Mitotic phosphorylation of the microtubule nucleator γ-tubulin regulates spindle assembly

The assembly of cellular microtubules relies heavily on γ-tubulin ring complex (γTuRC), a macromolecular assembly of γ-tubulin and associated proteins that serves as a nucleation template. Here, we identify that within γTuRC, γ-tubulin undergoes mitosis-specific phosphorylation at the conserved residue Ser364. This phosphorylation is mediated by Cdk1/cyclin B and occurs exclusively in cytoplasmic γTuRC, but not in γTuRC associated with mitotic spindles. Functionally, Ser364 phosphorylation strongly suppresses the microtubule-nucleating activity of γTuRC. Although γTuRC activity is essential for spindle microtubule assembly, disrupting Ser364 phosphorylation by expressing a non-phosphorylatable γ-tubulin mutant leads to defective spindle formation and chromosome segregation. Ser364 phosphorylation establishes spatial control over microtubule nucleation by inactivating cytoplasmic γTuRC, while spindle-associated γTuRC remains unphosphorylated and functionally active, consistent with the recently identified inhibitory control of spindle-localized Cdk1/cyclin B. This γTuRC regulation acts together with other Cdk1/cyclin B actions to eliminate non-spindle microtubules and support spindle assembly. Our findings reveal that Ser364 phosphorylation provides precise microtubule control for mitotic progression.