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. 2024 Jan 15;15:532. doi: 10.1038/s41467-024-44843-w

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© The Author(s) 2024

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 4 — a DUSP22 directly dephosphorylated UBR2. In vitro phosphatase assays were performed using Myc-tagged DUSP22 wild-type or phosphatase-dead mutant (C88S) immunocomplex incubated with Flag-tagged UBR2 immunocomplex (substrates). The reactant products were subjected to immunoblotting analyses. b The Ser1694, Tyr1697, and Ser476 residues were identified as the DUSP22-targeted dephosphorylation sites on UBR2. The MS/MS fragmentation spectra of the tryptic peptides of UBR2 contain the phosphorylation modifications. S(p) and Y(p) denote phosphorylated serine and tyrosine residues, respectively. The blue color denotes the peptides corresponding to the loss of phosphate group from these peptides. c The UBR2 phosphomimetic mutants (S1694D, Y1697D, or S1694D/Y1697D) were resistant to the DUSP22-induced degradation. Myc-DUSP22 plus individual Flag-UBR2 mutants (S476D, S1694D, Y1697D, or S1694D/Y1697D) plasmids were co-transfected into HEK293T cells. The levels of Flag-tagged UBR2 proteins were determined by immunoblotting analysis. Vinculin immunoblotting was performed by reprobing the Flag (UBR2) immunoblot membrane with anti-vinculin antibody. d The phosphorylation-deficient mutation of UBR2 (S1694A/Y1697F) decreased protein stability. Flag-UBR2 or Flag-UBR2 mutant (S1694A/Y1697F) plasmid was transfected into HEK293T (with or without 25 μM MG132, 4 h) or Jurkat T cells. The cell lysates were subjected to immunoblotting analysis using anti-Flag antibody. e Phosphomimetic double mutations (S1694D/Y1697D) of UBR2 blocked the DUSP22-induced UBR2–βTrCP interaction. Myc-βTrCP, GFP-DUSP22, and individual Flag-UBR2, Flag-UBR2 (S1694D/Y1697D), and Flag-UBR2 (S1694A/Y1697F) plasmids were co-transfected into HEK293T cells. Red fluorescence represents the interactions ( < 40 nm) of UBR2 with βTrCP. GFP fluorescence (green color) indicates the expression of GFP-DUSP22. Images were captured with 400X original magnification by confocal microscope (Leica TCS SP5 I). Cell nuclei were stained with DAPI. Scale bar, 25 or 50 μm. f Phosphomimetic mutation (S1694D/Y1697D) of UBR2 reduced UBR2 Lys48-linked ubiquitination. Myc-DUSP22 and His-Ub (K48) plus either Flag-UBR2 or Flag-UBR2 mutant (S1694D/Y1697D) plasmids were co-transfected into HEK293T cells. Cells were treated with or without 25 μM MG132 for 4 h. UBR2 wild-type (WT) or phosphomimetic mutant (S1694D/Y1697D) proteins were immunoprecipitated with anti-Flag antibody and then immunoblotted with anti-His (top panel), anti-ubiquitin (Lys48) (bottom panel), or anti-Flag antibody. Arrow, intact UBR2 protein; asterisk, degraded UBR2.