BACKGROUND 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 γ-tubulin ring complex (γ-TuRC) assembly. However, the molecular mechanisms underlying such function were not completely understood. RESULTS Here, we show that Aurora A site-specific phosphorylation in TACC3 regulates formation of astral microtubules by stabilizing γ-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 γ-tubulin ring complex (γ-TuRC) proteins at the spindle poles. Under similar condition, phospho-mimicking S558D mutation retains astral microtubules and the γ-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 γ-TuRC proteins and further, S558A mutation impairs the interaction. We further reveal that the mutation affects the assembly of γ-TuRC from the small complex components. CONCLUSIONS The results demonstrate that TACC3 phosphorylation stabilizes γ- 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.

中文翻译：

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Aurora A位点特异性TACC3磷酸化通过稳定γ-微管蛋白环复合物来调节星状微管装配。

背景技术从有丝分裂中心体发出的星状微管在定义细胞分裂轴和组织形态发生中起关键作用。先前的研究表明，人类转化的酸性卷曲螺旋3（TACC3）是最保守的TACC家族蛋白，可通过影响γ-微管蛋白环复合物（γ-TuRC）装配来调节脊椎动物细胞中心体中星状微管的形成。但是，尚未完全了解这种功能的分子机制。结果在这里，我们表明TACC3中的Aurora A位点特异性磷酸化通过稳定人细胞中的γ-TuRC装配体来调节星状微管的形成。最保守的Aurora A定位网站的突变，TACC3中的558丙氨酸丝氨酸558（S558A）导致星状微管的大量丢失，并破坏了纺锤极的γ-微管蛋白环复合物（γ-TuRC）蛋白的定位。在类似条件下，模仿磷酸化S558D的突变会以与野生型TACC3表达的对照细胞相似的方式保留星状微管和γ-TuRC蛋白。延时成像显示，S558A突变会导致纺锤极的定位出现缺陷，从而导致中期到后期过渡的延迟。生化结果确定，Ser 558-磷酸化的TACC3与γ-TuRC蛋白相互作用，此外，S558A突变削弱了该相互作用。我们进一步揭示了该突变会影响小复合物组件组装的γ-TuRC。结论结果表明，TACC3磷酸化可稳定γ-微管蛋白环复合物的装配，从而调节中心体紫的形成。它们还暗示了TACC3磷酸化在中心体/纺锤体极的功能完整性中的潜在作用。