Cathy Tournier

Targeted Deletion of mek5 Causes Early Embryonic Death and Defects in the Extracellular Signal-Regulated Kinase 5/Myocyte Enhancer Factor 2 Cell Survival Pathway

ABSTRACT 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 signal-regulated 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.

Exploring the function of the JNK (c-Jun N-terminal kinase) signalling pathway in physiological and pathological processes to design novel therapeutic strategies

JNK (c-Jun N-terminal kinase) is a member of the MAPK (mitogen-activated protein kinase) family that regulates a range of biological processes implicated in tumorigenesis and neurodegenerative disorders. For example, genetic studies have demonstrated that the removal of specific Jnk genes can reduce neuronal death associated with cerebral ischaemia. As such, targeting JNK signalling constitutes an obvious opportunity for therapeutic intervention. However, MAPK inhibitors can display toxic effects. Consequently, dual-specificity MKKs (MAPK kinases) may represent more attractive targets. In particular, evidence that blocking JNK activation by removing MKK4 offers an effective therapy to treat pathological conditions has started to emerge. MKK4 was the first JNK activator identified. The remaining level of JNK activity in cells lacking MKK4 expression led to the discovery of a second activator of JNK, named MKK7. Distinct phenotypic abnormalities associated with the targeted deletion of Mkk4 and Mkk7 in mice have revealed that MKK4 and MKK7 have non-redundant function in vivo. Further insights into the specific functions of the JNK activators in cancer cells and in neurons will be of critical importance to validate MKK4 and MKK7 as promising drug targets.

The requirement of uncoordinated 51-like kinase 1 (ULK1) and ULK2 in the regulation of autophagy

Autophagy is an evolutionarily conserved physiological process of self-digestion by a cell to adapt to various stresses, including starvation. Its molecular basis involves the concerted activation of proteins encoded by the family of autophagy-related (Atg) genes. The best characterized is the serine/threonine protein kinase Atg1 in yeast which appears to be essential at the early stage of autophagy. In mammals, five Atg1 homologues have been identified as uncoordinated (UNC) 51-like kinase 1 to 4 and STK36. ULK1 and ULK2 are the most closely related members of the family, sharing 78% homology within their protein kinase domains. However, the specific function of ULK1 and ULK2 in mammalian autophagy is not fully understood. Here, we demonstrate that ULK1 and ULK2 are functionally redundant protein kinases required to mediate autophagy under nutrient-deprived conditions in fibroblasts. In contrast, ULK1, but not ULK2, is critical to induce the autophagic response of cerebellar granule neurons (CGN) to low potassium concentration in serum-free conditions. Furthermore, we found that ULK1 has a cytoprotective function in neurons. Together, these results provide strong genetic evidence that ULK1 is an essential component of the autophagic signaling pathway. The ability of ULK2 to compensate for the loss of ULK1 function is cell-type specific.

The regulation of Bax by c-Jun N-terminal protein kinase (JNK) is a prerequisite to the mitochondrial-induced apoptotic pathway.

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 jnk−/− 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.

JNK and PTEN cooperatively control the development of invasive adenocarcinoma of the prostate

The c-Jun NH2-terminal kinase (JNK) signal transduction pathway is implicated in cancer, but the role of JNK in tumorigenesis is poorly understood. Here, we demonstrate that the JNK signaling pathway reduces the development of invasive adenocarcinoma in the phosphatase and tensin homolog (Pten) conditional deletion model of prostate cancer. Mice with JNK deficiency in the prostate epithelium (ΔJnk ΔPten mice) develop androgen-independent metastatic prostate cancer more rapidly than control (ΔPten) mice. Similarly, prevention of JNK activation in the prostate epithelium (ΔMkk4 ΔMkk7 ΔPten mice) causes rapid development of invasive adenocarcinoma. We found that JNK signaling defects cause an androgen-independent expansion of the immature progenitor cell population in the primary tumor. The JNK-deficient progenitor cells display increased proliferation and tumorigenic potential compared with progenitor cells from control prostate tumors. These data demonstrate that the JNK and PTEN signaling pathways can cooperate to regulate the progression of prostate neoplasia to invasive adenocarcinoma.

The 2 Faces of JNK Signaling in Cancer

c-Jun NH2-terminal kinase (JNK) was discovered almost 20 years ago as the protein kinase responsible for phosphorylating c-Jun at Ser-63 and Ser-73. These sites had previously been demonstrated to be essential for the stimulation of c-Jun activity and for cooperation with Ha-ras in oncogenic transformation. This led to the idea that JNK was a positive regulator of cellular transformation. However, the analysis of jnk gene deletion in various mouse models of cancer has produced conflicting findings, with some studies supporting the pro-oncogenic function of JNK and others providing evidence that JNK acts as a tumor suppressor. This review will discuss how these unexpected findings have increased our understanding of the role of JNK signaling in cancer and have provided a source of new working hypotheses.

