Functional analysis of the nucleotide binding domain of membrane-associated guanylate kinases
Functional analysis of the nucleotide binding domain of membrane-associated guanylate kinases
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DOI：
10.1074/jbc.m210165200
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发表时间：
2003-02-28
影响因子：
4.8
通讯作者：
Bredt, DS
中科院分区：
文献类型：
--
作者：
Olsen, O;Bredt, DS
膜相关鸟苷酸激酶 (MAGUK) 调节细胞间接触部位的细胞粘附和信号转导。 MAGUK 由模块化蛋白质-蛋白质相互作用基序组成，包括 L27、PDZ、Src 同源 (SH) 3 和聚集粘附分子和受体的鸟苷酸激酶结构域。遗传分析表明，MAGUK 的致死突变通常发生在鸟苷酸激酶结构域中，表明该结构域的关键作用。在这里，我们探讨了 GMP 与鸟苷酸激酶结构域的结合是否调节 MAGUK 功能。令人惊讶的是，与之前发表的研究相反，我们未能检测到 GMP 与 MAGUK 突触后密度 95 (PSD-95) 和 CASK 的结合。 GMP 中的两个氨基酸残基。 MAGUK 和真正的鸟苷酸激酶之间不同的结合口袋解释了这种结合的缺乏，因为交换这些残基很大程度上阻止了 GMP 与酵母鸟苷酸激酶的结合。相反，这些突变恢复了 GMP 结合，但不恢复对 PSD-95 的催化活性。 PSD-95 鸟苷酸激酶结构域的蛋白配体、鸟苷酸激酶相关蛋白 (GKAP) 和 MAP1A 似乎不与规范的 GMP 结合袋相互作用，并且 GMP 结合不会影响 PSD-95 内的分子内 SH3/鸟苷酸激酶 (GK) 相互作用。这些研究表明 MAGUK 蛋白已失去对 GMP 的亲和力，但可能保留了鸟苷酸激酶结构以适应相关的调节配体。
Membrane-associated guanylate kinases (MAGUKs) regulate cellular adhesion and signal transduction at sites of cell-cell contact. MAGUKs are composed of modular protein-protein interaction motifs including L27, PDZ, Src homology (SH) 3, and guanylate kinase domains that aggregate adhesion molecules and receptors. Genetic analyses reveal that lethal mutations of MAGUKs often occur in the guanylate kinase domain, indicating a critical role for this domain. Here, we explored whether GMP binding to the guanylate kinase domain regulates MAGUK function. Surprisingly, and in contrast to previously published studies, we failed to detect GMP binding to the MAGUKs postsynaptic density-95 (PSD-95) and CASK. Two amino acid residues in the GMP. binding pocket that differ between MAGUKs and authentic guanylate kinase explain this lack of binding, as swapping these residues largely prevent GMP binding to yeast guanylate kinase. Conversely, these mutations restore GMP binding but not catalytic activity to PSD-95. Protein ligands for the PSD-95 guanylate kinase domain, guanylate kinase-associated protein (GKAP) and MAP1A, appear not to interact with the canonical GMP binding pocket, and GMP binding does not influence the intramolecular SH3/guanylate kinase (GK) interaction within PSD-95. These studies indicate that MAGUK proteins have lost affinity for GMP but may have retained the guanylate kinase structure to accommodate a related regulatory ligand.