Collaboration of JNKs and ERKs in Nerve Growth Factor Regulation of the Neurofilament Light Chain Promoter in PC12 Cells

doi:10.1074/jbc.M107824200

Collaboration of JNKs and ERKs in Nerve Growth Factor Regulation of the Neurofilament Light Chain Promoter in PC12 Cells*

From the Department of Medicine, University of Colorado Health Sciences Center, Denver, Colorado 80262

Abstract

Nerve growth factor (NGF) induces transcription-dependent neural differentiation of PC12 cells, and the ERK family of MAPKs has been implicated as the dominant signal pathway that mediates this response. We employed a neurofilament light chain (NFLC) promoter-luciferase (NFLC-Luc) reporter to define the role of the ERKs as well as additional MAPK pathways in NGF induction of this neural specific gene. Constitutive active forms of c-Raf-1, MEKK1 and MKK6, proximal regulators of the ERKs, JNKs, and p38 MAPKs, respectively, all stimulated NFLC-Luc activity. NFLC-Luc activity stimulated by NGF, however, was partially (∼50%) inhibited by the MEK inhibitor, PD098059, or by co-transfection of kinase-inactive MEK1 but not by the p38 MAPK inhibitor, SB203580, indicating a role for the ERKs, but not the p38 MAPKs, in NGF regulation of the NFLC promoter. Importantly, a gain-of-function MKK7-JNK3 fusion protein stimulated NFLC-Luc and synergized with gain-of-function c-Raf-1 to activate the NFLC promoter. In addition, transfection of kinase-inactive forms of MEK1 and MKK7 produced an additive inhibition of NGF-stimulated NFLC-Luc relative to either inhibitor alone. These findings indicate that the ERK and JNK pathways collaborate downstream of the NGF receptor for regulation of the NFLC promoter. Truncation analysis and electromobility shift assays established the requirement for a cAMP-response element/activating transcription factor-like site in the NFLC promoter that minimally interacts with constitutively expressed cAMP-response element-binding protein and JunD as well as c-Jun which is induced by NGF in an ERK-dependent manner. Cumulatively, these findings indicate that the ERK pathway requires collaboration with the JNK pathway for maximal activation of the NFLC gene in PC12 cells through the integrated control of c-Jun function.

Footnotes

↵* This work was supported by National Institutes of Health Grants GM61718 and DK59756.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵‡ To whom correspondence should be addressed: Division of Renal Medicine, C-281, University of Colorado Health Sciences Center, 4200 E. Ninth Ave., Denver, CO 80262. Tel.: 303-315-6065; Fax: 303-315-4852; E-mail: Lynn.Heasley@UCHSC.edu.
Published, JBC Papers in Press, December 3, 2001, DOI 10.1074/jbc.M107824200
Abbreviations:

MAP

mitogen-activated protein

NGF

nerve growth factor

EGF

epidermal growth factor

IGF-1

insulin-like growth factor-1

NFLC

neurofilament light chain

Luc

luciferase

MAPK

mitogen-activated protein kinase

ERK

extracellular signal-regulated kinase

JNK

c-Jun N-terminal kinase

MKK

MAPK kinase

MEK

MAP/ERK kinase

MEKK

MEK kinase

c-Jun-DBD

c-Jun DNA binding domain

PLCγ

phospholipase Cγ

DTT

dithiothreitol

DMEM

Dulbecco's modified Eagle's medium

PMSF

phenylmethylsulfonyl fluoride

RLU

relative light units

β-Gal

β-galactosidase

GAPDH

glyceraldehyde-3-phosphate dehydrogenase

CRE

cAMP-response element

CREB

CRE-binding protein

ATF

activating transcription factor

βPDGF

platelet-derived growth factor-β
- Received August 14, 2001.
- Revision received October 19, 2001.
The American Society for Biochemistry and Molecular Biology, Inc.