Aurora A site specific TACC3 phosphorylation regulates astral microtubule assembly by stabilizing [gamma]-tubulin ring complex

Astral microtubules emanating from the mitotic centrosomes play pivotal roles in defining cell division axis and tissue morphogenesis. Previous studies have demonstrated that human transforming acidic coiled-coil 3 (TACC3), the most conserved TACC family protein, regulates formation of astral microtubules at centrosomes in vertebrate cells by affecting [gamma]-tubulin ring complex ([gamma]-TuRC) assembly. However, the molecular mechanisms underlying such function were not completely understood. Here, we show that Aurora A site-specific phosphorylation in TACC3 regulates formation of astral microtubules by stabilizing [gamma]-TuRC assembly in human cells. Mutation of the most conserved Aurora A targeting site, Ser 558 to alanine (S558A) in TACC3 results in robust loss of astral microtubules and disrupts localization of the [gamma]-tubulin ring complex ([gamma]-TuRC) proteins at the spindle poles. Under similar condition, phospho-mimicking S558D mutation retains astral microtubules and the [gamma]-TuRC proteins in a manner similar to control cells expressed with wild type TACC3. Time-lapse imaging reveals that S558A mutation leads to defects in positioning of the spindle-poles and thereby causes delay in metaphase to anaphase transition. Biochemical results determine that the Ser 558- phosphorylated TACC3 interacts with the [gamma]-TuRC proteins and further, S558A mutation impairs the interaction. We further reveal that the mutation affects the assembly of [gamma]-TuRC from the small complex components. The results demonstrate that TACC3 phosphorylation stabilizes [gamma]- tubulin ring complex assembly and thereby regulates formation of centrosomal asters. They also implicate a potential role of TACC3 phosphorylation in the functional integrity of centrosomes/spindle poles.