Role of Apoptosis Signal-Regulating Kinase in Regulation of the c-Jun N-Terminal Kinase Pathway and Apoptosis in Sympathetic Neurons

Abstract

We have previously shown that nerve growth factor (NGF) withdrawal-induced death requires the activity of the small GTP-binding protein Cdc42 and that overexpression of an active form of Cdc42 is sufficient to mediate neuronal apoptosis via activation of the c-Jun pathway. Recently, a new mitogen-activated protein (MAP) kinase kinase kinase, apoptosis signal-regulating kinase 1 (ASK1) which activates both the c-Jun N-terminal kinase (JNK) and p38 MAP kinase pathways and plays pivotal roles in tumor necrosis factor- and Fas-induced apoptosis, has been identified. Therefore, we investigated the role of ASK1 in neuronal apoptosis by using rat pheochromocytoma (PC12) neuronal cells and primary rat sympathetic neurons (SCGs). Overexpression of ASK1-ΔN, a constitutively active mutant of ASK1, activated JNK and induced apoptosis in differentiated PC12 cells and SCG neurons. Moreover, in differentiated PC12 cells, NGF withdrawal induced a four- to fivefold increase in the activity of endogenous ASK1. Finally, expression of a kinase-inactive ASK1 significantly blocked both NGF withdrawal- and Cdc42-induced death and activation of c-jun. Taken together, these results demonstrate that ASK1 is a crucial element of NGF withdrawal-induced activation of the Cdc42–c-Jun pathway and neuronal apoptosis.

ACKNOWLEDGMENTS

We thank M. Kato and D. Goto for providing modified pTet vectors; A. Hall for the Cdc42 plasmid; and M. Fujii, I. Sato, and J. Miyazaki for the adenovirus vectors. We also thank M. Saitoh and H. Nishitoh for valuable discussions.

This work was supported by Grants-in-Aid for scientific Research from the Ministry of Education, Science and Culture of Japan, and grants provided by the Mochida Memorial Foundation for Medical and Pharmaceutical Research and the Ichiro Kanehara Foundation.

T.K., M.M., and K.T. contributed equally to this work.