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

DUSP22 is a dual-specificity phosphatase that inhibits T cell activation by inactivating the kinase Lck. Here we show that the E3 ubiquitin ligase UBR2 is a positive upstream regulator of Lck during T-cell activation. DUSP22 dephosphorylates UBR2 at specific Serine residues, leading to ubiquitin-mediated UBR2 degradation. UBR2 is also modified by the SCF E3 ubiquitin ligase complex via Lys48-linked ubiquitination at multiple Lysine residues. Single-cell RNA sequencing analysis and UBR2 loss of function experiments showed that UBR2 is a positive regulator of proinflammatory cytokine expression. Mechanistically, UBR2 induces Lys63-linked ubiquitination of Lck at Lys99 and Lys276 residues, followed by Lck Tyr394 phosphorylation and activation as part of TCR signalling. Inflammatory phenotypes induced by TCR-triggered Lck activation or knocking out DUSP22, are attenuated by genomic deletion of UBR2. UBR2-Lck interaction and Lck Lys63-linked ubiquitination are induced in the peripheral blood T cells of human SLE patients, which demonstrate the relevance of the UBR2-mediated regulation of inflammation to human pathology. In summary, we show here an important regulatory mechanism of T cell activation, which finetunes the balance between T cell response and aggravated inflammation.


Statistics
For all statistical analyses, confirm that the following items are present in the figure legend, table legend, main text, or Methods section.

n/a Confirmed
The exact sample size (n) for each experimental group/condition, given as a discrete number and unit of measurement A statement on whether measurements were taken from distinct samples or whether the same sample was measured repeatedly The statistical test(s) used AND whether they are one-or two-sided Only common tests should be described solely by name; describe more complex techniques in the Methods section.

A description of all covariates tested
A description of any assumptions or corrections, such as tests of normality and adjustment for multiple comparisons A full description of the statistical parameters including central tendency (e.g.means) or other basic estimates (e.g.regression coefficient) AND variation (e.g. standard deviation) or associated estimates of uncertainty (e.g.confidence intervals) For null hypothesis testing, the test statistic (e.g.F, t, r) with confidence intervals, effect sizes, degrees of freedom and P value noted Give P values as exact values whenever suitable.
For Bayesian analysis, information on the choice of priors and Markov chain Monte Carlo settings For hierarchical and complex designs, identification of the appropriate level for tests and full reporting of outcomes Estimates of effect sizes (e.g.Cohen's d, Pearson's r), indicating how they were calculated Our web collection on statistics for biologists contains articles on many of the points above.

Software and code
Policy information about availability of computer code Data collection For proteomics, Gel-extracted proteins were digested with trypsin and subjected to LC-MS/MS analyses by LTQ-Orbitrap Elite hybrid mass spectrometer (for identifying DUSP22-interacting proteins and UBR2-interacting protein) or LTQ-Orbitrap Fusion hybrid mass spectrometer (for mapping UBR2 phosphorylated residues, UBR2 ubiquitinated residues, and Lck ubiquitinated residues) using approaches described previously (Cancer Res. 79, 4978-4993 (2019); EMBO Mol.Med.14, e15904 ( 2022)).The peptide data were analyzed by MASCOT MS/MS Ions Search (Matrix Science).FFor fluorescent signal in cells, images were detected by fluorescence microscope (DM2500; Leica Microsystems, Buffalo Grove, IL, USA) or confocal microscope Leica TCS SP5, and Leica TCS SP5 II.Images were analyzed by LAS X software (v3.7.4).For scRNA-seq, single-cell capture and cDNA synthesis were performed using the BD Rhapsody Single-Cell Analysis System (210966 v1.0).cDNA libraries were constructed combined with Sample Tag and BD AbSeq libraries (BD Biosciences, 23-21752-00).cDNA libraries of individual cells were constructed using BD Rhapsody WTA Amplification Kit (Cat.633801).Paired-end sequencing was performed on a HiSeq X Ten sequencer (Illumina).Data analysis and quality control were performed following the BD Biosciences Rhapsody pipeline.Uniform Manifold Approximation and Projection (UMAP) generation were conducted with the R package Seurat (v3.0).For flow cytometry, data were acquired with a FACS CantoII (BD Biosciences) and analyzed with FlowJo (v10.8.1) analytical software.For immunoblotting, signals were detected by Syngene and analyzed by Pxi9 Access GENESys software.Statistical analyses were performed by using Excel (v16.7) or BD SEQGEQ (v1.8.0).
For manuscripts utilizing custom algorithms or software that are central to the research but not yet described in published literature, software must be made available to editors and reviewers.We strongly encourage code deposition in a community repository (e.g.GitHub).See the Nature Portfolio guidelines for submitting code & software for further information.

Reporting on race, ethnicity, or other socially relevant groupings n/a
Population characteristics A total of 12 individuals, including 6 healthy individuals (age from 24 to 49 year old) and 6 SLE patients (age from 24 to 49 year old) were enrolled in this study.

Recruitment
For collecting peripheral blood samples, 6 health individuals and 6 SLE patients who underwent clinic were invited to participate in academic research by providing peripheral blood samples.The selection of SLE patients was based on a welldefined SLE diagnosis.

Ethics oversight
This study was conducted in accordance with the Helsinki Declaration.For collecting peripheral blood samples, 6 health individuals and 6 SLE patients who underwent clinic were invited to participate in academic research by providing peripheral blood samples.The selection of SLE patients was based on a well-defined SLE diagnosis.The peripheral blood collection, Tcell purification, and the experiments were approved by the ethics committees of Taipei Veterans General Hospital (2017-06-003BC) and National Taiwan University Hospital (109-008-E).All study participants provided written informed consent prior to enrollment.
Note that full information on the approval of the study protocol must also be provided in the manuscript.

Field-specific reporting
Please select the one below that is the best fit for your research.If you are not sure, read the appropriate sections before making your selection.

Life sciences
Behavioural & social sciences Ecological, evolutionary & environmental sciences For a reference copy of the document with all sections, see nature.com/documents/nr-reporting-summary-flat.pdf

Life sciences study design
All studies must disclose on these points even when the disclosure is negative.

Sample size
Sample sizes were determined from similar experiments in the literatures of Li et al Nature Communications, 5, 1-13 (2014), Chuang et al Nature Communications 5, 4602 (2014), and Chuang et al 12, 1113-1118(2011).Sample sizes and the number of independent experiments are stated in the Statistical analysis and reproducibility section of Methods.Three or more independent results were used to for statistical analysis.
Data exclusions No data were excluded.

Replication
The experimental results were consistently reproduced, and the figure legends specify the exact number (n) of biological replicates used in the study.
Randomization All samples of the same genotype were randomly assigned for the control and experimental groups, and all cells analyzed were randomly selected from in vivo and in vitro samples.

nature portfolio | reporting summary
April 2023 Field-collected samples No field collected samples were used in the study.