MassIVEapplication/xmlftp://massive.ucsd.edu/v01/MSV000080850/primaryOK2001000Irina Kratchmarovahttps://massive.ucsd.edu/ProteoSAFe/dataset.jsp?task=69cdfcff187645c1ab21f440fd45fba2ihk@bmb.sdu.dkMassIVE13MOD:00397 - "A protein modification that is produced by reaction with iodoacetamide, usually replacement of a reactive hydrogen with a methylcarboxamido group."MOD:00674 - "A protein modification that effectively replaces a carboxyl group with a carboxamido group."MOD:00696 - "A protein modification that effectively replaces a hydrogen atom with a phosphono group (H2PO3 or 'phosphate')."ProteomicsQ ExactiveHomo Sapiens (ncbitaxon:9606)Department of Biochemistry and Molecular Biology University of Southern DenmarkIt remains a paradox that IL-2 and IL-15 can differentially modulate the immune response using the same signaling receptors. We have previously dissected the phosphotyrosine-driven signaling cascades triggered by both cytokines in Kit225 T-cells, unveiling subtle differences that may contribute to their functional dichotomy. In this study, we aimed to decipher the receptor complex assembly in IL-2- and IL-15-activated T-lymphocytes that is highly orchestrated by site-specific phosphorylation events. Comparing the cytokine-induced interactome of the interleukin receptor beta and gamma subunits shared by the two cytokines, we defined the components of the early IL-2 and IL-15 receptor-associated complex discovering novel constituents. Additionally, phosphopeptide-directed analysis allowed us to detect several cytokine-dependent and -independent phosphorylation events within the activated receptor complex including novel phosphorylated sites located in the cytoplasmic region of IL-2 receptor β subunit (IL-2Rβ). We proved that the distinct phosphorylations induced by the cytokines serve for recruiting different types of effectors to the initial receptor/ligand complex. Overall, our study sheds new light into the initial molecular events triggered by IL-2 and IL-15 and constitutes a further step toward a better understanding of the early signaling aspects of the two closely related cytokines in T-lymphocytes.Characterization of Receptor-Associated Protein Complex Assembly in Interleukin (IL)-2- and IL-15-Activated T-Cell Lines.Osinalde Nerea N, Sanchez-Quiles Virginia V, Akimov Vyacheslav V, Aloria Kerman K, Arizmendi Jesus M JM, Blagoev Blagoy B, Kratchmarova Irina IIl-2, determination, AI503618, AP-1, Receptors, DELTA AND GAMMA, gamma-Ada, Visible Light, T-Lymphocyte, betaTub1, phosphorylation, binding site, Readability, B1t, T Lymphocyte, Interleukins Receptors, IL-2, GRP1, hnu, Grp1, Fs(3)Hor, foton, STATI2, DmelCG2684, aldesleukin, kus, beta1tub, CG9277, T-Cells, PTPSTEP, T, beta-particle, present in organism, proteins, W, NTef2, T Cells, 1t, e, e-, beta1Tub, SOCS-2, signaling process, l(2)k08110, AP-1[gamma], associated, Thymus Dependent Lymphocytes, fam59a, single organism signaling, gamma, GPH, Ligand, BETA 56D, Radiation, AP1gamma, Light, Fs(3)Sz11, IL15, LIGHT, l(1)G0108, T-Cell, Tyrosine O phosphate, 3.1.3.48, precocious, FLOWERING TIME CONTROL PROTEIN FCA ALPHA, region, FAM59A, Visible Radiations, beta-tub, Thymus-Dependent Lymphocyte., Visible Radiation, Aldesleukin, Interleukin, positional polypeptide feature, Beta <eudicots>, HVEML, Step, CG11628, IL-2 receptor activity, Thymus-Dependent, AP1gamma1, FCAALL.331, Striatum-enriched protein-tyrosine phosphatase, beta56D, DmelCG9277, Thymus-Dependent Lymphocyte, early, beta1-tub, beta(-), DTB2, beta-Tub, light quantum, STEP, T-cell growth factor, Horka, i6, DL4180C, Cells, dihydrogen phosphate (ester), CG2684, Fs(3)Horka, Tyrosine-O-phosphate, T Cell, Fam59a, Interleukin Receptors, beta-Tub56D, L-tyrosine, betaTub, Specbeta, beta1-Tubulin, biological signaling, advanced, Thymus-Dependent Lymphocytes, GRP1/cytohesin 1, region or site annotation, C86169, e(-), Interleukins, Gene, electron, Ly113, CG11633, cytohesin/GRP1, IL-15, B-spec, cg9113, Interleukin 15, betaspec, MOD_RES Phosphotyrosine, Gene Products, DmF2, light, C18orf11, CIS2, lod, study, positional, betatub(56D), Lichtquant, ligand, Elektron, SSI2, beta[[1]] tubulin, beta1t, l(2)SH2 0323, interleukin-15 receptor ligand, Visible, DmelCG9113, anon-EST:fe1B3, Neural-specific protein-tyrosine phosphatase, SPEC8, IL-2R, IL2, photon, site, TR2, CG5870, T