Determination of the critical region of KRAS-induced actin-interacting protein for the interaction with inositol 1,4,5-trisphosphate receptor
Determination of the critical region of KRAS-induced actin-interacting protein for the interaction with inositol 1,4,5-trisphosphate receptor
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DOI：
10.1016/j.bbrc.2011.04.016
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发表时间：
2011-05-06
影响因子：
3.1
通讯作者：
Shirasawa, Senji
中科院分区：
文献类型：
--
作者：
Fujimoto, Takahiro;Machida, Takashi;Shirasawa, Senji
kras诱导的肌动蛋白相互作用蛋白（KRAP）最初被定性为丝状肌动蛋白相互作用蛋白。我们最近发现KRAP是肌醇1,4,5-三磷酸受体（IP3R）的相关分子，对IP3R的亚细胞定位和功能至关重要。然而，KRAP调控IP3R的分子机制尚不清楚。在本报告中，为了确定KRAP蛋白调控IP3R的关键区域，我们生成了几个KRAP突变体，并使用共免疫沉淀和共聚焦成像检测了它们与IP3R的关系。使用缺失突变体的共免疫沉淀显示，KRAP的氨基酸残基1-218而不是1-199与IP3R相互作用，这表明19长度的氨基酸残基（200-218）是与IP3R关联所必需的。这一关键区域在人和小鼠KRAP之间高度保守。在关键区域内，小鼠KRAP中的两个苯丙氨酸残基（Phe202/Phe203）被丙氨酸取代导致与IP3R的关联失败，这表明两个连续的苯丙氨酸残基对于该关联是必不可少的。此外，KRAP敲除稳定的HeLa细胞表现出IP3R不适当的亚细胞定位，其中外源表达全长KRAP可以正常恢复IP3R的亚细胞定位，但不能恢复1-218或1-236突变体的亚细胞定位，这表明残余羧基末端区域也是KRAP-IP3R复合物正确亚细胞定位所必需的。这些结果为进一步了解IP3R调控的分子机制提供了新的思路，并为靶向IP3R的药物开发提供了有效的策略。(C) 2011爱思唯尔公司版权所有。
KRAS-induced actin-interacting protein (KRAP) was originally characterized as a filamentous-actin-interacting protein. We have recently found that KRAP is an associated molecule with inositol 1,4,5-trisphosphate receptor (IP3R) and is critical for the proper subcellular localization and function of IP3R. However, the molecular mechanisms underlying the regulation of IP3R by KRAP remain elusive. In this report, to determine the critical region of KRAP protein for the regulation of IP3R, we generate several mutants of KRAP and examine the association with IP3R using coimmunoprecipitation and confocal imaging assays. Coimmunoprecipitations using the deletion mutants reveal that amino-acid residues 1-218 but not 1-199 of KRAP interact with IP3R, indicating that the 19-length amino-acid residues (200-218) are essential for the association with IP3R. This critical region is highly conserved between human and mouse KRAP. Within the critical region, substitutions of two phenylalanine residues (Phe202/Phe203) in mouse KRAP to alanines result in failure of the association with IP3R, suggesting that the two consecutive phenylalanine residues are indispensable for the association. Moreover, the KRAP-knockdown stable HeLa cells exhibit the inappropriate subcellular localization of IP3R, in which exogenous expression of full-length of KRAP properly restores the subcellular localization of IP3R, but not the 1-218 or 1-236 mutant, indicating that the residual carboxyl-terminal region is also required for the proper subcellular localization of KRAP-IP3R complex. All these results provide insight into the understandings for the molecular mechanisms underlying the regulation of IP3R, and would reveal a potent strategy for the drug development targeting on IP3R. (C) 2011 Elsevier Inc. All rights reserved.