Activation of cGMP-dependent protein kinase by protein kinase C
Activation of cGMP-dependent protein kinase by protein kinase C
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DOI：
10.1074/jbc.m300045200
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发表时间：
2003-05-09
影响因子：
4.8
通讯作者：
Browning, DD
中科院分区：
文献类型：
--
作者：
Hou, YL;Lascola, J;Browning, DD
cGMP依赖性蛋白激酶（PKG）正在成为主流信号转导途径的重要组成部分。通过刺激可溶性鸟苷酸环化酶，一氧化氮诱导的cGMP形成通常被认为是PKG激活的最普遍机制。在本研究中，PKG被发现是佛波醇12-肉豆蔻酸酯13-乙酸酯（PMA）反应性蛋白激酶C（PKC）的靶点。PKG 1 α在用PMA刺激的HEK-293细胞中变得磷酸化，并且在体外使用纯化的组分。PKC依赖的磷酸化被发现激活PKG测量的血管扩张剂刺激的磷蛋白的磷酸化，并在体外激酶测定。尽管PKG 1 α中有11个潜在的PKC底物识别位点，但由于苏氨酸58接近假底物结构域，因此对其进行了检查。针对该区域的磷酸化形式产生的抗体用于证明响应于PMA处理的细胞的磷酸化，其动力学类似于血管扩张剂刺激的磷蛋白磷酸化。该位点的磷酸化模拟突变（T58 E）产生了对cGMP水平更敏感的部分活化PKG。一个磷酸无效突变（T58 A）表明，这个残基是重要的，但不足以激活PKC的PKG。总之，这些发现概述了一种新的信号转导途径，连接PKC刺激与环核苷酸非依赖性激活PKG。
The cGMP-dependent protein kinases (PKG) are emerging as important components of mainstream signal transduction pathways. Nitric oxide-induced cGMP formation by stimulation of soluble guanylate cyclase is generally accepted as being the most widespread mechanism underlying PKG activation. In the present study, PKG was found to be a target for phorbol 12-myristate 13-acetate (PMA)-responsive protein kinase C (PKC). PKG1alpha became phosphorylated in HEK-293 cells stimulated with PMA and also in vitro using purified components. PKC-dependent phosphorylation was found to activate PKG as measured by phosphorylation of vasodilator-stimulated phosphoprotein, and by in vitro kinase assays. Although there are 11 potential PKC substrate recognition sites in PKG1alpha, threonine 58 was examined due to its proximity to the pseudosubstrate domain. Antibodies generated against the phosphorylated form of this region were used to demonstrate phosphorylation in response to PMA treatment of the cells with kinetics similar to vasodilator-stimulated phosphoprotein phosphorylation. A phospho-mimetic mutation at this site (T58E) generated a partially activated PKG that was more sensitive to cGMP levels. A phospho-null mutation (T58A) revealed that this residue is important but not sufficient for PKG activation by PKC. Taken together, these findings outline a novel signal transduction pathway that links PKC stimulation with cyclic nucleotide-independent activation of PKG.