Journal: Nature Communications

Article Title: The phosphatase DUSP22 inhibits UBR2-mediated K63-ubiquitination and activation of Lck downstream of TCR signalling

doi: 10.1038/s41467-024-44843-w

Figure Lengend Snippet: a Identification of CUL1 as an UBR2-interacting protein by mass spectrometry-based proteomics. Identified CUL1 peptides were shown. b Immunoblotting of CUL1, Flag-tagged UBR2, Myc-tagged (DUSP22), and GAPDH proteins in Jurkat T cells co-transfected with Myc-CUL1, Flag-UBR2, and Myc-DUSP22 plasmids. c Immunoblotting of the endogenous CUL1, Flag-tagged UBR2, Myc-tagged DUSP22, and vinculin proteins in HEK293T cells co-transfected with Flag-UBR2, Myc-DUSP22, and CUL1 shRNA plasmids. d CUL1 knockdown inhibited DUSP22-induced UBR2 ubiquitination. Immunoprecipitation and immunoblotting analysis of Lys48-linked ubiquitination of UBR2, Flag-tagged UBR2, Myc-tagged DUSP22, and endogenous CUL1 proteins were performed using the lysates of HEK293T cells co-transfected with Flag-UBR2, Myc-DUSP22, and CUL1 shRNA #3 plasmids. The transfected cells were treated with 25 µM MG132 for 4 h. Arrow, UBR2 protein; asterisk, degraded UBR2 protein. e βTrCP overexpression plus suboptimal DUSP22 (0.8 μg) induced UBR2 degradation. Immunoblotting of Flag-tagged UBR2, Myc-tagged DUSP22, and tubulin proteins in Jurkat T cells co-transfected with Flag-UBR2 and Myc-DUSP22 plus different amounts (0.8 μg, 1.6 μg) of Flag-βTrCP plasmids. f UBR2 interacted with βTrCP. Immunoprecipitation and immunoblotting of Flag-tagged UBR2 with Myc-tagged-βTrCP proteins were performed using the lysates of HEK293T transfected with Flag-UBR2 and Myc-βTrCP plasmids. Anti-vinculin immunoblotting was performed by reprobing the anti-Flag (UBR2) immunoblot membrane. g Confocal microscopy analyses of PLA for the interaction between Flag-tagged UBR2 and Myc-tagged βTrCP proteins in HEK293T cells co-transfected with Flag-UBR2, Myc-βTrCP, and either GFP-DUSP22 or GFP-DUSP22 (C88S) plasmids. Red fluorescence represents the interactions of UBR2 with βTrCP proteins. Images were captured with 400X original magnification. Cell nuclei were stained with DAPI. Scale bar, 50 μm. h Immunoblotting of endogenous UBR2, βTrCP, or CUL1 proteins in Jurkat T cells transfected with scramble shRNAs, βTrCP shRNAs #1, or CUL1 shRNAs #3. T cells were stimulated with anti-CD3 antibody for indicated time periods. i CUL1-βTrCP E3 ligase complex induced UBR2 ubiquitination in vitro. Recombinant His-ubiquitin, E1 (UBA1), E2 (top panel, UBE2D3; bottom panel, CDC34), ATP, and SKP1-CUL1-βTrCP-RBX1 complex were co-incubated in the ubiquitination buffer with Flag-tagged UBR2 E3 ligase-inactive mutant (C1210/1213A) proteins.

Article Snippet: Anti-CD3ζ (Y142) antibody (Cat A02421Y142, Lot A02421Y142) was purchased from Boster Biotechnology.

Techniques: Mass Spectrometry, Western Blot, Transfection, shRNA, Knockdown, Ubiquitin Proteomics, Immunoprecipitation, Over Expression, Membrane, Confocal Microscopy, Fluorescence, Staining, In Vitro, Recombinant, Incubation, Mutagenesis

