Activation of the Drosophila MLK by Ceramide Reveals TNF-α and Ceramide as Agonists of Mammalian MLK3

Self Cite

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The transcription factor peroxisome proliferator-activated receptor ␥ (PPAR␥) belongs to the family of nuclear hormone receptors and consists of two isotypes, PPAR␥1 and PPAR␥2. Our earlier studies have shown that troglitazone (TZD)-mediated activation of PPAR␥2 in hepatocytes inhibits growth and attenuates cyclin D1 transcription via modulating CREB levels. Because this process of growth inhibition was also associated with an inhibition of ␤-catenin expression at a post-translational level, our aim was to elucidate the mechanism involved. ␤-Catenin is a multifunctional protein, which can regulate cell-cell adhesion by interacting with Ecadherin and other cellular processes via regulating target gene transcription in association with TCF/LEF transcription factors. Two adenomatous polyposis coli (APC)-dependent proteasomal degradation pathways, one involving glycogen synthase kinase 3␤ (GSK3␤) and the other involving p53-Siah-1, degrade excess ␤-catenin in normal cells. Our immunofluorescence and Western blot studies indicated a TZD-dependent decrease in cytoplasmic and membrane-bound ␤-catenin, indicating no increase in its membrane translocation. This was associated with a reduction in E-cadherin expression. PPAR␥2 activation inhibited GSK3␤ kinase activity, and pharmacological inhibition of GSK3␤ activity was unable to restore ␤-catenin expression following PPAR␥2 activation. Additionally, this ␤-catenin degradation pathway was operative in cells, with inactivating mutations of both APC and p53. Inhibition of the proteasomal pathway inhibited PPAR␥2-mediated degradation of ␤-catenin, and incubation with TZD increased ubiquitination of ␤-catenin. We conclude that PPAR␥2-mediated suppression of ␤-catenin levels involves a novel APC/ GSK3␤/p53-independent ubiquitination-mediated proteasomal degradation pathway.

Section: Methodsmentioning

confidence: 99%

Peroxisome Proliferator-activated Receptor γ Activation Can Regulate β-Catenin Levels via a Proteasome-mediated and Adenomatous Polyposis Coli-independent Pathway

Sharma

Pradeep

Wong

et al. 2004

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111

“…Recently, we have shown that TNF-␣ also induces MLK3 activity in Jurkat T Cells (22) and that TNF-␣-induced cell death was inhibited by CEP-11004, a specific mixed lineage kinase inhibitor in PC-12 cells (21). These observations along with our Immunoblotting with anti-GST antibody was done to check the expression of various constructs used in the assay.…”

Section: Fig 2 Endogenous and Recombinant Mlk3 Interact With Akt Anmentioning

confidence: 99%

Negative Regulation of Mixed Lineage Kinase 3 by Protein Kinase B/AKT Leads to Cell Survival

