Integrated activation of MAP3Ks balances cell fate in response to stress.

In vivo, tissues and organs are exposed to numerous stressors that require cells to respond appropriately for viability and homeostasis. Cells respond to these stressors, which range from UV irradiation, heat shock, chemicals, and changes in osmolality, to oxidative stress and inflammatory cytokines, by activating pathways that protect cells from damage. If the stress is too great, cells commit to undergo apoptosis. Such cell fate decisions involve the stress-mediated activation of mitogen-activated protein kinase (MAPK) networks, ultimately under the control of MAPK kinase kinases, or MAP3Ks. It is the MAP3Ks that coordinate the localization, duration and magnitude of MAPK activation in response to cell stress. A single stressor may activate several MAP3Ks, each of which impacts the balance between survival and apoptotic signaling. In this prospect article, we review the specific MAP3Ks that integrate the physiological response to cell stressors. The interrelationships among different stressors are discussed, with an emphasis on how the balance of signaling through MAP3Ks controls the MAPK response to determine cell fate.

Ablation of MEKK4 kinase activity causes neurulation and skeletal patterning defects in the mouse embryo.

Skeletal disorders and neural tube closure defects represent clinically significant human malformations. The signaling networks regulating normal skeletal patterning and neurulation are largely unknown. Targeted mutation of the active site lysine of MEK kinase 4 (MEKK4) produces a kinase-inactive MEKK4 protein (MEKK4(K1361R)). Embryos homozygous for this mutation die at birth as a result of skeletal malformations and neural tube defects. Hindbrains of exencephalic MEKK4(K1361R) embryos show a striking increase in neuroepithelial cell apoptosis and a dramatic loss of phosphorylation of MKK3 and -6, mitogen-activated protein kinase kinases (MKKs) regulated by MEKK4 in the p38 pathway. Phosphorylation of MAPK-activated protein kinase 2, a p38 substrate, is also inhibited, demonstrating a loss of p38 activity in MEKK4(K1361R) embryos. In contrast, the MEK1/2-extracellular signal-regulated kinase 1 (ERK1)/ERK2 and MKK4-Jun N-terminal protein kinase pathways were unaffected. The p38 pathway has been shown to regulate the phosphorylation and expression of the small heat shock protein HSP27. Compared to the wild type, MEKK4(K1361R) fibroblasts showed significantly reduced phosphorylation of p38 and HSP27, with a corresponding heat shock-induced instability of the actin cytoskeleton. Together, these data demonstrate MEKK4 regulation of p38 and that substrates downstream of p38 control cellular homeostasis. The findings are the first demonstration that MEKK4-regulated p38 activity is critical for neurulation.

Post-prenylation-processing enzymes as new targets in oncogenesis.

RAS and many other oncogenic proteins undergo a complex series of post-translational modifications that are initiated by the addition of an isoprenoid lipid through a process known as prenylation. Following prenylation, these proteins usually undergo endoproteolytic processing by the RCE1 protease and then carboxyl methylation by a unique methyltransferase known as isoprenylcysteine carboxyl methyltransferase (ICMT). Although inhibitors that have been designed to target the prenylation step are now in advanced-stage clinical trials, their utility and efficacy seem to be limited. Recent findings, however, indicate that the inhibition of these post-prenylation-processing steps--particularly that of ICMT-catalysed methylation--might provide a better approach to the control of cancer-cell proliferation.

A small-molecule inhibitor of isoprenylcysteine carboxyl methyltransferase with antitumor activity in cancer cells.

Many key regulatory proteins, including members of the Ras family of GTPases, are modified at their C terminus by a process termed prenylation. This processing is initiated by the addition of an isoprenoid lipid, and the proteins are further modified by a proteolytic event and methylation of the C-terminal prenylcysteine. Although the biological consequences of prenylation have been characterized extensively, the contributions of prenylcysteine methylation to the functions of the modified proteins are not well understood. This reaction is catalyzed by the enzyme isoprenylcysteine carboxyl methyltransferase (Icmt). Recent genetic disruption studies have provided strong evidence that blocking Icmt activity has profound consequences on oncogenic transformation. Here, we report the identification of a selective small-molecule inhibitor of Icmt, 2-[5-(3-methylphenyl)-1-octyl-1H-indol-3-yl]acetamide (cysmethynil). Cysmethynil treatment results in inhibition of cell growth in an Icmt-dependent fashion, demonstrating mechanism-based activity of the compound. Treatment of cancer cells with cysmethynil results in mislocalization of Ras and impaired epidermal growth factor signaling. In a human colon cancer cell line, cysmethynil treatment blocks anchorage-independent growth, and this effect is reversed by overexpression of Icmt. These findings provide a compelling rationale for development of Icmt inhibitors as another approach to anticancer drug development.

Targeting Ras signaling through inhibition of carboxyl methylation: an unexpected property of methotrexate.

The antifolate methotrexate is one of the most successful drugs in cancer chemotherapy. Although its efficacy is widely attributed to a decrease in nucleotide biosynthesis (1), methotrexate is known to increase homocysteine (2), a compound associated with an elevated risk of heart disease, Alzheimer's disease (3), and neural tube defects (4). A potential mechanism for the detrimental effects of homocysteine is cellular hypomethylation from an increase in S-adenosylhomocysteine (5), an inhibitor of methyltransferases including isoprenylcysteine carboxyl methyltransferase (Icmt). Among the substrates of Icmt is the monomeric G protein Ras, a critical component of many signaling pathways that regulate cell growth and differentiation. Because carboxyl methylation of Ras is important for proper plasma membrane localization and function (6), we investigated the role of Icmt in the antiproliferative effect of methotrexate. After methotrexate treatment of DKOB8 cells, Ras methylation is decreased by almost 90%. This hypomethylation is accompanied by a mislocalization of Ras to the cytosol and a 4-fold decrease in the activation of p44 mitogen-activated protein kinase and Akt. Additionally, cells lacking Icmt are highly resistant to methotrexate. Whereas cells expressing wild-type levels of Icmt are inhibited by methotrexate, stable expression of myristoylated H-Ras, which does not require carboxyl methylation for membrane attachment (7), confers resistance to methotrexate. These results suggest that inhibition of Icmt is a critical component of the antiproliferative effect of methotrexate, expanding our understanding of this widely used drug and identifying Icmt as a target for drug discovery.