Requirements for distinct steps of phospholipase Cgamma2 regulation, membrane-raft-dependent targeting and subsequent enzyme activation in B-cell signalling.
Requirements for distinct steps of phospholipase Cgamma2 regulation, membrane-raft-dependent targeting and subsequent enzyme activation in B-cell signalling.
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B 细胞信号传导中磷脂酶 Cgamma2 调节、膜筏依赖性靶向和随后的酶激活的不同步骤的要求。
DOI：
10.1042/bj20021778
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发表时间：
2003
期刊：
影响因子：
--
通讯作者：
M. Katan
中科院分区：
文献类型：
--
作者：
Rosie Rodriguez;M. Matsuda;A. Storey;M. Katan
PLC γ（磷脂酶C γ）的研究已经确定了一些信号所需的调节成分，然而，分子机制和事件之间的关系，导致易位和底物水解的增加还没有得到很好的定义。向许多信号转导子添加膜靶向标签导致组成性激活，表明这些过程可能紧密相连且难以解剖。本研究通过DT 40 B细胞中BCR（B细胞抗原受体）或H2 O2应激的交联对PLC γ 2进行调节，表明膜靶向是导致酶活化和底物水解的进一步变化的单独步骤。此外，我们已经定义了PLC γ 2的不同结构域的作用，并使用一组缺乏与PLC γ 2调节相关的组分的细胞系，参与每个步骤的信号分子。我们已经发现，与非特异性膜靶向不同，只有脂质筏靶向的Lyn-PLC γ 2构建体克服了对衔接蛋白BLNK（B细胞接头）的要求。Lyn-PLC γ 2的稳定表达并不伴随着静息细胞中底物水解的增加，其在刺激之后并且特别需要Syk、Btk、磷酸肌醇3-激酶而不是BLNK的存在和/或活化，如使用缺陷细胞系或特异性抑制剂所建立的。基于特异性酪氨酸残基[Tyr 753-->Phe（Y 753 F）/Y 759 F]和SH 2的突变分析，（Src同源2）结构域（R564 A/R672 A）在Lyn-PLC γ 2的背景下，我们发现Tyr 753/Tyr 759是必需的，而PLC γ 2 SH 2结构域在林恩的瞬时激活中没有重要作用。PLC γ 2，但可用于稳定持续活化中的活化形式。
Studies of PLCgamma (phospholipase Cgamma) have identified a number of regulatory components required for signalling; however, molecular mechanisms and the relationship between events leading to translocation and an increase of substrate hydrolysis have not been well defined. The addition of a membrane-targeting tag to many signal transducers results in constitutive activation, suggesting that these processes could be closely linked and difficult to dissect. The present study of PLCgamma2 regulation by cross-linking of the BCR (B-cell antigen receptor) or H2O2 stress in DT40 B-cells, demonstrated that the membrane targeting is a separate step from further changes that result in enzyme activation and substrate hydrolysis. Furthermore, we have defined the roles of different domains of PLCgamma2 and, using a panel of cell lines deficient in components linked to PLCgamma2 regulation, the involvement of signalling molecules with respect to each of the steps. We have found that only the lipid-raft-targeted Lyn-PLCgamma2 construct, unlike non-specific membrane targeting, overcame the requirement for the adapter protein BLNK (B-cell linker). The stable expression of Lyn-PLCgamma2 was not accompanied by an increase in substrate hydrolysis in resting cells, which followed stimulation and specifically required the presence and/or activation of Syk, Btk, phosphoinositide 3-kinase but not BLNK, as established using deficient cell lines or specific inhibitors. Based on mutational analysis of the specific tyrosine residues [Tyr753-->Phe (Y753F)/Y759F] and SH2 (Src homology 2) domains (R564A/R672A) in the context of Lyn-PLCgamma2, we found that Tyr753/Tyr759 were essential, whereas the PLCgamma2 SH2 domains did not have an important role in the transient activation of Lyn-PLCgamma2 but may serve to stabilize an activated form in sustained activation.
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DOI：
10.1006/excr.2001.5241
发表时间：
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期刊：
Experimental cell research.
影响因子：
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作者：
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发表时间：
2000-06
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DOI：
--
发表时间：
1992
期刊：
The Journal of biological chemistry
影响因子：
--
作者：
Wahl,MI;Jones,GA;Nishibe,S;Rhee,SG;Carpenter,G
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