Journal: Nature Communications

Article Title: The phosphatase DUSP22 inhibits UBR2-mediated K63-ubiquitination and activation of Lck downstream of TCR signalling

doi: 10.1038/s41467-024-44843-w

Figure Lengend Snippet: a Schematic diagram of the murine UBR2 wild-type (WT) alleles and the targeted UBR2 mutant alleles. P1 and P2, the primers for PCR. b PCR analyses of wild-type and UBR2 mutant alleles using genomic DNAs from mouse tails. The PCR product of the upper band (579 bp) denotes the wild-type allele, and the lower band (511 bp) denotes the UBR2 mutant allele. c Immunoblotting analyses of UBR2 protein levels in T cells of wild-type or UBR2 knockout (KO) mice. d UMAP plot showed dimensional reduction of the distribution of 3061 cells. T cells derived from each group are shown in different colors (blue: wild-type; red: UBR2 knockout). e UMAP plot showed seven major clusters of the 3061 individual T cells. The number denotes the cluster frequency in parent population. f Volcano plot showed the differential expression transcripts between UBR2 knockout and wild-type T cells. Fold change represents the gene expression in UBR2 knockout T cells versus wild-type T cells. g Violin plots showed the expression levels of IFN-γ, TNF-α, IL-2, IL-4, and IL-6 genes in wild-type T cells under different UBR2 expression levels. The mRNA levels of UBR2 and cytokines were detected as unique molecular identifier (UMI) counts. h ELISAs of IFN-γ, TNF-α, and IL-17A levels in culture supernatants from peripheral blood T cells treated with plate-bound anti-CD3 plus anti-CD28 costimulation for 72 h. Total murine primary T cells were used in Fig. 5c-h.

Article Snippet: Anti-CD3ζ (Y142) antibody (Cat A02421Y142, Lot A02421Y142) was purchased from Boster Biotechnology.

Techniques: Mutagenesis, Western Blot, Knock-Out, Derivative Assay, Quantitative Proteomics, Gene Expression, Expressing

Journal: Nature Communications

Article Title: The phosphatase DUSP22 inhibits UBR2-mediated K63-ubiquitination and activation of Lck downstream of TCR signalling

doi: 10.1038/s41467-024-44843-w

Figure Lengend Snippet: a Immunoprecipitations of the endogenous Lck with either UBR2 or DUSP22 proteins in the lysates of anti-CD3-stimulated murine primary T cells. b TCR signaling stimulated Lys63-linked ubiquitination and Tyr394 phosphorylation of Lck in murine primary T cells. Endogenous Lck immunocomplexes in the lysates of anti-CD3-stimulated T cells were immunoprecipitated with anti-Lck antibody and then subjected to immunoblotting. c Confocal microscopy analyses of PLA for the ubiquitinated and phosphorylated Lck in TCR-stimulated T cells of wild-type (WT) or UBR2 knockout mice using anti-Lck antibody plus either anti-ubiquitin (Lys63) or anti-phospho-Lck (Tyr394) antibodies. Red fluorescence represents the endogenous Lck proteins containing Lys63-linked ubiquitination or Tyr394 phosphorylation. Cell nuclei were stained with DAPI. d UBR2 induced Lck ubiquitination in vitro. Recombinant His-ubiquitin, E1 (UBE1), E2 (UBE2D3), Flag-Lck, and ATP were co-incubated in the ubiquitination buffer with E3 (Flag-tagged UBR2 or Flag-tagged UBR2 ligase-inactive mutant (C1210/1213A)) proteins. e The Lys99 and Lys276 residues were identified as ubiquitination sites of Lck by mass spectrometry analysis. K(ub), ubiquitinated lysine residue. f Immunoprecipitations of ubiquitinated Flag-tagged Lck with Myc-tagged UBR2 proteins in the lysates of indicated HEK293T transfectants. Flag-tagged Lck proteins were immunoprecipitated with anti-Flag antibody and then immunoblotted with anti-ubiquitin (Lys63) antibody or anti-Flag antibody. g Immunoprecipitation and immunoblotting analysis of Flag-tagged Lck with Tyr394 phosphorylated Lck proteins in the lysates of Jurkat T cells transfected with Flag-Lck (WT) or Flag-Lck (K99/276R) plasmid. h Confocal microscopy analyses of PLA for the ubiquitinated Lck and Tyr394-phosphorylated Lck in TCR-stimulated Jurkat transfectants using anti-Lck plus anti-ubiquitin (Lys63) and anti-phospho-Lck (Tyr394) antibodies, respectively. Red fluorescence represents the endogenous Lck proteins containing Lys63-linked ubiquitination or Tyr394 phosphorylation. Cell nuclei were stained with DAPI. i Double mutations (K99/276R) of Lck inhibited phosphorylation of Lck at Tyr394 residue by in vitro kinase assay. j In vitro ubiquitin E3 ligase assay in combination with kinase assay showed that ubiquitination-deficient Lck inhibited Lys63-linked polyubiquitination-induced trans-autophosphorylation of Lck by UBR2. Arrow, intact UBR2 protein; asterisk, degraded UBR2 protein.

