We previously demonstrated that the marine toxin and skin tumor promoter palytoxin activates the stress-activated protein kinase/c-Jun N-terminal kinase (JNK), but not the extracellular signal-regulated kinase (ERK), which is typically activated by mitogenic agents. JNK, ERK, and p38, another stress-activated protein kinase, are members of the mitogen-activated protein (MAP) kinase family of serine/threonine kinases, which coordinate the transmission of various signals through the cell. The Na+,K+-ATPase is the putative palytoxin receptor. Therefore, we hypothesized that the Na+,K+-ATPase inhibitor ouabain might also stimulate signaling pathways that activate MAP kinases. Using HeLa and COS7 cells, we found that, although there are similarities between the protein kinase cascades by which palytoxin and ouabain activate JNK, there are also significant differences between the activation of specific MAP kinases by palytoxin and ouabain. Transient expression of dominant negative mutants indicates that ouabain, like palytoxin, activates JNK through a protein kinase cascade that involves the JNK kinase SEK1 but does not require the GTPase Ras. Palytoxin activates JNK and p38 to a greater extent than ouabain. By contrast, ouabain activates ERK to a greater extent than palytoxin. Ouabain blocked palytoxin-stimulated activation of JNK and p38, but not anisomycin-stimulated activation of these kinases, supporting the conclusion that ouabain and palytoxin bind to the same site on the Na+,K+-ATPase. These results suggest that the Na+,K+-ATPase can differentially mediate the activation of MAP kinases by two diverse ligands, palytoxin and ouabain.
Bibliographical noteFunding Information:
We thank Gary S. Bignami (Hawaii Biotechnology Group, Inc.) for his gift of palytoxin, Dr. Benoit Dérijard (Centre de Biochimie) for his gift of pGEX-3X-ATF-2, Dr. Audrey Minden (Columbia University) for her gift of pSRαHA-JNK1 and pSRαRas(N17), Dr. Daniel Mueller and Dr. Wei Li (University of Minnesota) for their gift of pGEX-2T-c-Jun, Dr. Leonard Zon (Harvard Medical School) for his gift of pEBG-GST-SEK1(K-R), and Dr. Lisa Peterson and Dr. Timothy Church (University of Minnesota), and Dr. Diane Wotta (R & D Systems) for insightful discussions. These studies were supported in part by National Institutes of Health Grant CA72498 and by a Pharmaceutical Research and Manufacturers of America Foundation Research Starter Grant.