The effect of 12-hydroxyeicosatetraenoic acid (12-HETE), an arachidonic acid metabolite of 12-lipoxygenase, to activate p21Rac/Cdc42-activated kinase (PAK1) was studied in a Chinese hamster ovary fibroblast cell line overexpressing the rat vascular type-1a angiotensin II receptor (CHO-AT1a). 12-HETE (0.1 μM) treatment induced a time-dependent activation of PAK1, with a peak effect at 10 min (335±16% of control; n = 3, P < 0.001). The stimulatory effect of 12-HETE on PAK1 activity was dose-dependent, with the maximal activation at 0.01 μM (350±15% of control; n = 3, P < 0.001). A PAK1 fragment encoding the Cdc42/Rac binding domain (amino acid residues 67-150 of hPAK1 termed PBD), was transfected into CHO-AT1a cells. PBD transfection markedly reduced 12-HETE-induced PAK1 activation. Furthermore, transfection of dominant negative Cdc42 and Rac1 inhibited 12-HETE-induced PAK1, strongly suggesting that Cdc42 and Rac1 are the upstream activators of 12-HETE-induced PAK1 activation. Low concentrations (1.5 μM) of LY294002, a highly specific inhibitor of phosphoinositide 3-kinase (PI-3K), abolished 12-HETE-induced PAK1 activation, suggesting that PI-3K activation is upstream of 12-HETE-induced PAK1 activation. Transfection of dominant negative PAK1 blocked 12-HETE-induced PAK1, cJun N-terminal kinase (JNK1) and extracellular-signal-regulated kinase (ERK) activity, while transfection of constitutively active PAK1 stimulated PAK1, JNK1 and ERK activity, suggesting that PAK1 is an upstream activator of 12-HETE-induced JNK1 and ERK activation in these cells. We conclude that 12-HETE can activate Cdc42, Rac1 and PI-3K, which then participate as upstream signalling molecules for PAK1 and JNK1 activation.
Evidence that 12-lipoxygenase product 12-hydroxyeicosatetraenoic acid activates p21-activated kinase
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Yeshao WEN, Jiali GU, Ulla G. KNAUS, Lisa THOMAS, Noe GONZALES, Jerry L. NADLER; Evidence that 12-lipoxygenase product 12-hydroxyeicosatetraenoic acid activates p21-activated kinase. Biochem J 15 July 2000; 349 (2): 481–487. doi: https://doi.org/10.1042/bj3490481
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