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Bender"],"technology_type":["Data-dependent acquisition","Mass Spectrometry","Bottom-up proteomics"],"software":[""],"submitter_keywords":["cd20","Rhoa/rock1 pathway","Phosphoproteomics","Microtubule network","B lymphocyte","Microtubule-to-actin switch","Pkcδ","B lymphocyte; cd20","Silac","Mass spectrometry"],"full_dataset_link":["https://www.ebi.ac.uk/pride/archive/projects/PXD063667"],"tissue":["B Cell","Cell Culture"],"sample_protocol":["SILAC labeling of cells: Ramos B-cells were labelled using stable isotope labeling by amino acids in cell culture (SILAC). Light labeling was performed using 12C6-L-arginine/12C6-L-lysine. For medium-heavy and heavy labeling 13C6-L-arginine/D4-L-lysine and 13C615N4-L-arginine/13C615N2-L-lysine were used, respectively.  Generation of the Ramos PKC (PRKCD) gene KO: The sequence AACCCAATCATAGCAGAGC or GCTGAGTTCAGTGAGTGC encoding for the specific gRNA was inserted into a lentivirus CRISPR vector (Addgene, Plasmid #52961). The constructs were co-transfected with an mCherry expression vector into Ramos cells. The cells were incubated at 37°C for 24 hours in a humidified CO2 incubator. The following day, single cells were sorted for mCherry reporter gene expression with the BD FACSAria™ III Cell Sorter. The sorted single cells were cultured for 3-4 weeks in complete RPMI medium supplied with 10% FBS in a humidified CO2 incubator at 37°C. The expected PRKCD gene deficiency was verified by Sanger sequencing and the loss of PKC protein expression by Western blotting.  Phosphopeptide enrichment: SILAC-labeled Ramos B-cells (wild-type and knockout) were starved by serum depletion (FCS) for 30 min. Following starvation, cells were harvested by centrifugation at 1,200 rpm for 5 min, washed twice with starvation medium. As a control, mock treatment was performed using PBS. After activation, cells were rapidly snap-frozen in liquid nitrogen to preserve their signaling status. Cell lysis was performed using GdmHCl buffer (6 M GdmHCl, 100 mM Tris-HCl pH 8.5, 10 mM TCEP, and 40 mM chloroacetamide). The cell pellets were resuspended in the lysis buffer, followed by sonication (two cycles of 30 seconds). Protein denaturation was achieved by incubating the lysates at 95°C for 5 min. To remove cellular debris, the lysates were centrifuged at 3,500 x g for 30 min at 4°C. Protein concentration was determined using a Bradford assay. To precipitate proteins, four volumes of ice-cold acetone were added to the lysate. The protein precipitates were collected by centrifugation and washed with ice-cold acetone to remove residual contaminants. Proteins were digested using a combined Lys-C / trypsin digestion. Phosphopeptides were enriched using the Easyphos workflow. The phosphopeptide-enriched eluates were desalted using C18 StageTips (3M Company), pooled, and stored at -80°C until further analysis.  Quantitative mass spectrometry: Peptide mixtures, reconstituted in 0.1% TFA, were analyzed by nano HPLC-ESI-MS/MS using an UltiMate 3000 RSLCnano HPLC system (Thermo Fisher Scientific, Dreieich, Germany) online coupled to an QExactive Plus mass spectrometer (Thermo Fisher Scientific, Bremen, Germany). The RSLC system was equipped with C18 trap columns (nanoEase M/Z Symmetry C18 Trap; 20 mm length, 180 µm inner diameter, 5 µm particle size, 100 Å pore size, Waters Corporation, Milford, MA) and an analytical C18 reversed-phase nano LC column (nanoEase M/Z HSS C18 T3; 250 mm length, 75 mm inner diameter, 1.8 µm particle size, 100 Å pore size, Waters Corporation, Milford, MA).  