In vitro kinase assay Pim1/GBP1
Ontology highlight
ABSTRACT:
INSTRUMENT(S): Orbitrap Fusion
ORGANISM(S): Homo Sapiens (human)
SUBMITTER: 
Fabian Wendt
LAB HEAD: Bernd Wollscheid
PROVIDER: PXD030010 | Pride | 2023-09-26
REPOSITORIES: Pride
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| Phospho__STY_Sites.txt | Txt | |||
| checksum.txt | Txt | |||
| wendtf_Z2105_012.raw | Raw | |||
| wendtf_Z2105_013.raw | Raw | |||
| wendtf_Z2105_014.raw | Raw |
Items per page: 5 1 - 5 of 8 |
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Project description:Pathogen virulence factors interfere with various cellular activities, a disruption that can trigger innate immune responses. Interferon-gamma (IFNγ)-inducible antimicrobial factors, such as the guanylate binding proteins (GBPs), promote cell-intrinsic defense against intracellular pathogens by directly attacking intracellular pathogens and by committing the infected host cell to programmed cell death. GBPs are large GTPases that traffic to pathogens in the cytosol or that reside within membrane-bound compartments and restrict microbial growth. We discovered that ectopic expression of the prototypical IFNγ-inducible GTPase, Guanylate Binding Protein 1 (GBP1), in the absence of IFNγ kills human macrophages, accompanied by fragmentation of the Golgi apparatus. Exposure to IFNγ improved host cell survival via the activity of PIM1 kinase. PIM1 phosphorylates GBP1 and imposes its sequestration by 14-3-3σ to prevent membrane association of GBP1. Analysis by cryo-EM showed that a 14-3-3σ-dimer trapped GBP1 in an inactive monomeric state. This control mechanism becomes evident during Toxoplasma gondii infection, where the effector protein TgIST interferes with IFNγ-signaling and thereby unintentionally depletes the short-lived kinase PIM1. This, in turn, increases GBP1-driven control of the pathogen. Uninfected bystander cells are spared during inflammation, while the infected cells restrain the pathogen in a rapid GBP1-dependent manner. Our work establishes PIM1 as a bait for pathogen virulence factors, thus safeguarding the integrity of IFNγ-signaling. This work presents a new paradigm of IFNγ-dependent protection of uninfected cells against self-inflicted innate immune damage.
2023-07-26 | PXD029463 | Pride
Project description:Background: PIM1 is a constitutively active serine-threonine kinase regulating cell survival and proliferation. Increased PIM1 expression has been correlated with cancer metastasis by facilitating migration and anti-adhesion. Endothelial cells play a pivotal role in these processes by contributing a barrier to the blood stream. Here, we investigated whether PIM1 regulates mouse aortic endothelial cell (MAEC) monolayer integrity. Methods: Pim1-/-MAEC were isolated from Pim1 knockout mice and used in trypsinization-, wound closure assays, electrical cell-substrate sensing, immunostaining, cDNA transfection and as RNA source for microarray analysis. Results: Pim1-/-MAEC displayed decreased migration, slowed cell detachment and increased electrical resistance across the endothelial monolayer. Reintroduction of Pim1- cDNA into Pim1-/-MAEC significantly restored wildtype adhesive characteristics. Pim1-/--MAEC displayed enhanced focal adhesion and adherens junction structures containing vinculin and M-NM-2-catenin, respectively. Junctional molecules such as Cadherin 13 and matrix components such as Collagen 6a3 were highly upregulated in Pim1-/- cells. Intriguingly, extracellular matrix deposited by Pim1-/- cells alone was sufficient to induce the hyperadhesive phenotype in wildtype endothelial cells. Conclusion: Loss of Pim1 induces a strong adhesive phenotype by enhancing endothelial cell-cell and cell-matrix adhesion by the deposition of a specific extracellular matrix. Targeting PIM1 function therefore might be important to promote endothelial barrier integrity. Pim-1-/- mouse aortic endothelial cells were compared to wildtype cells
2012-10-29 | E-GEOD-32059 | biostudies-arrayexpress
Project description:We previously identified 14-3-3 ζ S64 as a substrate of PIM1 in an in vivo chemical genetic screen. To verify this phosphorylation event and determine whether S64 is the predominant phosphorylation site, we performed in vitro phosphorylation of 14-3-3 ζ by PIM1.
