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Activation of the small GTPase Rac 1 by cGMP-dependent protein kinase

机译：cGMP依赖性蛋白激酶激活小GTPase Rac 1

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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.
机译：依赖环-GMP的蛋白激酶（PKG）在多种细胞类型中具有多种作用，因此广受赞赏。许多报道表明PKG可能通过激活信号蛋白的促分裂原活化蛋白激酶（MAPK）家族成员来调节细胞功能。在这项研究中，发现一氧化氮（NO）刺激HEK-293细胞可诱导磷酸化的p38 MAPK迅速积累。通过共转染显性阴性PKG构建体（G1alphaR-GFP）可以证实PKG参与了这一过程，该构建体能够阻断cGMP诱导的p38 MAPK激活。转染表达显性负性Rac1（T17N）的细胞也能够剂量依赖性地阻断cGMP刺激的p38 MAPK激活，因此表明了该途径在PKG下游的重要性。 GST-PDB亲和沉淀实验表明，用一氧化氮或8-Br-cGMP刺激HEK293细胞会导致Rac1迅速而短暂地激活，其动力学与p38 MAPK磷酸化相似。此外，使用体外激酶测定法发现，cGMP还刺激Rac1效应子Pak1的活性。通过用G1alphaR-GFP转染细胞，完全消除了8-Br-cGMP对Rac1和Pak1的激活。 Rac1（T17N）突变体的表达抑制了PAK1的PKG依赖性激活，表明Rac1在该途径中在PAK1的上游起作用。免疫荧光实验表明，在膜褶皱和支持功能相互作用的动态膜区域中，PKG和Rac1明显共定位。但是，体外激酶测定表明Rac1不是PKG的底物，表明存在间接激活机制。这些数据合在一起证明了Rac1 / Pak1途径被激活的新型PKG依赖性途径。此外，我们证明该途径是这些细胞中PKG激活p38 MAPK的关键。 （C）2004 Elsevier Inc.保留所有权利。

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