Selective Regulation of c-jun Gene Expression by Mitogen-Activated Protein Kinases via the 12-O-Tetradecanoylphorbol-13-Acetate- Responsive Element and Myocyte Enhancer Factor 2 Binding Sites

ABSTRACT To further understand how the mitogen-activated protein kinase (MAPK) signaling pathways regulate AP-1 activity, we have elucidated the physiological role of these cascades in the regulation of c-jun gene expression. c-Jun is a crucial component of AP-1 complexes and has been shown in vitro to be a point of integration of numerous signals that can differentially affect its expression as well as its transcriptional activity. Our strategy was based on the use of (i) genetically modified fibroblasts deficient in components of the MAPK cascades and (ii) pharmacological reagents. The results demonstrate that c-Jun NH2-terminal protein kinase (JNK) is essential for a basal level of c-Jun expression and for c-Jun phosphorylation in response to stress. In addition to JNK, p38 MAPK or ERK1/2 and ERK5 are required for mediating UV radiation- or epidermal growth factor (EGF)-induced c-Jun expression, respectively. Further studies indicate that p38 MAPK inhibits the activation of JNK in response to EGF, causing a down-regulation of c-Jun. Overall, these data provide important insights into the mechanisms that ultimately determine the function of c-Jun as a regulator of cell fate.

Targeted Deletion of the Mitogen-Activated Protein Kinase Kinase 4 Gene in the Nervous System Causes Severe Brain Developmental Defects and Premature Death

ABSTRACT The c-Jun NH2-terminal protein kinase (JNK) is a mitogen-activated protein kinase (MAPK) involved in the regulation of various physiological processes. Its activity is increased upon phosphorylation by the MAPK kinases MKK4 and MKK7. The early embryonic death of mice lacking an mkk4 or mkk7 gene has provided genetic evidence that MKK4 and MKK7 have nonredundant functions in vivo. To elucidate the physiological role of MKK4, we generated a novel mouse model in which the mkk4 gene could be specifically deleted in the brain. At birth, the mutant mice were indistinguishable from their control littermates, but they stopped growing a few days later and died prematurely, displaying severe neurological defects. Decreased JNK activity in the absence of MKK4 correlated with impaired phosphorylation of a subset of physiologically relevant JNK substrates and with altered gene expression. These defects resulted in the misalignment of the Purkinje cells in the cerebellum and delayed radial migration in the cerebral cortex. Together, our data demonstrate for the first time that MKK4 is an essential activator of JNK required for the normal development of the brain.

Activation of extracellular signal-regulated protein kinase 5 downregulates FasL upon osmotic stress.

Extracellular signal-regulated protein kinase (ERK) 5 is a mitogen-activated protein kinase (MAPK) that is activated by dual phosphorylation via a unique MAPK/ERK kinase 5, MEK5. The physiological importance of this signaling cascade is underscored by the early embryonic death caused by the targeted deletion of the erk5 or the mek5 genes in mice. Here, we have found that ERK5 is required for mediating the survival of fibroblasts under basal conditions and in response to sorbitol treatment. Increased Fas ligand (FasL) expression acts as a positive feedback loop to enhance apoptosis of ERK5- or MEK5-deficient cells under conditions of osmotic stress. Compared to wild-type cells, erk5−/− and mek5−/− fibroblasts treated with sorbitol display a reduced protein kinase B (PKB) activity associated with increased Forkhead box O3a (Foxo3a) activity. Based on these results, we conclude that the ERK5 signaling pathway promotes cell survival by downregulating FasL expression via a mechanism that implicates PKB-dependent inhibition of Foxo3a downstream of phosphoinositide 3 kinase.

Regulation of neuronal survival by the extracellular signal-regulated protein kinase 5

The extracellular signal-regulated protein kinase 5 (ERK5) is a mitogen-activated protein kinase that phosphorylates and regulates various transcription factors in response to growth factors and extracellular stresses. To address its biological function during the development of the peripheral nervous system (PNS), we have engineered a novel model of sympathetic neurons in which the erk5 gene can be deleted in vitro. Our data provide for the first time genetic evidence that ERK5 is required to mediate the survival response of neurons to nerve growth factor. Increased cell death associated with the loss of ERK5 is caused by elevated expression of the BH3-only members of the Bcl-2 family, Bad and Bim. Further investigation indicated that ERK5 suppresses the transcription of the bad and the bim genes by Ca2+/cAMP response element-binding protein and Forkhead box O3a, respectively. Consistently, we found that the phosphorylation of both p90 ribosomal S6 kinase and protein kinase B is impaired in neurons lacking ERK5. Together these findings reveal a novel signaling mechanism that promotes neuronal survival during the development of the PNS.