Involvement of alpha-PAK-interacting exchange factor in the PAK1-c-Jun NH2-terminal kinase 1 activation and apoptosis induced by benzo[a]pyrene

Benzo[a]pyrene [B(a)P], a potent procarcinogen found in combustion products such as diesel exhaust and cigarette smoke, has been recently shown to activate the c-jun NH2-terminal kinase 1 (JNK1) and induce caspase-3-mediated apoptosis in Hepa1cIc7 cells. However, the molecules of the signaling pathway that control the mitogen-activated protein kinase cascades induced by B(a)P and the interaction between those and apoptosis by B(a)P have not been well defined. We report here that B(a)P promoted Cdc42/Rac1, p21-activated kinase 1 (PAK1), and JNK1 activities in 293T and HeLa cells. Moreover, alpha-PAK-interacting exchange factor (alpha PIX) mRNA and its protein expression were upregulated by B(a)P. While overexpression of an active mutant of alpha PIX (Delta CH) facilitated B(a)P-induced activation of Cdc42/Rac1, PAK1, and JNK1, overexpression of mutated alpha PIX (L383R, L384S), which lacks guanine nucleotide exchange factor activity, SH3 domain deleted alpha PIX (Delta SH3), which lacks the ability to bind PAK, kinase-negative PAK1 (K299R), and kinase negative SEK1 (K220A, K224L) inhibited B(a)P-triggered JNK1 activation. Interestingly, over expression of alpha PIX (Delta CH) and a catalytically active mutant PAK1 (T423E) accelerated B(a)P-induced apoptosis in HeLa cells, whereas alpha PIX (Delta SH3), PAK1 (K299R), and SEK 1 (K220A, K224L) inhibited B(a)P-initiated apoptosis. Finally, a preferential caspase inhibitor, Z-Asp-CH2-DCB, strongly blocked the alpha PIX (Delta CH)-enhanced apoptosis in cells treated with B(a)P but did not block PAK1/JNK1 activation. Taken together, these results indicate that alpha PIX plays a crucial role in B(a)P-induced apoptosis through activation of the JNK1 pathway kinases.