Involvement of Alpha-PAK-Interacting Exchange Factor in the PAK1–c-Jun NH₂-Terminal Kinase 1 Activation and Apoptosis Induced by Benzo[a]pyrene

Abstract

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 Hepa1c1c7 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 (α PIX) mRNA and its protein expression were upregulated by B(a)P. While overexpression of an active mutant of α PIX (ΔCH) facilitated B(a)P-induced activation of Cdc42/Rac1, PAK1, and JNK1, overexpression of mutated αPIX (L383R, L384S), which lacks guanine nucleotide exchange factor activity, SH3 domain-deleted αPIX (Δ 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, overexpression of αPIX (Δ CH) and a catalytically active mutant PAK1 (T423E) accelerated B(a)P-induced apoptosis in HeLa cells, whereas αPIX (Δ 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 αPIX (Δ CH)-enhanced apoptosis in cells treated with B(a)P but did not block PAK1/JNK1 activation. Taken together, these results indicate that αPIX plays a crucial role in B(a)P-induced apoptosis through activation of the JNK1 pathway kinases.

ACKNOWLEDGMENTS

We thank Bruce J. Mayer, Department of Genetics and Developmental Biology, University of Connecticut Health Center, Farmington, Yutaka Eguchi and Yoshihide Tsujimoto, Osaka University Graduate School of Medicine, and Takahiro Nagase, Kazusa DNA Research Institute, for providing us with plasmids used in these experiments. Technical support by Research Equipment Center, Hamamatsu University School of Medicine, is acknowledged.

This work is supported by grant-in-aids from the Ministry of Education, Science, Culture, Sports, and Technology of Japan (B,C-2:12218215), the Ministry of Health and Welfare for Comprehensive 10-Year Strategy for Cancer Control, and the Smoking Research Foundation.