Article Snippet: Anti-CD3ζ (Y142) antibody (Cat A02421Y142, Lot A02421Y142) was purchased from Boster Biotechnology.

Techniques: Ubiquitin Proteomics, Phospho-proteomics, Immunoprecipitation, Western Blot, Confocal Microscopy, Knock-Out, Fluorescence, Staining, In Vitro, Recombinant, Incubation, Mutagenesis, Mass Spectrometry, Residue, Transfection, Plasmid Preparation, Kinase Assay

Journal: Nature Communications

Article Title: The phosphatase DUSP22 inhibits UBR2-mediated K63-ubiquitination and activation of Lck downstream of TCR signalling

doi: 10.1038/s41467-024-44843-w

Figure Lengend Snippet: a Immunoblotting of phospho-Tyr394 Lck, Lck, and GAPDH proteins from purified peripheral blood murine T cells of wild-type (WT), DUSP22 knockout (KO), or DUSP22/UBR2 double knockout (dKO) mice upon anti-CD3 stimulation. Quantification of p-Lck proteins (normalized to Lck proteins) is shown at the bottom of p-Lck panel. b , c Confocal microscopy analyses of PLA for ubiquitinated Lck ( b ) and Tyr394 phosphorylated Lck ( c ) proteins in TCR-stimulated T cells of wild-type, DUSP22 knockout, or DUSP22/UBR2 double knockout (dKO) mice using anti-Lck antibody plus anti-ubiquitin (Lys63) antibody ( b ) and anti-phospho-Lck (Tyr394) antibody ( c ), respectively. Red fluorescence represents the endogenous Lck proteins containing Lys63-linked ubiquitination or Tyr394 phosphorylation. Cell nuclei were stained with DAPI. The red signal intensity was plotted in the lower panels. d Serum levels of IFN-γ, TNF-α, and IL-17A in wild-type, DUSP22 knockout (KO), and DUSP22/UBR2 double knockout (dKO) mice were determined by ELISAs. e Hematoxylin and eosin (H&E)-stained sections of the kidney, liver, and lung from 10- to 11-month-old wild-type, DUSP22 knockout (KO) and DUSP22/UBR2 double knockout (dKO) mice. Scale bar, 50 μm (kidney and liver) and 100 μm (lung). f Western blotting analysis of UBR2, p-Lck, and DUSP22 proteins in peripheral blood T cells of human SLE patients and healthy controls (HC). Anti-GAPDH immunoblotting was performed by reprobing the anti-Lck immunoblot membrane. g , h Confocal microscopy analyses of PLA for the interaction between the endogenous UBR2 and Lck proteins or Lys63-linked ubiquitination of Lck in peripheral blood T cells of a representative human SLE patient and a representative healthy control (HC). T-cell nuclei were stained with DAPI. i ELISAs of IFN-γ, TNF-α, and IL-17A levels in culture supernatants from MOG-restimulated T cells. Mice were immunized with MOG peptide emulsified in CFA, followed by injection of pertussis at day 0, 1, and 2. To determine the activation of Ag-specific T cells, T cells from lymph nodes of the immunized mice were cultured in the presence of 0, 20, and 50 μg/ml MOG for 72 h, and cytokine levels of IFN-γ, TNF-α, and IL-17A were analyzed by ELISA. n.s., not significant.

Article Snippet: Anti-CD3ζ (Y142) antibody (Cat A02421Y142, Lot A02421Y142) was purchased from Boster Biotechnology.

Techniques: Western Blot, Purification, Knock-Out, Double Knockout, Confocal Microscopy, Ubiquitin Proteomics, Fluorescence, Phospho-proteomics, Staining, Membrane, Control, Injection, Activation Assay, Cell Culture, Enzyme-linked Immunosorbent Assay