Loss of phosphatidylinositol 3-phosphate binding by the C-terminal Tiam-1 pleckstrin homology domain prevents in vivo Rac1 activation without affecting membrane targeting
Loss of phosphatidylinositol 3-phosphate binding by the C-terminal Tiam-1 pleckstrin homology domain prevents in vivo Rac1 activation without affecting membrane targeting
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DOI：
10.1074/jbc.m211901200
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发表时间：
2003-03-28
影响因子：
4.8
通讯作者：
Chou, MM
中科院分区：
文献类型：
--
作者：
Baumeister, MA;Martinu, L;Chou, MM
Rho 家族小 GTP 酶的 Dbl 家族鸟嘌呤核苷酸交换因子 (GEF) 始终包含紧随其 Dbl 同源 (DH) 结构域的普莱克斯特林同源 (PH) 结构域。尽管 DH 结构域负责 GEF 活性，但 PH 结构域的作用尚不清楚。我们之前报道过，来自几个 Dbl 家族成员的 PH 结构域以非常低的亲和力（K-d 值在 10 pm 范围内）结合磷酸肌醇。这表明，与其他几个 PH 结构域不同，来自 Dbl 蛋白的 PH 结构域不会作为独立的膜靶向模块发挥作用。为了确定低亲和力磷酸肌醇结合的功能相关性，我们突变了 Tiam-1 的相应 PH 结构域，以消除其与磷脂酰肌醇 3-磷酸的弱特异性结合。我们首先在体外证实，突变损害了分离的 DH/PH 结构域的磷酸肌醇结合，但内在的 GEF 活性不受影响。然后，我们将 PH 结构域突变引入全长 Tiam-1，发现其在体内激活 Rac1 或血清反应因子的能力被废除。免疫荧光研究表明 Tiam-1 的膜靶向基本上不受 C 端 PH 结构域突变的影响。因此，我们的研究表明，C 端 PH 结构域的低亲和力磷脂酰肌醇 3-磷酸结合可能对于 Tiam-1 的体内调节和活性至关重要，但 PH 结构域在不改变膜靶向的情况下发挥其调节作用。相反，我们建议配体与 PH 结构域的结合会诱导膜表面的构象和/或方向变化，这是其相邻 DH 结构域的最大交换活性所必需的。
Dbl family guanine nucleotide exchange factors (GEFs) for Rho family small GTPases invariably contain a pleckstrin homology (PH) domain that immediately follows their Dbl homology (DH) domain. Although the DH domain is responsible for GEF activity, the role of the PH domain is less clear. We previously reported that PH domains from several Dbl family members bind phosphoinositides with very low affinity (K-d values in the 10 pm range). This suggests that, unlike several other PH domains, those from Dbl proteins will not function as independent membrane-targeting modules. To determine the functional relevance of low affinity phosphoinositide binding, we mutated the corresponding PH domain from Tiam-1 to abolish its weak, specific binding to phosphatidylinositol 3-phosphate. We first confirmed in vitro that phosphoinositide binding by the isolated DH/PH domain was impaired by the mutations but that intrinsic GEF activity was unaffected. We then introduced the PH domain mutations into full-length Tiam-1 and found that its ability to activate Rac1 or serum response factor in vivo was abolished. Immunofluorescence studies showed that membrane targeting of Tiam-1 was essentially unaffected by mutations in the C-terminal PH domain. Our studies therefore indicate that low affinity phosphatidylinositol 3-phosphate binding by the C-terminal PH domain may be critical for in vivo regulation and activity of Tiam-1 but that the PH domain exerts its regulatory effects without altering membrane targeting. We suggest instead that ligand binding to the PH domain induces conformational and/or orientational changes at the membrane surface that are required for maximum exchange activity of its adjacent DH domain.