Evidence that 12-lipoxygenase product 12-hydroxyeicosatetraenoic acid activates p21-activated kinase

The effect of 12-hydroxyeicosatetraenoic acid (12-HETE), an arachidonic acid metabolite of 12-lipoxygenase, to activate p21^Rac/Cdc42-activated kinase (PAK1) was studied in a Chinese hamster ovary fibroblast cell line overexpressing the rat vascular type-1a angiotensin II receptor (CHO-AT_1a). 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-AT_1a 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.