Independent activation of endogenous p21-activated protein kinase-3 (PAK3) and JNK by thrombin in CCL39 fibroblasts.
Thrombin, a potent mitogen for CCL39 hamster lung fibroblasts, activates the seven membrane-spanning receptor PAR1. To better understand the signaling pathways controlled by this receptor we analyzed a potential downstream effector, p21-activated protein kinase (PAK). Thrombin and PAR1 agonist peptide, as well as serum and lysophosphatidic acid, were found to stimulate HA-mPAK3 activity in CCL39 cells transfected with a plasmid encoding the epitope-tagged kinase. Similar results were obtained using antibodies developed against the endogenous kinase. PAK3 activation is sensitive to pertussis toxin, but insensitive to LY 294002, an inhibitor of phosphatidylinositol 3'-kinase. Thrombin and serum also activate c-jun amino terminal kinase (JNK). Similar to PAK3 activation, thrombin-stimulated JNK activity is inhibited by pertussis toxin, but not by LY 294002. In a CCL39-derived cell line expressing constitutively active mPAK3 in a tetracyline-dependent manner, induction of PAK activity does not lead to corresponding increases in JNK activity. Our findings indicate that PAK3 is responsive to thrombin and other G protein-coupled receptor systems. Furthermore, our data suggest that in CCL39 cells, JNK activation by thrombin occurs independently of PAK3.
Malcolm
K C
KC
Centre de Biochimie, Centre National de la Recherche Scientifique, Nice, France.
Chambard
J C
JC
Grall
D
D
Pouysségur
J
J
van Obberghen-Schilling
E
E
D002478:Cells, Cultured; D004789:Enzyme Activation; D004790:Enzyme Induction; D005347:Fibroblasts / Q000201:enzymology*; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000502:physiology; D006801:Humans; D048031:JNK Mitogen-Activated Protein Kinases; D048670:MAP Kinase Kinase 4; D020929:Mitogen-Activated Protein Kinase Kinases / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D013012:Sorbitol / Q000494:pharmacology; D013917:Thrombin / Q000494:pharmacology*; D054462:p21-Activated Kinases
D013012:Sorbitol; C515911:PAK3 protein, human; D017346:Protein Serine-Threonine Kinases; D054462:p21-Activated Kinases; D048031:JNK Mitogen-Activated Protein Kinases; D048670:MAP Kinase Kinase 4; D020929:Mitogen-Activated Protein Kinase Kinases; D013917:Thrombin; D019206:GTP-Binding Protein alpha Subunits, Gi-Go