The JNKK2-JNK1 Fusion Protein Acts As a Constitutively Active c-Jun Kinase That Stimulates c-Jun Transcription Activity

doi:10.1074/jbc.274.41.28966

The JNKK2-JNK1 Fusion Protein Acts As a Constitutively Active c-Jun Kinase That Stimulates c-Jun Transcription Activity*

From the ^‡Department of Pathology and ^§Gene Therapy Center, University of Alabama at Birmingham, Birmingham, Alabama 35294 and the ^¶Molecular Biology and Virology Laboratory, The Salk Institute for Biological Studies, La Jolla, California 92037

Abstract

c-Jun N-terminal protein kinase (JNK), a member of the mitogen-activated protein (MAP) kinase family, regulates gene expression in response to various extracellular stimuli. JNK is activated by JNK-activating kinase (JNKK1 and JNKK2), a subfamily of the dual specificity MAP kinase kinase (MEK) family, through phosphorylation on threonine (Thr) 183 and tyrosine (Tyr) 185 residues. The physiological functions of the JNK pathway, however, are not completely understood. A major obstacle is the lack of specific and activated kinase components that can stimulate the JNK pathway in the absence of any stimulus. Here we show that fusion of JNK1 to its upstream activator JNKK2 resulted in its constitutive activation. In HeLa cells, the JNKK2-JNK1 fusion protein showed significant JNK activity, which was comparable with that of JNK1 activated by many stimuli and activators, including EGF, TNF-α, anisomycin, UV irradiation, MEKK1, and small GTP binding proteins Rac1 and Cdc42Hs. Immunoblotting analysis indicated that JNK1 was phosphorylated by JNKK2 in the fusion protein on both Thr¹⁸³ and Tyr¹⁸⁵ residues. Like JNKK2, the JNKK2-JNK1 fusion protein was highly specific for the JNK pathway and did not activate either p38 or ERK2. Transient transfection assays demonstrated that the JNKK2-JNK1 fusion protein was sufficient to stimulate c-Jun transcriptional activity in the absence of any stimulus. Immunofluorescence analysis revealed that the JNKK2-JNK1 fusion protein was predominantly located in the nucleus of transfected HeLa cells. These results indicate that the JNKK2-JNK1 fusion protein is a constitutively active Jun kinase, which will facilitate the investigation of the physiological roles of the JNK pathway.

Footnotes

↵* This work was supported by National Institutes of Health Grant CA73740 and American Heart Association Scientist Development Grant 9630261N (to A. L.).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. Tel.: 205-975-9225; Fax: 205-934-1775; E-mail: lin@vh.path.uab.edu.
↵2 C. Zheng and A. Lin, unpublished results.
Abbreviations:

JNK

c-Jun N-terminal protein kinase

EGF

epidermal growth factor

TNF

tumor necrosis factor

MAP

mitogen-activated protein

MEK

MAP kinase kinase

JNKK

JNK-activating kinase

MEKK

MAP kinase kinase kinase

ERK

extracellular signal-regulated kinase

GST

glutathioneS-transferase

PBS

phosphate-buffered saline

NES

nuclear export sequence
- Received June 21, 1999.
The American Society for Biochemistry and Molecular Biology, Inc.