SILAC-based phosphoproteomics reveals an inhibitory role of KSR1 in p53 transcriptional activity via modulation of DBC1

BACKGROUND We have previously identified kinase suppressor of ras-1 (KSR1) as a potential regulatory gene in breast cancer. KSR1, originally described as a novel protein kinase, has a role in activation of mitogen-activated protein kinases. Emerging evidence has shown that KSR1 may have dual functions as an active kinase as well as a scaffold facilitating multiprotein complex assembly. Although efforts have been made to study the role of KSR1 in certain tumour types, its involvement in breast cancer remains unknown. METHODS A quantitative mass spectrometry analysis using stable isotope labelling of amino acids in cell culture (SILAC) was implemented to identify KSR1-regulated phosphoproteins in breast cancer. In vitro luciferase assays, co-immunoprecipitation as well as western blotting experiments were performed to further study the function of KSR1 in breast cancer. RESULTS Of significance, proteomic analysis reveals that KSR1 overexpression decreases deleted in breast cancer-1 (DBC1) phosphorylation. Furthermore, we show that KSR1 decreases the transcriptional activity of p53 by reducing the phosphorylation of DBC1, which leads to a reduced interaction of DBC1 with sirtuin-1 (SIRT1); this in turn enables SIRT1 to deacetylate p53. CONCLUSION Our findings integrate KSR1 into a network involving DBC1 and SIRT1, which results in the regulation of p53 acetylation and its transcriptional activity

Enhanced TH17 responses in patients with IL10 receptor deficiency and infantile-onset IBD

Background IL10 receptor (IL10R) deficiency causes severe infantile-onset IBD. Intact IL10R-dependent signals have been shown to be important for innate and adaptive immune cell function in mice. We have previously reported a key role of IL10 is the generation and function of human anti-inflammatory macrophages. Independent of innate immune cell defects, the aim of the current study was to determine the role of IL10R signaling in regulating human CD4+ T cell function. Methods Peripheral blood mononuclear cells and intestinal biopsies cells were collected from IL10/IL10R-deficient patients and controls. Frequencies of CD4+ T cell subsets, naïve T cell proliferation, regulatory T cell (Treg)-mediated suppression and Treg and TH17 generation were determined by flow cytometry. Transcriptional profiling was performed by NanoString and quantitative real-time PCR. RNA in situ hybridization was used to determine the quantities of various transcripts in intestinal mucosa. Results Analysis of 16 IL10- and IL10R-deficient patients demonstrated similar frequencies of peripheral blood and intestinal Tregs, compared to control subjects. In addition, in vitro Treg suppression of CD4+ T cell proliferation and generation of Treg were not dependent on IL10R signaling. However, IL10R-deficient T naïve cells exhibited significantly higher proliferative capacity, a strong TH17 signature and an increase in polarization towards TH17 cells, compared to controls. Moreover, the frequency of TH17 cells was increased in the colon and ileum of IL10R-deficient patients. Finally, we show that stimulation of IL10R-deficient Tregs in the presence of IL1 leads to enhanced production of IL17A. Conclusions IL10R signaling regulates TH17 polarization and T cell proliferation in humans, but is not required for the generation and in vitro suppression of Tregs. Therapies targeting the TH17 axis might be beneficial for IL10- and IL10R-deficient patients as a bridge to allogeneic hematopoietic stem cell transplantation

Benomyl-resistant Beauveria bassiana (Hypocreales: Clavicipitaceae) mutants induced by ion beams

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