Enhanced JNK activation by NESK without kinase activity upon caspase-mediated cleavage during apoptosis.
Nck-interacting kinase-like embryo-specific kinase (NESK) is a protein kinase that is predominantly expressed in skeletal muscle during the late stages of mouse embryogenesis. NESK belongs to the germinal center kinase (GCK) family and selectively activates the c-Jun N-terminal kinase (JNK) pathway when overexpressed in cultured cells. Some members of the GCK family have been shown to be proteolytically cleaved and activated during apoptosis. Here, we report that NESK is also proteolytically cleaved during apoptosis. Treatment of NESK-transfected HeLa cells with TNF-alpha in the presence of cycloheximide or with staurosporine induced proteolytic cleavage of NESK. The cleavage of NESK occurred at two sites, generating three fragments: an N-terminal fragment containing a kinase domain, an intermediate fragment and a C-terminal fragment containing a regulatory CNH domain. These two cleavages occurred in a stepwise manner and were dependent on a caspase activity. The cleavage sites were identified as aspartic acid residues at 868 and 1091. The N-terminal fragment had less kinase activity than the full-length NESK and did not activate the JNK pathway. In contrast, the C-terminal fragment activated the JNK pathway more strongly than the full-length NESK and promoted TNF-alpha-induced apoptotic cell death. These results implicate NESK in the JNK pathway-mediated promotion of apoptosis through its C-terminal regulatory domain generated by proteolytic cleavage during apoptosis, in a unique manner different from other GCK family kinases.
Kakinuma
Hisaya
H
Department of Biological Sciences, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, Midori-ku, Yokohama 226-8501, Japan.
Inomata
Hidehiko
H
Kitamura
Naomi
N
D017209:Apoptosis; D020169:Caspases / Q000378:metabolism*; D002460:Cell Line; D003513:Cycloheximide / Q000494:pharmacology; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D048670:MAP Kinase Kinase 4 / Q000378:metabolism*; D009154:Mutation; D010766:Phosphorylation; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D015398:Signal Transduction; D019311:Staurosporine / Q000494:pharmacology; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
D004791:Enzyme Inhibitors; D047908:Intracellular Signaling Peptides and Proteins; D014409:Tumor Necrosis Factor-alpha; D003513:Cycloheximide; C411848:Nik related kinase; D017346:Protein Serine-Threonine Kinases; D048670:MAP Kinase Kinase 4; D020169:Caspases; D019311:Staurosporine