Barthwal¹,

Sathyanarayana²,

Kundu³

et al. 2003

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Mixed lineage kinase 3 (MLK3) is a mitogen-activated protein kinase kinase kinase (MAPKKK) that activates c-jun N-terminal kinase (JNK) and can induce cell death in neurons. By contrast, the activation of phosphatidylinositol 3-kinase and AKT/protein kinase B (PKB) acts to suppress neuronal apoptosis. Here, we report a functional interaction between MLK3 and AKT1/PKB␣. Endogenous MLK3 and AKT1 interact in HepG2 cells, and this interaction is regulated by insulin. The interaction domain maps to the C-terminal half of MLK3 (amino acids 511-847), and this region also contains a putative AKT phosphorylation consensus sequence. Endogenous JNK, MKK7, and MLK3 kinase activities in HepG2 cells are significantly attenuated by insulin treatment, whereas the phosphatidylinositol 3-kinase inhibitors LY294002 and wortmannin reversed the effect. Finally, MLK3-mediated JNK activation is inhibited by AKT1. AKT phosphorylates MLK3 on serine 674 both in vitro and in vivo. Furthermore, the expression of activated AKT1 inhibits MLK3-mediated cell death in a manner dependent on serine 674 phosphorylation. Thus, these data provide the first direct link between MLK3-mediated cell death and its regulation by a cell survival signaling protein, AKT1.The cellular decision to undergo either cell death or cell survival is determined by the integration of multiple survival and death signals. Mixed lineage kinase 3 (MLK3) 1 is a member of a growing family of mixed lineage kinases (1). Recently, it has been shown that overexpression of MLK3 or NGF withdrawal leads to neuronal cell death, which can be prevented by treatment with a small molecule inhibitor of MLKs, CEP-1347 (2). Similarly, CEP-11004, an analog of CEP-1347, has also been shown to prevent neuronal cell death upon NGF withdrawal (3). These results indicate a significant and direct involvement of MLKs in regulating cell death; however, the detailed mechanism by which MLKs are regulated is still unknown.The c-jun-N-terminal kinase/stress-activated protein kinase (JNK/SAPK) is stimulated by proinflammatory cytokines, oxidative stress, heat shock, UV, ␥-irradiation, and by other cellular stresses (4, 5). The signals in stress-activated JNK pathway are transmitted through three core modules: MAP3Ks such as members of the mixed lineage kinases or MEKK members, a MAP2K such as SEK1/MKK4 or MKK7, and MAPK such as JNK family members (4, 5). The activated MAP3K phosphorylates and activates MKK7 or SEK1, which in turn phosphorylates and activates JNK. JNKs phosphorylate several nuclear transcription factors that include ELK1, c-Jun, and ATF2 (4, 5). In several cell types, the activation of JNKs is directly linked to cell death (6 -8). Therefore, one mechanism of cell survival could be to block JNK pathway induction. The activation of phosphatidylinositol 3-kinase (PI3K) correlates with increased cell survival, and this effect is largely mediated through the activation of a serine/threonine kinase, AKT (also known as PKB). PI3K agonists such as insulin and insulin-like growth factor-1 (IGF-1) ...

“…MLK3-induced JNK activation is implicated in apoptosis of neuronal cells in response to trophic factor withdrawal (4 -7). MLK3 also activates JNK in Jurkat T lymphocytes (8) and MCF-7 breast cancer cells (9) in response to tumor necrosis factor ␣ treatment. MLK3 activates the p38 pathway through phosphorylation of MKK3/6 (10), although this activity may be dependent upon the scaffold JIP2 (11,12).…”

mentioning

confidence: 99%

Cdc42 Induces Activation Loop Phosphorylation and Membrane Targeting of Mixed Lineage Kinase 3

Du¹,

Böck²,

Schachter³

et al. 2005

Mixed lineage kinase 3 (MLK3) functions as a mitogen-activated protein kinase kinase kinase to activate multiple mitogen-activated protein kinase pathways. Our current studies demonstrate that lack of MLK3 blocks signaling of activated Cdc42 to c-Jun N-terminal kinase, giving strong support for the idea that Cdc42 is a physiological activator of MLK3. We show herein that Cdc42, in a prenylationdependent manner, targets MLK3 from a perinuclear region to membranes, including the plasma membrane. Cdc42-induced membrane targeting of MLK3 is independent of MLK3 catalytic activity but depends upon an intact Cdc42/Rac-interactive binding motif, consistent with MLK3 membrane translocation being mediated through direct binding of Cdc42. Phosphorylation of the activation loop of MLK3 requires MLK3 catalytic activity and is induced by Cdc42 in a prenylation-independent manner, arguing that Cdc42 binding is sufficient for activation loop autophosphorylation of MLK3. However, membrane targeting is necessary for full activation of MLK3 and maximal signaling to JNK. We previously reported that MLK3 is autoinhibited through an interaction between its N-terminal SH3 domain and a proline-containing sequence found between the leucine zipper and the CRIB motif of MLK3. Thus we propose a model in which GTP-bound Cdc42/Rac binds MLK3 and disrupts SH3-mediated autoinhibition leading to dimerization and activation loop autophosphorylation. Targeting of this partially active MLK3 to membranes likely results in additional phosphorylation events that fully activate MLK3 and its ability to maximally signal through the JNK pathway.