Self-association of the single-KH-domain family members Sam68, GRP33, GLD-1, and Qk1: Role of the KH domain
Self-association of the single-KH-domain family members Sam68, GRP33, GLD-1, and Qk1: Role of the KH domain
复制标题
DOI：
10.1128/mcb.17.10.5707
复制
发表时间：
1997-10-01
影响因子：
5.3
通讯作者：
Richard, S
中科院分区：
文献类型：
--
作者：
Chen, TP;Damaj, BB;Richard, S
Sam68 是不断增长的蛋白质家族的一员，该蛋白质家族包含嵌入在类似于 170 个氨基酸的较大保守结构域中的单个 KH 结构域，该 KH 结构域家族的环 1 和 4 比其他 RH 结构域中的相应环更长，并且包含保守残基。 RH 结构域是参与 RNA 结合的蛋白质基序，通常以多个拷贝的形式存在。在这里，我们通过免疫共沉淀研究证明 Sam68 自关联，并且该关联需要细胞 RNA。删除研究表明 Sam68 KH 结构域环 1 和 4 是自关联所必需的。 Sam68 相互作用也在酿酒酵母中通过双杂交系统观察到。哺乳动物细胞中的原位化学交联研究表明 Sam68 在体内寡聚。这些 Sam68 复合物在体外结合同聚 RNA 以及 p59(fyn) 和磷脂酶 C gamma 1 的 SH3 结构域，证明 Sam68 作为多聚体与 RNA 和信号分子结合。 Sam68 复合物的形成受到 p59(fyn) 的抑制，表明酪氨酸磷酸化调节 Sam68 寡聚化。其他 Sam68 家族成员，包括卤虫 GRP33、秀丽隐杆线虫 GLD-1 和小鼠 Qk1 也发生寡聚化。此外，Sam68、GRP33、GLD-1 和 Qk1 与其他 KH 结构域蛋白（例如 Bicaudal C）相关。这些观察结果表明，Sam68 家族中发现的单个 KH 结构域除了介导蛋白质-RNA 相互作用之外，还介导蛋白质-蛋白质相互作用。
Sam68 is a member of a growing family of proteins that contain a single KH domain embedded in a larger conserved domain of similar to 170 amino acids, Loops 1 and 4 of this KH domain family are longer than the corresponding loops in other RH domains and contain conserved residues. RH domains are protein motifs that are involved in RNA binding and are often present in multiple copies. Here we demonstrate by coimmunoprecipitation studies that Sam68 self-associated and that cellular RNA was required for the association. Deletion studies demonstrated that the Sam68 KH domain loops 1 and 4 were required for self-association. The Sam68 interaction was also observed in Saccharomyces cerevisiae by the two-hybrid system, In situ chemical cross-linking studies in mammalian cells demonstrated that Sam68 oligomerized in vivo. These Sam68 complexes bound homopolymeric RNA and the SH3 domains of p59(fyn) and phospholipase C gamma 1 in vitro, demonstrating that Sam68 associates with RNA and signaling molecules as a multimer. The formation of the Sam68 complex was inhibited by p59(fyn), suggesting that tyrosine phosphorylation regulates Sam68 oligomerization. Other Sam68 family members including Artemia salina GRP33, Caenorhabditis elegans GLD-1, and mouse Qk1 also oligomerized. In addition, Sam68, GRP33, GLD-1, and Qk1 associated,vith other KH domain proteins such as Bicaudal C. These observations indicate that the single KH domain found in the Sam68 family, in addition to mediating protein-RNA interactions, mediates protein-protein interactions.