Characterization of Phospholipase Cγ Enzymes with Gain-of-Function Mutations
Characterization of Phospholipase Cγ Enzymes with Gain-of-Function Mutations
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DOI：
10.1074/jbc.m109.019265
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发表时间：
2009-08-21
影响因子：
4.8
通讯作者：
Katan, Matilda
中科院分区：
文献类型：
--
作者：
Everett, Katy L.;Bunney, Tom D.;Katan, Matilda
磷脂酶C γ同工酶（PLC γ 1和PLC γ 2）在调节多种细胞功能中具有关键作用。这两种酶也涉及异常细胞反应的信号传导事件。使用N-乙基-N-亚硝基脲（ENU）诱变，我们最近确定了单点突变的小鼠PLC γ 2，导致自发性炎症和自身免疫。在这里，我们进一步描述了两个功能获得性突变，D993 G和Y 495 C，命名为ALI 5和ALI 14的机制表征。在ALI 5中突变的残基Asp-993是PLC酶催化结构域中的保守残基。PLC γ 1和PLC γ 2与该残基的点突变的分析表明，去除负电荷增强PLC活性响应于EGF刺激或激活的Rac。PLC活性在体外的测量和膜结合的分析表明，ALI 5型突变促进膜相互作用，而不损害底物结合和水解。ALI 14中突变的残基（Tyr-495）位于spPH结构域内。该残基的替换对结构域的折叠没有影响，并且在不增加Rac结合的情况下增强PLC γ 2的Rac活化。重要的是，ALI 14-PLC γ 2和相应的PLC γ 1变体的激活响应于EGF刺激而增强，并且绕过了关键酪氨酸残基磷酸化的需要。ALI 5和ALI 14型突变影响基础活性只有轻微的，但是，它们的组合导致了组成型活性PLC。基于这些数据，我们认为，每个突变可以妥协在失活PLC的自抑制，促进激活过程;此外，ALI 5型突变可以增强激活状态下的膜相互作用。
Phospholipase C gamma isozymes (PLC gamma 1 and PLC gamma 2) have a crucial role in the regulation of a variety of cellular functions. Both enzymes have also been implicated in signaling events underlying aberrant cellular responses. Using N-ethyl-N-nitrosourea (ENU) mutagenesis, we have recently identified single point mutations in murine PLC gamma 2 that lead to spontaneous inflammation and autoimmunity. Here we describe further, mechanistic characterization of two gain-of-function mutations, D993G and Y495C, designated as ALI5 and ALI14. The residue Asp-993, mutated in ALI5, is a conserved residue in the catalytic domain of PLC enzymes. Analysis of PLC gamma 1 and PLC gamma 2 with point mutations of this residue showed that removal of the negative charge enhanced PLC activity in response to EGF stimulation or activation by Rac. Measurements of PLC activity in vitro and analysis of membrane binding have suggested that ALI5-type mutations facilitate membrane interactions without compromising substrate binding and hydrolysis. The residue mutated in ALI14 (Tyr-495) is within the spPH domain. Replacement of this residue had no effect on folding of the domain and enhanced Rac activation of PLC gamma 2 without increasing Rac binding. Importantly, the activation of the ALI14-PLC gamma 2 and corresponding PLC gamma 1 variants was enhanced in response to EGF stimulation and bypassed the requirement for phosphorylation of critical tyrosine residues. ALI5- and ALI14-type mutations affected basal activity only slightly; however, their combination resulted in a constitutively active PLC. Based on these data, we suggest that each mutation could compromise auto-inhibition in the inactive PLC, facilitating the activation process; in addition, ALI5-type mutations could enhance membrane interaction in the activated state.