A binary solvent system consisting of 0.1% (v/v) formic acid (FA) as solvent A and 80% (v/v) acetonitrile (ACN)/0.1% (v/v) FA as solvent B was employed for peptide separation. Peptide mixtures were loaded, washed and preconcentrated on the pre-column for 5 min using solvent A and a flow rate of 10 μL/min. A gradient was then applied at a flow rate of 300 nL/min ranging from 4 to 39% B in 140 min, 39–54% B in 15 min, 54%-95% in 5 min and 3 min at 95% B. Eluted peptides were transferred to a fused silica emitter for electrospray ionization enabled by a Nanospray Flex ion source with DirectJunction adaptor (Thermo Fisher Scientific), applying a spray voltage of 1.6 kV and a capillary temperature of 250°C. MS/MS data were acquired in data-dependent mode using the following parameters: MS precursor scans at m/z 370–1700 with a resolution of 70,000 (at m/z 400); automatic gain control (AGC) of 3 × 106 ions; a maximum injection time (IT) of 60 ms; a top 12 method for higher-energy collisional dissociation of multiply charged precursor ions with a normalized collision energy of 28%. MS/MS scans from 200 to 2000 m/z were recorded at a resolution of 35,000. The AGC for MS/MS scans was set to 1 × 105 with a maximum IT of 120 ms and a dynamic exclusion time of 45 s."],"repository":["Pride"],"quantification_method":[""],"modification":[""],"data_protocol":["MaxQuant  (version 2.6.7.0) with its integrated Andromeda search engine  was employed. MS/MS data were searched against the human proteome set with isoforms downloaded from UniProt (105300 entries). Protein identification was performed using MaxQuant default settings (including carbamidomethylation of cysteine residues as fixed modification and N-terminal acetylation and methionine oxidation as variable modifications), with the following exceptions: ‘Arg6;Lys4’ and ‘Arg10;Lys8’ were specified as medium and heavy modification labels, respectively; a maximum of three missed cleavages was allowed; ‘Phospho (STY)’, ‘Oxidation (M)’ and  ‘Acetyl (Protein N-term)’ were selected as variable modifications; and the options ‘match between runs’ and ‘requantify’ were activated. Ratios between labels (normalized by MaxQuant) were extracted from the Phospho(STY)Sites.txt file, reverse entries and potential contaminants were removed, and the list was filtered for at least two unique peptides per phosphosite. Phosphosite ratios were normalized to the respective ratio at the protein group level using the median ratio of all protein group ratios referenced in the ‘Protein group IDs’ column of the Phospho(STY)Sites.txt file. Resulting phospho site ratios were filtered so that only sites with valid values in >=3 of 4 replicates were retained. Significance of differential regulation was tested using linear models implemented in the LIMMA package and the resulting fold changes and significance level were plotted. All original code for MS data analysis has been deposited at Zenodo (https://doi.org/10.5281/zenodo.15350913)."],"omics_type":["Proteomics"],"labhead":["Bettina Warscheid"],"instrument_platform":[""],"labhead_affiliation":["Chair of Biochemistry II, Theodor Boveri-Institute, Biocenter, University of Würzburg, Würzburg, Germany"],"submission_type":["COMPLETE"],"species":["Homo Sapiens (human)"],"submitter_mail":["julian.bender@uni-wuerzburg.de"],"publication":["10.1038/S44318-026-00781-5"],"submitter_affiliation":["University of Wuerzburg\nChair of Biochemistry II\nAm Hubland\n97074 Würzburg"],"submitter_country":["Germany"],"doi":["10.6019/PXD063667"],"additional_accession":[]},"is_claimable":false,"name":"Microtubule anchoring and coupling of CD20 to the RhoA/Rock1 pathway (PKCdelta KO SILAC