NEKL-3 kinase directly phosphorylates and inhibits OSM-3 motility, influencing OSM-3 motility properties in vitro. (A) Tandem mass spectrum of the S96-phosphorylated peptide of OSM-3. The y and b series indicate fragments at amide bonds of the peptide. (B and C) Schematic of hydrogen bond between R10 and S96 (pink) at the AMPPNP (yellow)-binding pocket of OSM-3 (green). (PDB: 7A5E) (C) Relative ATPase activity of OSM-3^WT reacted with NEKL-3 or heat-denatured NEKL-3 (dNEKL-3). The assays were performed as described in methods following the established protocols. The mean activity of kinesin heavy chain (KHC) was set to 100%. The results were obtained from three independent experiments. Statistical significance, compared with the control with a matching color code, is based on Student’s t test. n.s., not significant; *P < 0.05; ***P < 0.001. Data are mean ± SD. (D) Representative kymographs showing the motion of phosphorylated or unphosphorylated OSM-3^G444E along the MT. Horizontal scale bar, 5 µm; vertical bar, 10 s. (E) Quantification of the number of processive events per micrometer of MT per minute in C. The total numbers of events analyzed were 316 (OSM-3^G444E), 80 (OSM-3^G444E+NEKL-3), and 235 (OSM-3^G444E+dNEKL-3). Statistical significance, compared with the control with matching color codes, is by Mann–Whitney nonparametric test. n.s., not significant; ***P < 0.001. Data are mean ± SD. (F) Statistics of motility properties of OSM-3 under different conditions. † OSM-3^G444E was used for single-molecule motility assay. ‡ OSM-3^WT was used in MT-gliding assay. Statistical significance is compared with the control. n.s., not significant; **P < 0.01; ***P < 0.001. ND, not detected. Data are mean ± SD. (G) Quantification of the number of detachment events per micrometer of MT per minute during MT-gliding assay. N ≥ 20. Statistical significance, compared with the control with matching color codes, is by Mann–Whitney nonparametric test. n.s., not significant; ***P < 0.001. Data are mean ± SD.