CDK1-dependent N-terminal NuMA phosphorylation promotes dynein-dynactin-NuMA assembly for accurate chromosome segregation

The microtubule-based motor dynein and its cofactor dynactin are activated by various adaptors to fulfill essential functions throughout the cell cycle, including organelle transport and mitotic spindle assembly. NuMA is a mitotic adaptor that interacts with dynein-dynactin via its N-terminal region (NuMA-N). However, how NuMA-N binds and activates dynein-dynactin in mitosis remains unclear. Here, we combine a membrane-tethering assay, quantitative proteomics, and live-cell analyses to show that mitotic phosphorylation of NuMA-N drives dynein-dynactin-NuMA (DDN) assembly. We find that CDK1-Cyclin B1 phosphorylates NuMA-N, primarily at its conserved serine 203, which stimulates dynein activation in vitro. Replacing endogenous NuMA with phosphorylation-deficient mutants further reveals that NuMA-N phosphorylation, together with its dynein-binding site and Spindly-like motif, is required to form stable DDN complexes for functional spindle assembly. These results highlight CDK1-dependent N-terminal NuMA phosphorylation as a crucial mitotic phospho-switch that ensures stable multivalent interactions between dynein-dynactin and NuMA for accurate chromosome segregation.