Regulation of protein kinase C βII by its C2 domain
Regulation of protein kinase C βII by its C2 domain
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DOI：
10.1021/bi9718752
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发表时间：
1997-12-16
期刊：
影响因子：
2.9
通讯作者：
Newton, AC
中科院分区：
文献类型：
--
作者：
Edwards, AS;Newton, AC
C2结构域在包括常规蛋白激酶Cs的不同蛋白质组中充当膜靶向模块。这项工作探讨了C2结构域靶向蛋白激酶C膜的机制。分子模型确定了两个高度带电的表面上的C2结构域的蛋白激酶C β II：钙离子结合位点，其中包含5个谷氨酸和一个基本面位于后面的钙离子位点，其中包含7个赖氨酸残基。这两个表面突变，以评估其在Ca2+依赖性膜结合的作用。令人惊讶的是，去除四个正电荷的基本面上没有影响蛋白激酶C的脂质或Ca2+的敏感性，揭示了基本面不提供参与脂质结合的决定因素，也不是定位足够接近膜，以提高非特异性招聘其正电面。相反，在Ca2+结合位点用两个正电荷替换两个负电荷会使蛋白激酶C对Ca2+和阴离子脂质的亲和力降低几个数量级。由这种突变引起的负电性电位的急剧降低在没有Ca 2+的情况下并没有增加蛋白激酶C对酸性膜的亲和力，这表明简单的电荷中和并不能解释Ca 2+如何增加蛋白激酶C对阴离子膜的亲和力。我们的数据表明：（1）C2结构域的膜相互作用表面定位于Ca 2+结合位点，其正表面远离膜定位，以及（2）Ca 2+位点不充当简单的静电开关。
The C2 domain serves as a membrane-targeting module in a diverse group of proteins that includes the conventional protein kinase Cs. This work examines the mechanism by which the C2 domain targets protein kinase C to membranes. Molecular modeling identified two highly-charged surfaces on the C2 domain of protein kinase C beta II: the Ca2+ binding site which contains five aspartates and a basic face positioned behind the Ca2+ site that contains seven lysine residues. Both surfaces were mutated to assess their role in Ca2+-dependent membrane binding. Surprisingly, removal of four positive charges on the basic face had no effect on protein kinase C's lipid or Ca2+ sensitivity, revealing that the basic face does not provide determinants involved in lipid binding, nor is it positioned close enough to the membrane to enhance nonspecific recruitment by its electropositive face. In contrast, replacement of two negative charges with two positive charges in the Ca2+ binding site decreased protein kinase C's affinity both for Ca2+ and for anionic lipids by several orders of magnitude. The dramatic reduction in electronegative potential resulting from this mutation did not increase protein kinase C's affinity for acidic membranes in the absence of Ca2+, revealing that simple charge neutralization does not account for how Ca2+ increases protein kinase C's affinity for anionic membranes. Our data suggest that (1) the membrane interaction surface of the C2 domain is localized to the Ca2+-binding site, with the positive face positioned away from the membrane, and (2) the Ca2+ site does not serve as a simple electrostatic switch.