Independent activation of endogenous p21-activated protein kinase-3 (PAK3) and JNK by thrombin in CCL39 fibroblasts

• Published in: Journal of cellular physiology 2000-11, Vol.185 (2), p.235-243
• Main Authors: Malcolm, Kenneth C., Chambard, Jean-Claude, Grall, Dominique, Pouysségur, Jacques, van Obberghen-Schilling, Ellen
• Format: Article
• Language: English
• Subjects: Cells, Cultured; Enzyme Activation; Enzyme Induction; Fibroblasts - enzymology; GTP-Binding Protein alpha Subunits, Gi-Go - physiology; Humans; JNK Mitogen-Activated Protein Kinases; MAP Kinase Kinase 4; Mitogen-Activated Protein Kinase Kinases - metabolism; p21-Activated Kinases; Protein-Serine-Threonine Kinases - metabolism; Sorbitol - pharmacology; Thrombin - pharmacology
• ISSN: 0021-9541; 1097-4652
• DOI: 10.1002/1097-4652(200011)185:2<235::AID-JCP8>3.0.CO;2-D

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. J. Cell. Physiol. 185:235–243, 2000. © 2000 Wiley‐Liss, Inc.