Abstract
The c-Jun N-terminal protein kinases (JNKs), also called stress-activated protein kinases, are members of the growing family of serine/threonine kinases in the mitogen-activated protein (MAP) kinase superfamily. Like other MAP kinases, JNKs are activated via phosphorylation on adjacent threonine and tyrosine residues and can be inactivated by a unique family of dual specificity phosphatases, called MAP kinase phosphatases (MKPs). MKPs are encoded by immediate early genes and induced in response to environmental stressors and growth factor stimulation. Two prevalent isoforms of MKP, MKP1 and MKP2, are co-expressed in a wide variety of cell types. In this study, we examined the actions of MKP1 and MKP2 on JNK1 and JNK2. JNK1 phosphorylation and activation was inhibited by expression of both MKP1 and MKP2, although MKP1 selectivity toward JNK1 appeared significantly higher than that of MKP2. In contrast, JNK2 activity was inhibited by either phosphatase to similar degrees. Both MKP1 and MKP2 were highly effective at blocking the activation of the physiological target of JNK activation, the transcription factor c-Jun. In PC12 cells, MKP1 and MKP2 are transcriptionally induced following stimulation by nerve growth factor. In these cells, UV light-evoked JNK activation was reduced by pretreatment with nerve growth factor. Therefore, JNKs may be selective targets of MKP action in certain cells.
Highlights
Mitogen-activated protein (MAP)1 kinases are a unique family of serine/threonine kinases that are activated via reversible phosphorylation and mediate signal transduction for multiple extracellular stimuli
At least two MAP kinase pathways have been extensively characterized: the extracellular signal-regulated protein kinase (ERK) cascade, which is responsible for signal transduction involving growth and differentiation, and the stress-activated protein kinase [1] or c-Jun N-terminal protein kinase (JNK) cascade, which mediates the cellular responses to proinflammatory cytokines and genotoxic stress [2]
To confirm the efficacy of COS-7 cells as a model for MAP kinase phosphatases (MKPs) activity in vivo, we examined the ability of MKP1 and MKP2 to inactivate ERK2 following
Summary
Materials—The anti-ERK2 (C-14) antibody, anti-JNK1 (FL) antibody, and the anti-MEK kinase (C-22) antibody were purchased from Santa Cruz Biotechnology. Other samples containing 100 g of total protein were immunoprecipitated overnight with an anti-JNK1 antibody, and the immunoprecipitate was washed in LiCl and assay buffers as described for the JNK immune complex assay above. These samples were resolved on a 12% SDS-polyacrylamide gel and transferred to Immobilon P membrane and probed with anti-phosphotyrosine antibody (diluted at 1:1000). Jun/Gal Assay—As described previously, COS-7 cells were co-transfected using the calcium phosphate method with the various combinations of the following plasmids: 1 g of pCMV5-MEKK, 5–20 g of pcDNA3-MKP1, 5–20 g of pcDNA3-MKP2, and 10 g of pcDNA3-JNK1. Protein assays were performed on the remaining supernatant by the method of Bradford [31], and all raw data were normalized to light units/g of total protein
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