Functional and structural characterization of allosteric activation of phospholipase Cε by Rap1A.
Functional and structural characterization of allosteric activation of phospholipase Cε by Rap1A.
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RAP1A对磷脂酶Cε的变构激活的功能和结构表征。
DOI：
10.1074/jbc.ra120.015685
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发表时间：
2020-12-04
期刊：
影响因子：
--
通讯作者：
Lyon AM
中科院分区：
文献类型：
--
作者：
Sieng M;Selvia AF;Garland-Kuntz EE;Hopkins JB;Fisher IJ;Marti AT;Lyon AM
磷脂酶 Cε (PLCε) 通过与小型 GTP 酶（包括 Rap1A 和 Ras）直接相互作用，在 G 蛋白偶联受体和受体酪氨酸激酶下游被激活。尽管据报道 Ras 可以变构激活脂肪酶，但尚不清楚 Rap1A 是否具有相同的能力或其分子机制是什么。 Rap1A 响应 β-肾上腺素能受体的刺激而激活 PLCε，将复合物易位至核周膜。由于 PLCε 的 C 端 Ras 关联 (RA2) 结构域被认为是 Rap1A 的主要结合位点，因此我们首先使用纯化的蛋白质证实 RA2 结构域确实对于 Rap1A 的激活至关重要。然而，我们还表明，PLCε pleckstrin 同源 (PH) 结构域和前两个 EF 手 (EF1/2) 是 Rap1A 激活所必需的，并鉴定出 RA2 结构域表面上的疏水残基也是必需的。小角度 X 射线散射表明，Rap1A 结合诱导并稳定了可由 GTPase 激活的 PLCε 变体中的离散构象状态。这些数据与 PLCε 催化活性片段的最新结构一起，提供了第一个证据，表明 Rap1A 以及扩展的 Ras 通过促进和稳定 RA2 结构域和 PLCε 核心之间的相互作用来变构激活脂肪酶。
Phospholipase Cε (PLCε) is activated downstream of G protein–coupled receptors and receptor tyrosine kinases through direct interactions with small GTPases, including Rap1A and Ras. Although Ras has been reported to allosterically activate the lipase, it is not known whether Rap1A has the same ability or what its molecular mechanism might be. Rap1A activates PLCε in response to the stimulation of β-adrenergic receptors, translocating the complex to the perinuclear membrane. Because the C-terminal Ras association (RA2) domain of PLCε was proposed to the primary binding site for Rap1A, we first confirmed using purified proteins that the RA2 domain is indeed essential for activation by Rap1A. However, we also showed that the PLCε pleckstrin homology (PH) domain and first two EF hands (EF1/2) are required for Rap1A activation and identified hydrophobic residues on the surface of the RA2 domain that are also necessary. Small-angle X-ray scattering showed that Rap1A binding induces and stabilizes discrete conformational states in PLCε variants that can be activated by the GTPase. These data, together with the recent structure of a catalytically active fragment of PLCε, provide the first evidence that Rap1A, and by extension Ras, allosterically activate the lipase by promoting and stabilizing interactions between the RA2 domain and the PLCε core.