Dominant suppression of inflammation via targeted mutation of the mRNA destabilizing protein tristetraprolin

In myeloid cells, the mRNA-destabilizing protein tristetraprolin (TTP) is induced and extensively phosphorylated in response to LPS. To investigate the role of two specific phosphorylations, at serines 52 and 178, we created a mouse strain in which those residues were replaced by nonphosphorylatable alanine residues. The mutant form of TTP was constitutively degraded by the proteasome and therefore expressed at low levels, yet it functioned as a potent mRNA destabilizing factor and inhibitor of the expression of many inflammatory mediators. Mice expressing only the mutant form of TTP were healthy and fertile, and their systemic inflammatory responses to LPS were strongly attenuated. Adaptive immune responses and protection against infection by Salmonella typhimurium were spared. A single allele encoding the mutant form of TTP was sufficient for enhanced mRNA degradation and underexpression of inflammatory mediators. Therefore, the equilibrium between unphosphorylated and phosphorylated TTP is a critical determinant of the inflammatory response, and manipulation of this equilibrium may be a means of treating inflammatory pathologies

Regulation of the activity and expression of ERK8 by DNA damage

AbstractWe have investigated the agonists that activate transfected extracellular signal-regulated kinase 8 (ERK8) in cells, and have found that the most potent activators are hydrogen peroxide, DNA alkylating and cross-linking agents and the poly (ADP-ribose) polymerase inhibitor KU-0058948. The feature shared by all these agents is that they lead to the accumulation of single strand breaks in DNA, suggesting a role for ERK8 in the response to, or repair of, DNA single strand breaks. The DNA alkylating agent MMS also induced the disappearance of endogenous ERK8 by a proteasome-dependent mechanism

Modification des tRNA: Elongator promeut la formation de métastases dans le cancer du sein

Insight into the role of Elp3 in breast cancer of Elp3 in breast cance

NF-kappaB-induced KIAA1199 promotes survival through EGFR signalling.

Constitutive activation of EGFR- and NF-kappaB-dependent pathways is a hallmark of cancer, yet signalling proteins that connect both oncogenic cascades are poorly characterized. Here we define KIAA1199 as a BCL-3- and p65-dependent gene in transformed keratinocytes. KIAA1199 expression is enhanced on human papillomavirus (HPV) infection and is aberrantly expressed in clinical cases of cervical (pre)neoplastic lesions. Mechanistically, KIAA1199 binds Plexin A2 and protects from Semaphorin 3A-mediated cell death by promoting EGFR stability and signalling. Moreover, KIAA1199 is an EGFR-binding protein and KIAA1199 deficiency impairs EGF-dependent Src, MEK1 and ERK1/2 phosphorylations. Therefore, EGFR stability and signalling to downstream kinases requires KIAA1199. As such, KIAA1199 promotes EGF-mediated epithelial-mesenchymal transition (EMT). Taken together, our data define KIAA1199 as an oncogenic protein induced by HPV infection and constitutive NF-kappaB activity that transmits pro-survival and invasive signals through EGFR signalling

Characterization of the reversible phosphorylation and activation of ERK8

ERK8 (extracellular-signal-regulated protein kinase 8) expressed in Escherichia coli or insect cells was catalytically active and phosphorylated at both residues of the Thr-Glu-Tyr motif. Dephosphorylation of the threonine residue by PP2A (protein serine/threonine phosphatase 2A) decreased ERK8 activity by over 95% in vitro, whereas complete dephosphorylation of the tyrosine residue by PTP1B (protein tyrosine phosphatase 1B) decreased activity by only 15–20%. Wild-type ERK8 expressed in HEK-293 cells was over 100-fold less active than the enzyme expressed in bacteria or insect cells, but activity could be increased by exposure to hydrogen peroxide, by incubation with the protein serine/threonine phosphatase inhibitor okadaic acid, or more weakly by osmotic shock. In unstimulated cells, ERK8 was monophosphorylated at Tyr-177, and exposure to hydrogen peroxide induced the appearance of ERK8 that was dually phosphorylated at both Thr-175 and Tyr-177. IGF-1 (insulin-like growth factor 1), EGF (epidermal growth factor), PMA or anisomycin had little effect on activity. In HEK-293 cells, phosphorylation of the Thr-Glu-Tyr motif of ERK8 was prevented by Ro 318220, a potent inhibitor of ERK8 in vitro. The catalytically inactive mutants ERK8[D154A] and ERK8[K42A] were not phosphorylated in HEK-293 cells or E. coli, whether or not the cells had been incubated with protein phosphatase inhibitors or exposed to hydrogen peroxide. Our results suggest that the activity of ERK8 in transfected HEK-293 cells depends on the relative rates of ERK8 autophosphorylation and dephosphorylation by one or more members of the PPP family of protein serine/threonine phosphatases. The major residue in myelin basic protein phosphorylated by ERK8 (Ser-126) was distinct from that phosphorylated by ERK2 (Thr-97), demonstrating that, although ERK8 is a proline-directed protein kinase, its specificity is distinct from ERK1/ERK2

