The kinase domain of MEKK1 induces apoptosis by dysregulation of MAP kinase pathways.
MAP kinase pathways comprise a group of parallel protein phosphorylation cascades, which are involved in signaling triggered by a variety of stimuli. Previous findings suggested that the ERK and the JNK pathways have opposing roles in regulating proliferation and survival or apoptosis and that apoptosis can be promoted by inhibiting the ERK pathway or by activation of the JNK pathway. In order to test this hypothesis and explore whether it can be exploited as a strategy for killing human cancer cells, we used gene transfer experiments with a range of cancer cell lines. We expressed the catalytic fragment of human MEKK1 to activate JNK and the Ras-binding domain (RBD) of Raf-1 to inhibit the Ras-ERK pathway. In addition, we designed several RBD-MEKK1 fusion proteins aiming to simultaneously activate the JNK and block the ERK pathway. We found that the MEKK1 proteins as well as the RBD alone could reduce colony formation in all cell lines. The survival time of MEKK1-expressing cells depended on the cell line. In HeLa cells, survival could be prolonged by inhibition of caspases but not by coexpression of the anti-apoptotic protein Bcl-2. Due to a lower kinase activity the RBD-MEKK1 fusion proteins were less effective in apoptosis induction than the MEKK1 kinase domain alone. Using mutant forms of Ras and Raf-1 we could show that the reduced kinase activity of RBD-MEKK1 fusion proteins was caused by binding to the Ras protein. The expression of lethal doses of MEKK1 resulted in a strong activation of all three major MAP kinase families JNK, ERK, and p38. Blocking these pathways either by coexpressing a dominant negative form of MKK4 or with inhibitors of MEK or p38 failed to inhibit apoptosis. This suggests that MEKK1 induces apoptosis by causing a general deregulation of MAP kinase signaling rather than by the activation of a single pathway.
Boldt
Simone
S
Beatson Institute for Cancer Research, Cancer Research UK, Garscube Estate, Switchback Road, Bearsden, Glasgow G61 1BD, UK. sboldt@beatson.gla.ac.uk
Weidle
Ulrich H
UH
Kolch
Walter
W
D017209:Apoptosis / Q000187:drug effects / Q000235:genetics*; D002455:Cell Division / Q000187:drug effects / Q000502:physiology*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005057:Eukaryotic Cells / Q000187:drug effects / Q000201:enzymology*; D015971:Gene Expression Regulation, Enzymologic / Q000502:physiology; D015972:Gene Expression Regulation, Neoplastic / Q000502:physiology; D049452:Green Fluorescent Proteins; D006801:Humans; D008164:Luminescent Proteins; D048670:MAP Kinase Kinase 4; D048728:MAP Kinase Kinase Kinase 1; D020935:MAP Kinase Signaling System / Q000187:drug effects / Q000502:physiology*; D048055:Mitogen-Activated Protein Kinase 8; D020929:Mitogen-Activated Protein Kinase Kinases / Q000235:genetics / Q000378:metabolism; D020928:Mitogen-Activated Protein Kinases / Q000235:genetics / Q000378:metabolism; D009369:Neoplasms / Q000188:drug therapy / Q000201:enzymology*; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism* / Q000627:therapeutic use; D017434:Protein Structure, Tertiary / Q000502:physiology; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism / Q000627:therapeutic use; D014407:Tumor Cells, Cultured; D018631:ras Proteins / Q000235:genetics / Q000378:metabolism
D004791:Enzyme Inhibitors; D008164:Luminescent Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D017346:Protein Serine-Threonine Kinases; D048055:Mitogen-Activated Protein Kinase 8; D020928:Mitogen-Activated Protein Kinases; D048728:MAP Kinase Kinase Kinase 1; C482576:MAP3K1 protein, human; D048670:MAP Kinase Kinase 4; C482559:MAP2K4 protein, human; D020929:Mitogen-Activated Protein Kinase Kinases; D018631:ras Proteins