The PN2-3 Domain of Centrosomal P4.1-associated Protein Implements a Novel Mechanism for Tubulin Sequestration
The PN2-3 Domain of Centrosomal P4.1-associated Protein Implements a Novel Mechanism for Tubulin Sequestration
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DOI：
10.1074/jbc.m808249200
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发表时间：
2009-03-13
影响因子：
4.8
通讯作者：
Curmi, Patrick A.
中科院分区：
文献类型：
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作者：
Cormier, Anthony;Clement, Marie-Jeanne;Curmi, Patrick A.
微管是参与多种细胞功能（例如有丝分裂、运动或细胞内运输）的细胞骨架成分。在体内，这些由 αβ-微管蛋白制成的聚合物主要从中心体成核，建立相间微管网络，或者在有丝分裂过程中建立有丝分裂纺锤体。中心体 P4.1 相关蛋白（CPAP；也称为 CENPJ）是一种参与中心粒组装的中心体蛋白，对中心体功能很重要。该蛋白含有一个称为 PN2-3 的微管不稳定区域。在这里，我们在分子水平上解密了 PN2-3 的微管去稳定活性，并表明它是由 PN2-3 在不可聚合的 1:1 复合物中螯合微管蛋白引起的。我们还在 PN2-3 序列和微管蛋白表面上绘制了微管蛋白/PN2-3 相互作用图。溶液中游离 PN2-3 的 NMR 和 CD 数据表明，这是一种本质上非结构化的蛋白质，包含 23 个氨基酸残基 α 螺旋。该螺旋嵌入 76 个残基区域，与微管蛋白强烈相互作用。 PN2-3 与已明确表征的微管蛋白特性（即 GTP 酶活性、核苷酸交换、长春花碱诱导的自组装和 stathmin 家族蛋白结合）的干扰，突出显示了当微管蛋白嵌入微管中作为微管蛋白/PN2-3 相互作用区域时位于分子间纵向界面的 β 亚基表面。这些发现将 CPAP 的 PN2-3 片段描述为一种蛋白质，具有不同于 Stathmin 家族蛋白质的前所未有的微管蛋白隔离机制。
Microtubules are cytoskeletal components involved in multiple cell functions such as mitosis, motility, or intracellular traffic. In vivo, these polymers made of alpha beta-tubulin nucleate mostly from the centrosome to establish the interphasic microtubule network or, during mitosis, the mitotic spindle. Centrosomal P4.1-associated protein (CPAP; also named CENPJ) is a centrosomal protein involved in the assembly of centrioles and important for the centrosome function. This protein contains a microtubule-destabilizing region referred to as PN2-3. Here we decrypt the microtubule destabilization activity of PN2-3 at the molecular level and show that it results from the sequestration of tubulin by PN2-3 in a non-polymerizable 1: 1 complex. We also map the tubulin/PN2-3 interaction both on the PN2-3 sequence and on the tubulin surface. NMR and CD data on free PN2-3 in solution show that this is an intrinsically unstructured protein that comprises a 23-amino acid residue alpha-helix. This helix is embedded in a 76-residue region that interacts strongly with tubulin. The interference of PN2-3 with well characterized tubulin properties, namely GTPase activity, nucleotide exchange, vinblastine-induced self-assembly, and stathmin family protein binding, highlights the beta subunit surface located at the intermolecular longitudinal interface when tubulin is embedded in a microtubule as a tubulin/PN2-3 interaction area. These findings characterize the PN2-3 fragment of CPAP as a protein with an unprecedented tubulin sequestering mechanism distinct from that of stathmin family proteins.