Phosphorylation of Ewing's sarcoma protein (EWS) and EWS-Fli1 in response to DNA damage

In Ewing\u27s sarcomas, chromosomal translocations cause the N-terminal domain of the EWS (Ewing\u27s sarcoma protein) to fuse with the DNA-binding domains of the Ets (E26 transformation-specific) family of transcription factors. Here we show that EWS and EWS-Fli1 (Friend leukaemia virus integration 1), the fusion most frequently found in Ewing\u27s sarcomas, become phosphorylated at Thr(79) in response to either mitogens or DNA-damaging agents. The much weaker mitogen-induced phosphorylation of EWS is catalysed by the MAPKs (mitogen-activated protein kinases) ERK1 (extracellular signal-regulated kinase 1) and ERK2, whereas the much stronger phosphorylation of EWS induced by the DNA alkylating agent MMS (methyl methanesulphonate) can be catalysed by JNK (c-Jun N-terminal kinase) and at least one other protein kinase distinct from ERK1/ERK2. In contrast, the phosphorylation of EWS-Fli1 induced by MMS was largely mediated by p38alpha/p38beta MAPKs. MMS induced a much stronger phosphorylation of EWS-Fli1 than EWS in heterodimers comprising both proteins

Elp3 drives Wnt-dependent tumor initiation and regeneration in the intestine

Tumor initiation in the intestine can rapidly occur from Lgr5(+) crypt columnar stem cells. Dclk1 is a marker of differentiated Tuft cells and, when coexpressed with Lgr5, also marks intestinal cancer stem cells. Here, we show that Elp3, the catalytic subunit of the Elongator complex, is required for Wnt-driven intestinal tumor initiation and radiation-induced regeneration by maintaining a subpool of Lgr5(+)/Dclk1(+)/Sox9(+) cells. Elp3 deficiency dramatically delayed tumor appearance in Apc-mutated intestinal epithelia and greatly prolonged mice survival without affecting the normal epithelium. Specific ablation of Elp3 in Lgr5(+) cells resulted in marked reduction of polyp formation upon Apc inactivation, in part due to a decreased number of Lgr5(+)/Dclk1(+)/Sox9(+) cells. Mechanistically, Elp3 is induced by Wnt signaling and promotes Sox9 translation, which is needed to maintain the subpool of Lgr5(+)/Dclk1(+) cancer stem cells. Consequently, Elp3 or Sox9 depletion led to similar defects in Dclk1(+) cancer stem cells in ex vivo organoids. Finally, Elp3 deficiency strongly impaired radiation-induced intestinal regeneration, in part because of decreased Sox9 protein levels. Together, our data demonstrate the crucial role of Elp3 in maintaining a subpopulation of Lgr5-derived and Sox9-expressing cells needed to trigger Wnt-driven tumor initiation in the intestine

The Prosurvival IKK-Related Kinase IKK epsilon Integrates LPS and IL17A Signaling Cascades to Promote Wnt-Dependent Tumor Development in the Intestine

Constitutive Wnt signaling promotes intestinal cell proliferation, but signals from the tumor microenvironment are also required to support cancer development. The role that signaling proteins play to establish a tumor microenvironment has not been extensively studied. Therefore, we assessed the role of the proinflammatory Ikk-related kinase Ikke in Wnt-driven tumor development. We found that Ikke was activated in intestinal tumors forming upon loss of the tumor suppressor Apc. Genetic ablation of Ikke in beta-catenin-driven models of intestinal cancer reduced tumor incidence and consequently extended survival. Mechanistically, we attributed the tumor-promoting effects of Ikk epsilon to limited TNF-dependent apoptosis in transformed intestinal epithelial cells. In addition, Ikk epsilon was also required for lipo-polysaccharide (LPS) and IL17A-induced activation of Akt, Mek1/2, Erk1/2, and Msk1. Accordingly, genes encoding proinflammatory cytokines, chemokines, and anti-microbial peptides were downregulated in Ikk epsilon-deficient tissues, subsequently affecting the recruitment of tumor-associated macrophages and IL17A synthesis. Further studies revealed that IL17A synergized with commensal bacteria to trigger Ikk epsilon phosphorylation in transformed intestinal epithelial cells, establishing a positive feedback loop to support tumor development. Therefore, TNF, LPS, and IL17A-dependent signaling pathways converge on Ikk epsilon to promote cell survival and to establish an inflammatory tumor microenvironment in the intestine upon constitutive Wnt activation. (C) 2016 AACR