To elucidate the physiological significance of MEK5 in vivo, we have examined the effect of mek5 gene elimination in mice. Heterozygous mice appear to be healthy and were fertile. However, mek5 ؊/؊ embryos die at approximately embryonic day 10.5 (E10.5). The phenotype of the mek5 ؊/؊ embryos includes abnormal cardiac development as well as a marked decrease in proliferation and an increase in apoptosis in the heart, head, and dorsal regions of the mutant embryos. The absence of MEK5 does not affect cell cycle progression but sensitizes mouse embryonic fibroblasts (MEFs) to the ability of sorbitol to enhance caspase 3 activity. Further studies with mek5 ؊/؊ MEFs indicate that MEK5 is required for mediating extracellular signalregulated kinase 5 (ERK5) activation and for the regulation of the transcriptional activity of myocyte enhancer factor 2. Overall, this is the first study to rigorously establish the role of MEK5 in vivo as an activator of ERK5 and as an essential regulator of cell survival that is required for normal embryonic development.The mitogen-activated protein kinase (MAPK) cascades constitute a complex network of signaling pathways that are involved in the regulation of numerous cell functions (9). They consist of the sequential activation of protein kinases that include MAPKs, MAPK/extracellular signal-regulated kinase (ERK) kinases (MEKs or MKKs), and MEK kinases (MEKKs) (9). MAPKs are activated by dual phosphorylation on threonine (T) and tyrosine (Y) residues within a T-X-Y motif by MEKs. MEKs are activated by MEKKs. Two main mechanisms have been proposed to ensure specific transmission of the signals from upstream kinases to MAPKs (38, 40): (i) scaffold proteins that assemble the different components of a cascade; (ii) physical interactions between the components of a cascade. Both mechanisms may operate in parallel and allow different responses of the same MAPK signaling pathways to different stimuli.At least four MAPK subfamilies have been identified: ERK1/2, ERK5, c-Jun NH 2 -terminal protein kinases (JNKs), and p38 MAPKs. MAPK activators include MEK1 and MEK2 for ERK1/2, MEK5 for ERK5, MKK4 and MKK7 for JNKs, and MKK3 and MKK6 for p38 MAPKs (9). Targeted deletion of the mapk and mek/mkk genes has contributed substantially to our increased understanding of the physiological role of these pathways in development and pathogenesis. In particular, the recent elimination of the erk5 gene in mice has provided genetic evidence that ERK5 is required for normal cardiac development (31,34,42). ERK5, also known as big MAPK, is almost twice the size (815 amino acids) of the other MAPKs (45). Its unique COOH-terminal tail contains a myocyte enhancer factor 2 (MEF2)-interacting domain and a potent transcriptional activation domain (12). The ERK5 catalytic NH 2 -terminal domain is 50% identical to ERK2. The activity of a number of transcription factors has been shown to be regulated by ERK5, including MEF2, c-Fos and Fra-1, Sap1, c-Myc, and NF-B (6,11,13,15,28,37). In vitro, the ERK5 signaling pathway has...

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The signaling mechanism by which JNK affects mitochondria is critical to initiate apoptosis. Here we show that the absence of JNK provides a partial resistance to the toxic effect of the heavy metal cadmium. Both wild type and jnkÀ/À fibroblasts undergoing death exhibit cytosolic cytochrome c but, unlike wild type cells, the JNK-deficient fibroblasts do not display increased caspase activity and DNA fragmentation. The absence of apoptotic death correlates with a specific defect in activation of Bax. We conclude that JNK-dependent regulation of Bax is essential to mediate the apoptotic release of cytochrome c regardless of Bid and Bim activation.

Axl is a receptor tyrosine kinase (RTK) upregulated in various tumors including cutaneous squamous cell carcinoma (SCC). Axl expression correlates with poor prognosis and induction of epithelial-mesenchymal transition (EMT), hence we hypothesized that Axl is involved in the disruption of cell-cell adhesion to allow invasion and chemotherapy resistance of the cancer stem cell population. Cutaneous SCC cell lines with stable knockdown of Axl were generated using retroviral vectors. Axl depletion altered expression of intercellular junction molecules increasing cell-cell adhesion with downregulation of Wnt and TGFβR signaling. Furthermore, Axl expression correlated with the expression of putative cancer stem cell markers, CD44 and ALDH1, increased resistance to chemotherapy drugs, enhanced sphere formation ability and expression of EMT features by cancer stem cells. Axl depletion resulted in loss of tumor formation in an in vivo zebrafish xenograft model. In conclusion, these data suggest that abrogation of Axl results in loss of cancer stem cell properties indicating a role for Axl as a therapeutic target in chemotherapy-resistant cancer.