2021-11-25 | PXD023623 | Pride
Project description:It has been estimated that about 11% of cellular genes present a functional E box to which MYC can associate on the genome. MYC silencing demonstrated that MYC recruits PIM1 at specific MYC binding sites and confocal microscopy following growth factor treatment, showed an elevated degree of nuclear co-localization of PIM1 with nascent transcripts and with MYC suggesting that PIM1 is recruited by MYC to a large number of sites. To understand the actual extension of PIM1 and MYC co-operation in gene transcription we performed expression profile analysis of 293 cells silenced either for MYC or PIM1 at 120 minutes after serum treatment. MYC silencing affected the expression of 1026 genes of which 818 were up-regulated and 208 were down-regulated. Comparison of genes regulated by MYC with those regulated by PIM1, by RNAi silencing, showed that PIM1 contributes to the regulation of 207 genes out of the 1026 MYC-regulated genes. Thus, a subset of 20% of MYC-regulated genes, are also regulated by PIM1. The co-regulated includes genes involved in cell metabolism, protein synthesis, cycle progression, and oncogenesis. Interestingly, a large number of genes are transcriptional factors, which suggests that PIM1 participates in MYC-dependent regulatory networks. Experiment Overall Design: The gene expression analysis was performed by hybridizing RNA samples to the Whole Human Genome Oligo Microarray from Agilent Technologies. Samples were obtained from 293 cells, treated with serum for 120 minutes, expressing either two independent MYC shRNA (shMYC#1 and shMYC#2) or their relative scrambled shRNA (shsM#1 and shsM#2) for MYC expression analysis; two independent PIM1 shRNA (shPIM1#1 and shPIM1#2) or their relative scrambled shRNA (shsP#1 and shsP#2) for PIM1 expression analysis.
2008-06-15 | E-GEOD-7960 | biostudies-arrayexpress
Project description:It has been estimated that about 11% of cellular genes present a functional E box to which MYC can associate on the genome. MYC silencing demonstrated that MYC recruits PIM1 at specific MYC binding sites and confocal microscopy following growth factor treatment, showed an elevated degree of nuclear co-localization of PIM1 with nascent transcripts and with MYC suggesting that PIM1 is recruited by MYC to a large number of sites. To understand the actual extension of PIM1 and MYC co-operation in gene transcription we performed expression profile analysis of 293 cells silenced either for MYC or PIM1 at 120 minutes after serum treatment. MYC silencing affected the expression of 1026 genes of which 818 were up-regulated and 208 were down-regulated. Comparison of genes regulated by MYC with those regulated by PIM1, by RNAi silencing, showed that PIM1 contributes to the regulation of 207 genes out of the 1026 MYC-regulated genes. Thus, a subset of 20% of MYC-regulated genes, are also regulated by PIM1. The co-regulated includes genes involved in cell metabolism, protein synthesis, cycle progression, and oncogenesis. Interestingly, a large number of genes are transcriptional factors, which suggests that PIM1 participates in MYC-dependent regulatory networks. Keywords: expression profiles
2007-07-31 | GSE7960 | GEO
Project description:Background: PIM1 is a constitutively active serine-threonine kinase regulating cell survival and proliferation. Increased PIM1 expression has been correlated with cancer metastasis by facilitating migration and anti-adhesion. Endothelial cells play a pivotal role in these processes by contributing a barrier to the blood stream. Here, we investigated whether PIM1 regulates mouse aortic endothelial cell (MAEC) monolayer integrity. Methods: Pim1-/-MAEC were isolated from Pim1 knockout mice and used in trypsinization-, wound closure assays, electrical cell-substrate sensing, immunostaining, cDNA transfection and as RNA source for microarray analysis. Results: Pim1-/-MAEC displayed decreased migration, slowed cell detachment and increased electrical resistance across the endothelial monolayer. Reintroduction of Pim1- cDNA into Pim1-/-MAEC significantly restored wildtype adhesive characteristics. Pim1-/--MAEC displayed enhanced focal adhesion and adherens junction structures containing vinculin and β-catenin, respectively. Junctional molecules such as Cadherin 13 and matrix components such as Collagen 6a3 were highly upregulated in Pim1-/- cells. Intriguingly, extracellular matrix deposited by Pim1-/- cells alone was sufficient to induce the hyperadhesive phenotype in wildtype endothelial cells. Conclusion: Loss of Pim1 induces a strong adhesive phenotype by enhancing endothelial cell-cell and cell-matrix adhesion by the deposition of a specific extracellular matrix. Targeting PIM1 function therefore might be important to promote endothelial barrier integrity.
