The JNKK2-JNK1 fusion protein acts as a constitutively active c-Jun kinasethat stimulates c-Jun transcription activity

c-Jun N-terminal protein kinase (JNK), a member of the mitogen-activated pr otein (MAP) kinase family, regulates gene expression in response to various extracellular stimuli. JNK is activated by JNK-activating kinase (JNKK1 an d JNKK2), a subfamily of the dual specificity MAP kinase kinase (MEK) famil y, through phosphorylation on threonine (Thr) 183 and tyrosine (Tyr) 185 re sidues. The physiological functions of the JNK pathway, however, are not co mpletely understood. A major obstacle is the lack of specific and activated kinase components that: can stimulate the JNK pathway in the absence of an y stimulus. Here we show that fusion of JNK1 to its upstream activator JNKK 2 resulted in its constitutive activation. In HeLa cells, the JNKK2-JNK1 fu sion protein showed significant JNK activity, which was comparable with tha t of JNK1 activated by many stimuli and activators, including EGF, TNF-alph a, anisomycin, UV irradiation, MEKK1, and small GTP binding proteins Rad an d Cdc42Hs. Immunoblotting analysis indicated that JNK1 was phosphorylated b y JNKK2 in the fusion protein on both Thr(183) and Tyr(185) residues. Like JNKK2, the JNKK2-JNK1 fusion protein was highly specific for the JNK pathwa y and did not activate either p38 or ERK2. Transient transfection assays de monstrated that the JNKK2-JNK1 fusion protein was sufficient to stimulate c -Jun transcriptional activity in the absence of any stimulus. Immunofluores cence analysis revealed that the JNKK2-JNK1 fusion protein was predominantl y located in the nucleus of transfected HeLa cells. These results indicate that the JNKK2-JNK1 fusion protein is a constitutively active Jun kinase, w hich will facilitate the investigation of the physiological roles of the JN K pathway.