Activation of the small GTPase Rac 1 by cGMP-dependent protein kinase
Activation of the small GTPase Rac 1 by cGMP-dependent protein kinase
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DOI：
10.1016/j.cellsig.2004.03.002
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发表时间：
2004-09-01
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;Ye, RD;Browning, DD
环GMP依赖性蛋白激酶（PKG）被广泛认为在多种细胞类型中具有不同的作用。许多研究表明PKG可能通过激活丝裂原活化蛋白激酶（MAPK）家族的信号蛋白来调节细胞功能。在这项研究中，用一氧化氮（NO）刺激HEK-293细胞，发现诱导磷酸化p38 MAPK的快速积累。通过共转染显性负性PKG构建体（G1 α R-GFP）证实PKG参与了这一过程，该构建体能够阻断cGMP诱导的p38 MAPK活化。转染细胞表达显性阴性Rac 1（T17 N）也能够剂量依赖性地阻断cGMP刺激的p38 MAPK激活，从而表明PKG下游这一途径的重要性。GST-PDB亲和沉淀实验表明，用一氧化氮或8-Br-cGMP刺激HEK 293细胞导致Rac 1快速和短暂的激活，其动力学与p38 MAPK磷酸化相似。此外，使用体外激酶测定，发现cGMP也刺激Rac 1效应子Pak 1的活性。用G1 alphaR-GFP转染细胞可完全消除8-Br-cGMP对Rac 1和Pak 1的激活。Rac 1（T17 N）突变体的表达抑制PKG依赖的PAK 1激活，表明Rac 1在该途径中的PAK 1上游发挥作用。免疫荧光实验表明，PKG和Rac 1在膜皱褶和动态膜区域支持功能性相互作用的明确共定位。然而，体外激酶试验表明，Rac 1不是PKG的底物，表明间接激活机制。总之，这些数据证明了一种新的PKG依赖性途径，通过该途径Rac 1/Pak 1途径被激活。此外，我们证明，这一途径是中央的p38 MAPK的激活PKG在这些细胞。(C)2004年爱思唯尔公司All rights reserved.
Cyclic-GMP-dependent protein kinase (PKG) is widely appreciated as having diverse roles in a variety of cell types. Many reports have indicated that PKG might regulate cell function by activating members of the mitogen-activated protein kinase (MAPK) family of signaling proteins. In this study, stimulation of HEK-293 cells with nitric oxide (NO) was found to induce a rapid accumulation of phosphorylated p38 MAPK. The involvement of PKG in this process was confirmed by cotransfection of a dominant negative PKG construct (G1alphaR-GFP), which was able to block cGMP-induced p38 MAPK activation. Transfection of cells to express dominant negative Rac1 (T17N) was also able to dose-dependently block cGMP-stimulated activation of p38 MAPK, thus indicating the importance of this pathway downstream of PKG. GST-PDB affinity-precipitation experiments revealed that stimulation of HEK293 cells with either nitric oxide or 8-Br-cGMP resulted in a rapid and transient activation of Rac1 with similar kinetics to p38 MAPK phosphorylation. Moreover, using in vitro kinase assays it was found that cGMP also stimulated the activity of the Rac1 effector Pak1. The activation of both Rac1 and Pak1 by 8-Br-cGMP was completely abolished by transfection of the cells with G1alphaR-GFP. Expression of the Rac1 (T17N) mutant inhibited PKG-dependent activation of PAK1 indicating that Rac1 functions upstream of PAK1 in this pathway. Immunofluorescence experiments demonstrated clear colocalization of PKG and Rac1 in membrane ruffles and dynamic membrane regions supporting a functional interaction. However, in vitro kinase assays demonstrated that Rac1 is not a substrate for PKG suggesting an indirect activation mechanism. Taken together these data demonstrate a novel PKG-dependent pathway by which the Rac1/Pak1 pathway is activated. Furthermore, we demonstrate that this pathway is central to the activation of p38 MAPK by PKG in these cells. (C) 2004 Elsevier Inc. All rights reserved.