Conditional expression of MAP kinase phosphatase-2 protects against genotoxic stress-induced apoptosis by binding and selective dephosphorylation of nuclear activated c-jun N-terminal kinase.
MAP Kinase Phosphatase-2 (MKP-2) is a dual specific nuclear phosphatase which is selective for both ERK and JNK, MAP kinases implicated in the regulation of apoptosis in response to genotoxic stress. Here we report the conditional expression of MKP-2 in human embryonic kidney cells 293. We demonstrate that Flag-WT-MKP-2 is able to rescue cells from apoptotic commitment when subjected to UV-C or cisplatin treatment. We establish that upon stimulation all three major MAP kinase families (ERK, JNK and p38 MAP kinases) are activated. However, MKP-2 is surprisingly only able to deactivate JNK in vivo. Furthermore, whilst pre-treatment of cells with either the JNK inhibitor SP600125, or the MEK-1 inhibitor PD98059, also reverses UV-C and cisplatin-induced apoptosis, the anti-apoptotic effect of MKP-2 overexpression is not additive with SP600125 but is with PD098059, suggesting that MKP-2 is involved in specifically terminating JNK activity and not ERK. The inability of MKP-2 to dephosphorylate ERK in vivo is also not due to the inability of Flag-MKP-2 to bind both ERK and JNK; phosphorylated forms of each kinase are co-precipitated with both WT and CI-MKP-2. Immunofluorescence studies however demonstrate that ERK is exclusively cytosolic in origin and not translocated to the nucleus following UV-C and cisplatin treatment whilst JNK is principally nuclear. These studies demonstrate the in vivo specificity of MKP-2 for JNK and not ERK and show that nuclear-targeted JNK is involved in genotoxic stress-induced apoptosis.
Cadalbert
Laurence
L
Strathclyde Institute for Biomedical Sciences, Department of Physiology and Pharmacology, University of Strathclyde, 27 Taylor Street, G4 0NR, Glasgow, Scotland, UK. Laurence.Cadalbert@strath.ac.uk
Sloss
Callum M
CM
Cameron
Pamela
P
Plevin
Robin
R
D000873:Anthracenes / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects / Q000502:physiology* / Q000528:radiation effects; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D002945:Cisplatin / Q000494:pharmacology; D004249:DNA Damage / Q000502:physiology*; D004318:Doxycycline / Q000494:pharmacology; D054637:Dual-Specificity Phosphatases; D005419:Flavonoids / Q000494:pharmacology; D015870:Gene Expression / Q000187:drug effects; D006801:Humans; D048031:JNK Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D019950:Mitogen-Activated Protein Kinase 1 / Q000037:antagonists & inhibitors / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3 / Q000037:antagonists & inhibitors / Q000378:metabolism; D054639:Mitogen-Activated Protein Kinase Phosphatases; D009154:Mutation / Q000235:genetics; D009842:Oligopeptides; D010455:Peptides / Q000235:genetics; D010766:Phosphorylation / Q000187:drug effects / Q000528:radiation effects; D011485:Protein Binding; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D054648:Protein Phosphatase 2; D017027:Protein Tyrosine Phosphatases / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology* / Q000528:radiation effects; D014162:Transfection; D014466:Ultraviolet Rays; D048051:p38 Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism
D000873:Anthracenes; D005419:Flavonoids; D009842:Oligopeptides; D010455:Peptides; D047428:Protein Kinase Inhibitors; C432165:pyrazolanthrone; C085706:FLAG peptide; D048031:JNK Mitogen-Activated Protein Kinases; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D048051:p38 Mitogen-Activated Protein Kinases; D054639:Mitogen-Activated Protein Kinase Phosphatases; D054648:Protein Phosphatase 2; C490583:DUSP4 protein, human; D054637:Dual-Specificity Phosphatases; D017027:Protein Tyrosine Phosphatases; D004318:Doxycycline; D002945:Cisplatin; C093973:2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one