SPRK (also called PTK-1 and MLK-3), a member of the mixed lineage kinase subfamily of (Ser/Thr) protein kinases, encodes an amino-terminal SH 3 domain followed by a kinase catalytic domain, two leucine zippers interrupted by a short spacer, a Rac/Cdc42 binding domain, and a long carboxyl-terminal proline-rich region. We report herein that SPRK activates the stress-activated protein kinases (SAPKs) but not ERK-1 during transient expression in COS cells; the p38 kinase is activated modestly (1.3-2 fold) but consistently. SPRK also activates cotransfected SEK-1/MKK-4, a dual specificity kinase which phosphorylates and activates SAPK. Reciprocally, expression of mutant, inactive SEK-1 inhibits completely the basal and SPRK-activated SAPK activity. Immunoprecipitated recombinant SPRK is able to phosphorylate and activate recombinant SEK-1 in vitro to an extent comparable to that achieved by MEK kinase-1. These results identify SPRK as a candidate upstream activator of the stress-activated protein kinases, acting through the phosphorylation and activation of SEK-1.

α-synuclein dysregulation is a critical aspect of Parkinson's disease pathology. Recent studies have observed that α-synuclein aggregates are cytotoxic to cells in culture and that this toxicity can be spread between cells. However, the molecular mechanisms governing this cytotoxicity and spread are poorly characterized. Recent studies of viruses and bacteria, which achieve their cytoplasmic entry by rupturing intracellular vesicles, have utilized the redistribution of galectin proteins as a tool to measure vesicle rupture by these organisms. Using this approach, we demonstrate that α-synuclein aggregates can induce the rupture of lysosomes following their endocytosis in neuronal cell lines. This rupture can be induced by the addition of α-synuclein aggregates directly into cells as well as by cell-to-cell transfer of α-synuclein. We also observe that lysosomal rupture by α-synuclein induces a cathepsin B dependent increase in reactive oxygen species (ROS) in target cells. Finally, we observe that α-synuclein aggregates can induce inflammasome activation in THP-1 cells. Lysosomal rupture is known to induce mitochondrial dysfunction and inflammation, both of which are well established aspects of Parkinson's disease, thus connecting these aspects of Parkinson's disease to the propagation of α-synuclein pathology in cells.

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) ...

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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.

Mixed lineage kinases (MLKs) are MAPKKK members that activate JNK and reportedly lead to cell death. However, the agonist(s) that regulate MLK activity remain unknown. Here, we demonstrate ceramide as the activator of Drosophila MLK (dMLK) and identify ceramide and TNF-alpha as agonists of mammalian MLK3. dMLK and MLK3 are activated by a ceramide analog and bacterial sphingomyelinase in vivo, whereas a low nanomolar concentration of natural ceramide activates them in vitro. Specific inhibition of dMLK and MLK3 significantly attenuates activation of JNK by ceramide in vivo without affecting ceramide-induced p38 or ERK activation. In addition, TNF-alpha also activates MLK3 and evidently leads to JNK activation in vivo. Thus, the ceramide serves as a common agonist of dMLK and MLK3, and MLK3 contributes to JNK activation induced by TNF-alpha.

Pancreatic ductal adenocarcinoma (PDAC) remains remarkably lethal with a 5-year survival rate of 8%. This is mainly attributed to the late stage of presentation, as well as widespread resistance to conventional therapy. In addition, PDAC tumors are largely nonimmunogenic, and most patients have displayed incomplete responses to cancer immunotherapies. Our group has previously identified TGFb as a crucial repressor of antitumor immune function in PDAC, particularly with respect to cytotoxic T lymphocytes. However, pharmacologic inhibition of TGFb signaling has had limited efficacy in clinical trials, failing to promote a significant antitumor immune response. Hence, in this work, we extend our analysis to identify and circumvent the mechanisms of resistance to TGFb signal inhibition in PDAC. Consistent with our previous observations, adoptive transfer of TGFb-insensitive CD8 þ T cells led to the near complete regression of neoplastic disease in vivo. However, we demonstrate that this cannot be recapitulated via global reduction in TGFb signaling, through either genetic ablation or pharmacologic inhibition of TGFBR1. In fact, tumors with TGFb signal inhibition displayed increased PD-L1 expression and had no observable change in antitumor immunity. Using genetic models of advanced PDAC, we then determined that concomitant inhibition of both TGFb and PD-L1 receptors led to a reduction in the neoplastic phenotype, improving survival and reducing disease-associated morbidity in vivo. Combined, these data strongly suggest that TGFb and PD-L1 pathway inhibitors may synergize in PDAC, and this approach warrants clinical consideration. Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis):

Background: AKT kinases mediate insulin signaling downstream of phosphatidylinositol-3 kinase (PI3K). Results: AKT binds Sirt2 in insulin-responsive cells and Sirt2 inhibition blocks AKT activation, whereas Sirt2 overexpression sensitizes cells to insulin. Conclusion: Sirt2 deacetylase is an essential factor in AKT activation. Significance: Sirt2 modulators could be useful in treatment of diseases involving AKT, such as type 2 diabetes and cancer.