phosphoproteomics)","description":"CD20 is a B cell-specific four-helix transmembrane protein and a prominent target of successful therapeutic anti-CD20 antibodies such as rituximab (RTX) and GA101. We have recently described that CD20 is localized within a membrane nanocluster harboring the IgD class B cell antigen receptor (IgD-BCR) where it functions as a gatekeeper for the resting state of naïve human B lymphocytes. Loss of CD20 results in the remodeling of the IgD-BCR nanocluster and B cell activation. How CD20 exerts its gatekeeper function was not known so far. Using the Ramos B cell system and human peripheral blood B cells, we show here that another B cell gatekeeper, the serine/threonine kinase PKCδ, constitutively phosphorylates specific serine residues at the N- and C-terminal cytosolic tails of CD20. The phosphorylated CD20 becomes a target for 14-3-3 adaptor proteins that link CD20 to the RhoA GDP/GTP exchange factor GEF-H1 (ARHG2). The autoinhibited form of GEF-H1 couples CD20 to the microtubule network that controls the stability of the IgD-BCR nanocluster on resting B cells. Binding of anti-CD20 antibodies results in microtubule disassembly and the replacement of the GEF-H1/CD20 complex by a RhoA-GTP/ROCK1/CD20 complex, which drives actomyosin assembly and translocation of CD20 and the coreceptor CD19 to the IgM-BCR. The effect of anti-CD20 antibodies can be mimicked by exposing B cells to microtubule destabilizing drugs such as nocodazole (Noc) whereas microtubule stabilizing drugs such as Taxol (Tax) prevent the IgD-BCR nanocluster dissociation a finding that may alter therapeutic protocols of anti-CD20 treatments. Taken together, our study suggests that CD20 not only maintains the resting state, but also orchestrates the microtubule-actin switch in active B lymphocytes.","dates":{"publication":"2026-04-17","submission":"2025-05-06"},"accession":"PXD063667","cross_references":{"TAXONOMY":["NEWT:1773","NEWT:3555","NEWT:1182590","NEWT:10090","NEWT:749200","NEWT:35554","NEWT:4120","NEWT:5693","NEWT:347515","NEWT:1216979","NEWT:307972","NEWT:92867","NEWT:990346","NEWT:544496","NEWT:5334","NEWT:145953","NEWT:284812","NEWT:115104","NEWT:43330","NEWT:67825","NEWT:44544","NEWT:13076","NEWT:544404","NEWT:3702","NEWT:8839","NEWT:4232","NEWT:1736309","NEWT:4113","NEWT:7227","NEWT:11298","NEWT:885318","NEWT:4081","NEWT:876138","NEWT:554","NEWT:5691","NEWT:260710","NEWT:106592","NEWT:237561","NEWT:9913","NEWT:10036","NEWT:4100","NEWT:7574","NEWT:1351","NEWT:1076","NEWT:6763","NEWT:7215","NEWT:380394","NEWT:272563","NEWT:1639","NEWT:188229","NEWT:746360","NEWT:6239","NEWT:135588","NEWT:135622","NEWT:6915","NEWT:9986","NEWT:101510","NEWT:3880","NEWT:58002","NEWT:9103","NEWT:4577","NEWT:146479","NEWT:1000589","NEWT:145943","NEWT:85962","NEWT:160488","NEWT:317447","NEWT:3635","NEWT:7955","NCBITaxon:2","NEWT:7959","NEWT:2261","NEWT:3197","NEWT:9615","NEWT:884019","NEWT:4565","NEWT:1264690","NEWT:169963","NEWT:36329","NEWT:34305","NEWT:59729","NEWT:626528","NEWT:139927","NEWT:4558","NEWT:9606","NEWT:367830","NEWT:243230","NEWT:931281","NEWT:7029","NEWT:1283300","NEWT:334747","NEWT:470","NEWT:3218","NEWT:5759","NEWT:9838","NCBITaxon:9615","NEWT:1736231","NEWT:1193501","NEWT:6287","NEWT:6326","NEWT:9796","NEWT:2762","NEWT:5476","NEWT:562","NEWT:260707","NEWT:287","NEWT:10117","NEWT:10116","NEWT:1280","NEWT:1836","NEWT:29760","NEWT:260705","NEWT:1148","NEWT:4932","NEWT:70448","NEWT:9825","NEWT:3603","NEWT:698936","NEWT:39946","NEWT:11676","NEWT:9823","NEWT:100226","NCBITaxon:6073","NEWT:4896","NEWT:6279","NEWT:7370","NEWT:573","NEWT:6282","NEWT:7091"],"pubmed":[],"ORCID":["0000-0003-3316-9999"]}}