Functional Analysis of an Anchoring Protein CG-NAP in the Centrosome

中心体锚定蛋白 CG-NAP 的功能分析

• 批准号: 13680785
• 负责人: TAKAHASHI Mikiko
• 金额: $ 0.77万
• 依托单位: Biosignal Research Center, Kobe University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2001
• 资助国家: 日本
• 起止时间: 2001 至 2002
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-13680785/
• 关键词: Centrosome; Microtubule Organizing Center (MTOC); γ-Tubulin; Calmodulin; Microtubule; コイルドコイル蛋白質

Microtubule assembly is initiated by γ-tubulin ring complex (γ-TuRC). In yeast, microtubule is nucleated from γ-TuRC anchored to the amino terminus of the spindle pole body component SpcllOp, which interacts with Cmd1p (calmodulin) at the carboxyl terminus. However, mammalian protein that anchors γ-TuRC remains to be elucidated. A giant coiled-coil protein CG-NAP (c___entrosome and G___olgi localized PKN___-a___ssociated p___rotein) was localized to the centrosome via the carboxyl-terminal region. This region was found to interact with calmodulin by yeast two-hybrid screening, and shares high homology with the caroboxyl-terminal region of another centrosomal coiled-coil protein, kendrin. The amino-terminal region of either CG-NAP or kendrin indirectly associated with γ-tubulin through binding with γ-tubulin complex protein 2 (GCP2) and/or GCP3. Furthermore, endogenous CG-NAP and kendrin were coimmunoprecipitated each other, and with endogenous GCP2 and γ-tubulin, suggesting that CG-NAP and kendrin form complex and interact with γ-TuRC in vivo. These proteins were localized to the center of microtubule asters nucleated from isolated centrosomes. Pretreatment of the centrosomes by antibody to CG-NAP or kendrin moderately inhibited the microtubule aster formation, moreover, combination of these antibodies resulted in stronger inhibition. These results imply that CG-NAP and kendrin provide sites for microtubule nucleation in mammalian centrosome by anchoring γ-TuRC.

微管组装是由γ-微管蛋白环复合物（γ-TuRC）启动的。在酵母中，微管从锚定到纺锤体极体组分SpcllOp的氨基末端的γ-TuRC成核，SpcllOp在羧基末端与Cmd 1 p（钙调蛋白）相互作用。然而，锚定γ-TuRC的哺乳动物蛋白仍有待阐明。一个巨大的卷曲螺旋蛋白CG-NAP（c_entrosome and G_olgi localized PKN_-associated p_rotein）通过羧基端定位于中心体。通过酵母双杂交筛选发现该区域与钙调素相互作用，并且与另一个中心体卷曲螺旋蛋白Kendrin的羧基端区域具有高度同源性。CG-NAP或kendrin的氨基末端区域通过与γ-微管蛋白复合蛋白2（GCP 2）和/或GCP 3结合而与γ-微管蛋白间接结合。此外，内源性CG-NAP和kendrin之间存在免疫共沉淀，并与内源性GCP 2和γ-tubulin形成免疫共沉淀，表明CG-NAP和kendrin在体内与γ-TuRC形成复合物并相互作用。这些蛋白定位于中心体成核的微管星状体的中心。用抗CG-NAP抗体或抗Kendrin抗体预处理中心体，可抑制微管星形细胞的形成，且二者联合作用的抑制作用更强。这些结果表明，CG-NAP和kendrin通过锚定γ-TuRC在哺乳动物中心体中提供微管成核位点。

项目成果

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