Novel Features of DAG-Activated PKC Isozymes Reveal a Conserved 3-D Architecture
Novel Features of DAG-Activated PKC Isozymes Reveal a Conserved 3-D Architecture
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DOI：
10.1016/j.jmb.2015.11.001
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发表时间：
2016-01-16
影响因子：
5.6
通讯作者：
Leonard, Thomas K.
中科院分区：
文献类型：
--
作者：
Lucic, Iva;Truebestein, Linda;Leonard, Thomas K.
二酰基甘油 (DAG) 通过与蛋白激酶 C (PKC) 的 Cl 结构域结合来激活其八种传统和新型同工酶。处于部分激活构象的 PKC beta 1 的晶体结构显示了 C1B 结构域如何通过在激酶结构域的 C 端 AGC 延伸中夹住螺旋来调节活性。在这里，我们展示了传统 PKC 和新型 PKC 的整体三维形状是保守的，尽管它们的一级序列中的结构域顺序不同。 C1B 夹中突变体的膜易位表型在所有 DAG 激活的 PKC 中都是一致的，证明了该调节界面的保守性。我们现在确定了一种新的界面，该界面以膜不可接近的状态隔离 PKC beta ll 中的 Cl A 结构域，并将其推广到所有 DAG 激活的 PKC。在传统的 PKC 中，我们发现了其 C2 结构域的一个新元素，该元素还有助于非活性构象的稳定性。我们证明域间连接子在允许和稳定这种状态方面发挥着重要作用。我们提出了一种多步激活机制，其中调节结构域与膜的顺序和协作结合与 DAG 对激酶结构域的变构激活相结合，并且获得完整的催化活性需要 DAG 与 C1B 结构域结合。鉴于形状和分子内结构的保守性，我们认为这种机制对于所有 DAG 激活的 PKC 都是通用的。 (C) 2015 Elsevier Ltd. 保留所有权利。
Diacylglycerol (DAG) activates the eight conventional and novel isozymes of protein kinase C (PKC) by binding to their Cl domains. The crystal structure of PKC beta ll in a partially activated conformation showed how the C1B domain regulates activity by clamping a helix in the C-terminal AGC extension of the kinase domain. Here we show that the global three-dimensional shape of the conventional and novel PKCs is conserved despite differences in the order of the domains in their primary sequences. The membrane translocation phenotypes of mutants in the C1B clamp are consistent across all DAG-activated PKCs, demonstrating conservation of this regulatory interface. We now identify a novel interface that sequesters the Cl A domain in PKC beta ll in a membrane-inaccessible state and we generalize this to all DAG-activated PKCs. In the conventional PKCs, we identify a novel element of their C2 domains that additionally contributes to the stability of the inactive conformation. We demonstrate that the interdomain linkers play important roles in permitting and stabilizing this state. We propose a multi-step activation mechanism in which the sequential and cooperative binding of the regulatory domains to the membrane is coupled to allosteric activation of the kinase domain by DAG and that acquisition of full catalytic activity requires DAG binding to the C1B domain. In light of the conservation of shape and intramolecular architecture, we propose that this mechanism is common to all DAG-activated PKCs. (C) 2015 Elsevier Ltd. All rights reserved.