mutant recombinant Nm23-H1 proteins examined, P96S and S120G, exhibited reduced hexameric and increased dimeric oligomerization relative to the wild type. These correlative data suggest that the metastasis-suppressing activity of Nm23-H1 may depend on its oligomeric structure, but not on its NDP kinase activity.