The JNKK2-JNK1 fusion protein acts as a constitutively active c-Jun kinase that stimulates c-Jun transcription activity.
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-alpha, 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 Thr(183) and Tyr(185) 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.
Zheng
C
C
Department of Pathology, University of Alabama at Birmingham, Birmingham, Alabama 35294, USA.
Xiang
J
J
Hunter
T
T
Lin
A
A
D005455:Fluorescent Antibody Technique; D005786:Gene Expression Regulation / Q000235:genetics; D006367:HeLa Cells; D006801:Humans; D048031:JNK Mitogen-Activated Protein Kinases; D048688:MAP Kinase Kinase 7; D020929:Mitogen-Activated Protein Kinase Kinases; D020928:Mitogen-Activated Protein Kinases / Q000235:genetics* / Q000378:metabolism*; D009687:Nuclear Proteins / Q000032:analysis; D010766:Phosphorylation; D011494:Protein Kinases / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000235:genetics* / Q000378:metabolism; D015398:Signal Transduction; D014162:Transfection
D009687:Nuclear Proteins; D011993:Recombinant Fusion Proteins; D011494:Protein Kinases; D048031:JNK Mitogen-Activated Protein Kinases; D020928:Mitogen-Activated Protein Kinases; D048688:MAP Kinase Kinase 7; C482569:MAP2K7 protein, human; D020929:Mitogen-Activated Protein Kinase Kinases