2012-10-29 | GSE32059 | GEO
Project description:The Pim (proviral integration site for Moloney murine leukemia virus) proteins form a serine threonine kinase family that regulates cell proliferation, migration and cell survival. Here we demonstrate for the first time that a Pim1 kinase plays an essential role in antiviral innate immune responses. Specifically, our in vivo, in vitro and RNA-sequencing analyses showed that Pim1 was quickly upregulated after Toll-like receptor (TLR) stimulation in a NF-kB-dependent manner and then promoted IFN-b production by forming a cell-surface complex composed of TRIF-signaling molecules and IRF3 that promoted IRF3 phosphorylation, nuclear translocation, and IFN-b production. As shown by Pim1 knockdown and knockout, Pim1 was essential for this role but its kinase activity was not involved. Pim1-deficiency increased the susceptibility of mice to poly I:C-induced sepsis. Our study uncovers a previously unrecognized role for Pim1 in antiviral innate immune responses, thus providing a new target for controlling viral infection.
2022-12-14 | GSE195582 | GEO
Project description:PIM1 is constitutively active and involved in various cellular processes, including cell survival, proliferation, and differentiation by phosphorylating a wide range of substrates. We investigated whether PIM1 is involved in the pathogenesis of ulcerative colitis (UC).We performed RNA sequencing analysis in intestinal epithelial cells (IECs) from PIM1-deficient mice and WT mice to study the molecular mechanisms through which PIM1 regulates goblet cell numbers.
2024-07-30 | GSE234120 | GEO
Project description:PIM1 is constitutively active and involved in various cellular processes, including cell survival, proliferation, and differentiation by phosphorylating a wide range of substrates. We investigated whether PIM1 is involved in the pathogenesis of ulcerative colitis (UC).We performed RNA sequencing analysis in Caco2 cells infected with vector or PIM1 to study the molecular mechanisms through which PIM1 regulates goblet cell numbers.
2024-07-30 | GSE234122 | GEO
Project description:Background: In previous studies we showed that IL-27 shares several effects with IFN-γ in human cancer cells. To identify novel extracellular mediators, potentially involved in epithelial ovarian cancer (EOC) biology, we analyzed the effect of IL-27 or IFN-γ on the secretome of EOC cells in culture. Methods: The secretome of cytokine-treated or untreated SKOV3 cells was analyzed by nano-UHPLC-MS/MS and eluting peptides by an Orbitrap Fusion Tribrid mass spectrometer. Extracellular GBP1 was studied by ELISA, immunoprecipitation, GTP-agarose pull-down, and Western blotting. GBP and STAT proteins were analysed on cell lysates by Western blot. GBP1 was transfected using lipofectamine reagent and cell viability measured by MTT assay. Results: IL-27 and IFN-γ modulate the extracellular release of a limited fraction of proteins that were also induced in the whole cell. Among these proteins we focused our attention on GBP1, a guanylate-binding protein and GTPase, which is a mediator of several biological activities of IFNs. Interestingly GBP1, 2, and 5 were induced by cytokine treatment in EOC cells, but only GBP1 was secreted. ELISA and immuno-blotting analyses showed that cytokine-stimulated EOC cells release full-length GBP1 molecule in vitro, through non-classical pathways, not involving microvesicles. Moreover, soluble, full-length GBP1 accumulates in the ascites of most EOC patients, suggesting a potential role of extracellular GBP1 in the tumor environment. EOC cells enriched from ascites showed constitutive tyrosine-phosphorylated STAT1 and STAT3 proteins and GBP1 expression, supporting a role of STAT-activating cytokines in GBP1 induction in vivo. Data from the TCGA dataset of EOC indicate that high GBP1 gene expression correlates with a better overall survival. Accordingly GBP1 transfection reduced SKOV3 cell proliferation, suggesting a potential anti-tumor activity of GBP1. Conclusions: Our data show for the first time that soluble GBP1 is released by cytokine-stimulated EOC cells in vitro and accumulates in EOC patients’ ascites. GBP1 expression may have anti-tumor effects, in EOC, as suggested by TCGA dataset analysis and in vitro transfection experiments. Further studies to identify the biological role of soluble GBP1 in the tumor environment of EOC are warranted.
2020-05-27 | PXD016479 | Pride