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The Role of MAPK Pathways in Metastasis

By Robin Arnette
February 2009

Cidlowski introduced Johnson and monitored questions from the audience after the talk. (Photo courtesy of Steve McCaw)

Johnson said, "My lab's future work will involve targeting specific MKKKs or families of MKKKs with small molecules to selectively inhibit stimulus-specific activation of networks in gene expression." (Photo courtesy of Steve McCaw)

The mitogen-activated protein kinase (MAPK) pathways are signal transduction cascades that regulate gene expression, cell growth and development, and apoptosis or cell death. The NIEHS Distinguished Lecture Series on December 9, 2008 brought to NIEHS one of the nation's leading experts on the enzymes that make up the MAPK pathways in mammalian cells, Gary L. Johnson, Ph.D., professor and chair of the Department of Pharmacology at the University of North Carolina School of Medicine.

In his talk, Johnson explored the "Function of MAPK Signaling Networks: From Metastasis to Tissue Stem Cells." A group of principal investigators from the Laboratory of Signal Transduction - John Cidlowski, Ph.D., James Putney Jr., Ph.D., and Perry Blackshear, M.D., D.Phil. - hosted the event.

MAPK pathways are vitally important to living organisms, and Johnson ( Exit NIEHS pointed out that mutations in any of the proteins that comprise these pathways in humans may lead to illnesses such as cancer, cardiovascular disease and autoimmunity. He also explained that in humans there are 11 MAPKs, seven MAPK kinases (MKKs) and 21 MAPK kinase kinases (MKKKs). "One of the main questions I wanted to answer early in my research career was why there were so many MKKKs and fewer MKKs and MAPKs," Johnson said. "The reason is they have different functional protein interaction domains and different motifs, which means these enzymes have different functions depending on the cell type in which they are expressed."

The type of cellular stress also determines which enzymes are expressed. Johnson said that oxidative stress, DNA damage via radiation, or a pharmaceutical compound, for example, activates different kinase pathways. Research using UV radiation or osmotic stress on cells determined that six different MKKKs regulate other kinases within the signaling network.

Johnson's lab used gene targeting to characterize 13 of the 21 MKKKs, and he discussed one of these proteins, MEKK1, in detail. His team made MEKK1 knockouts and discovered that it was the only characterized kinase to date that had E3-ligase activity and was adjacent to a modified cysteine-rich region known as a SWIM domain. Johnson added MEKK1 back into the knockouts, and confocal imaging determined that MEKK1 was most abundant on actin fibers.

Since metastasis requires several steps such as vascularization, growth and proliferation of a primary tumor, MEKK1's localization to actin fibers seemed to corroborate other experimental findings. For instance, the team used a polyoma middle-T mouse model and found that MEKK1 regulated tumor cell invasiveness. Other studies demonstrated that MEKK1 regulated calpain, a caspase-like protease, which regulated two other proteins that are involved in focal adhesion and the association of the cytoskeleton with focal adhesion complexes. "MEKK1 is regulating through Jun kinase and externally regulated kinase (ERK)/calpain activity, and that's regulating the turnover of these focal adhesion complexes," Johnson explained.

Johnson also talked about another MKKK his lab characterized, MEKK4. It was highly expressed in the embryo and the trophoectoderm, the first differentiated tissue from the embryo and trophoblast stem cells. The MEKK4 stem cell knockout phenotypes were similar to those of MEKK1, but when Johnson's team mutated one amino acid in the active site, the new MEKK4 knock-in lacked kinase activity. Further studies indicated that Jun kinase or p38 inhibitors could mimic the up-regulation of a number of the genes that were altered in MEKK4 kinase-inactive trophoblast stem cells. "There are epigenetic changes in the MEKK4 knock-in trophoblast stem cells such as a loss of acetylation of lysine 5 at histone 2A and loss of acetylation of lysine 5 at histone 2B."

Johnson ended his talk by listing four major conclusions from his work with MKKKs:

  • They function as signaling hubs for the selective control of the MAP kinase network
  • They regulate gene expression much differently than just by the activation of a MAPK and phosphorylation of a substrate
  • They regulate the repertoire of required transcription machinery
  • They control epigenetic modification of chromatin

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