Activation of the small GTPase Rac1 by cGMP-dependent protein kinase.
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.
Hou
Yali
Y
Department of Biochemistry and Molecular Biology, Medical College of Georgia, 1120 15th Street, CB2510, Augusta, GA 30912-2100, USA.
Ye
Richard D
RD
Browning
Darren D
DD
D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D006152:Cyclic GMP / Q000031:analogs & derivatives* / Q000494:pharmacology; D017869:Cyclic GMP-Dependent Protein Kinases / Q000235:genetics / Q000378:metabolism*; D004789:Enzyme Activation; D006801:Humans; D016296:Mutagenesis; D009569:Nitric Oxide / Q000494:pharmacology; D010766:Phosphorylation / Q000187:drug effects; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D013379:Substrate Specificity; D014162:Transfection; D054462:p21-Activated Kinases; D048051:p38 Mitogen-Activated Protein Kinases / Q000378:metabolism; D020830:rac1 GTP-Binding Protein / Q000235:genetics / Q000378:metabolism*
C016276:8-bromocyclic GMP; D009569:Nitric Oxide; C515903:PAK1 protein, human; D017346:Protein Serine-Threonine Kinases; D054462:p21-Activated Kinases; D017869:Cyclic GMP-Dependent Protein Kinases; D048051:p38 Mitogen-Activated Protein Kinases; D020830:rac1 GTP-Binding Protein; D006152:Cyclic GMP