Lymphocytes, Lymphocyte, beta-tubulin56D, Spec-beta, Dmbeta1, Phosphotyrosine, Proteins, Phosphorylations, Tubulin, stepk, mKIAA4238, i168, CD258, Cell, Cytokine, Phosphopeptide, TCGF, Gm944, chemical analysis, Protein, betaSpec, lymphokine, Interleukin Receptor, b-Spec, l(2)SH0323, dgamma, interleukin-2 receptor ligand, Radiations, CYH1, DmelCG5870, l(1)G0074, l(1)G0198, negatron, CG9113, distinct, beta[[1]]-tubulin, HVEM-L, INSDC_feature:misc_binding, Photoradiation, b spectrin, Lymphocytes, beta1, Cish2, Understanding, Lds, signalling, beta, LTg, DmelCG11628, Tub, Protein Gene Products, Gene Proteins, Photoradiations, signalling process, binding_or_interaction_site, beta-spec, BETA, Receptor, assay, SSI-2, SpecIl-2, determination, AI503618, AP-1, Receptors, DELTA AND GAMMA, gamma-Ada, Visible Light, T-Lymphocyte, betaTub1, phosphorylation, binding site, Readability, B1t, T Lymphocyte, Interleukins Receptors, IL-2, GRP1, hnu, Grp1, Fs(3)Hor, foton, DmelCG2684, aldesleukin, kus, beta1tub, CG9277, T-Cells, PTPSTEP, T, beta-particle, NTef2, T Cells, 1t, e, e-, beta1Tub, signaling process, l(2)k08110, AP-1[gamma], associated, Thymus Dependent Lymphocytes, single organism signaling, gamma, GPH, Ligand, BETA 56D, Radiation, AP1gamma, Light, Fs(3)Sz11, IL15, LIGHT, l(1)G0108, T-Cell, Tyrosine O phosphate, 3.1.3.48, precocious, FLOWERING TIME CONTROL PROTEIN FCA ALPHA, region, Visible Radiations, beta-tub, Thymus-Dependent Lymphocyte., Visible Radiation, Aldesleukin, Interleukin, positional polypeptide feature, Beta <eudicots>, HVEML, Step, CG11628, Thymus-Dependent, AP1gamma1, FCAALL.331, Striatum-enriched protein-tyrosine phosphatase, beta56D, DmelCG9277, Thymus-Dependent Lymphocyte, early, beta1-tub, beta(-), DTB2, beta-Tub, light quantum, STEP, T-cell growth factor, Horka, i6, DL4180C, Cells, dihydrogen phosphate (ester), CG2684, Fs(3)Horka, Tyrosine-O-phosphate, T Cell, Interleukin Receptors, beta-Tub56D, L-tyrosine, betaTub, Specbeta, beta1-Tubulin, biological signaling, advanced, Thymus-Dependent Lymphocytes, GRP1/cytohesin 1, region or site annotation, e(-), Interleukins, electron, Ly113, CG11633, cytohesin/GRP1, IL-15, B-spec, cg9113, Interleukin 15, betaspec, MOD_RES Phosphotyrosine, DmF2, light, lod, study, positional, betatub(56D), Lichtquant, ligand, Elektron, beta[[1]] tubulin, beta1t, l(2)SH2 0323, interleukin-15 receptor ligand, Visible, DmelCG9113, anon-EST:fe1B3, Neural-specific protein-tyrosine phosphatase, SPEC8, IL2, photon, site, TR2, CG5870, T Lymphocytes, Lymphocyte, beta-tubulin56D, Spec-beta, Dmbeta1, Phosphotyrosine, Phosphorylations, Tubulin, stepk, i168, CD258, Cell, Cytokine, Phosphopeptide, TCGF, chemical analysis, betaSpec, lymphokine, Interleukin Receptor, b-Spec, l(2)SH0323, dgamma, interleukin-2 receptor ligand, Radiations, CYH1, DmelCG5870, l(1)G0074, l(1)G0198, negatron, CG9113, distinct, beta[[1]]-tubulin, HVEM-L, INSDC_feature:misc_binding, Photoradiation, b spectrin, Lymphocytes, beta1, Understanding, Lds, signalling, beta, LTg, DmelCG11628, Tub, Photoradiations, signalling process, binding_or_interaction_site, beta-spec, BETA, Receptor, assay, Speccellular macromolecule complex assembly, Thymus-Dependent Lymphocyte., Interleukin, Thymus-Dependent Lymphocytes, AI503618, T-Cells, Thymus-Dependent, protein complex formation, T, Lymphocytes, interleukin-15 receptor ligand, T-Lymphocyte, Cell, chaperone activity, T Cells, IL15, IL-15, T Lymphocyte, T-Cell, macromolecular complex assembly, Interleukin 15, cellular protein-containing complex assembly, cellular protein complex assembly, Cells, T Cell, associated, protein complex assembly, macromolecule complex assembly, T Lymphocytes, Thymus Dependent Lymphocytes, Lymphocytechaperone activity, cellular macromolecule complex assembly, IL15, Interleukin, IL-15, macromolecular complex assembly, Interleukin 15, AI503618, cellular protein-containing complex assembly, cellular protein complex assembly, Line, Cell Lines, protein complex formation, Cell., associated, interleukin-15 receptor ligand, protein complex assembly, macromolecule complex assembly, Cell, Lines1PXD002386trueCharacterization of receptor-associated protein complex assembly in Interleukin (IL)-2- and IL-15-activated T-lymphocytesDespite extensive investigation, it remains a paradox that IL-2 and IL-15 can differentially modulate the immune response using the same signaling receptors. In our previous work, we dissected the phosphotyrosine-driven signaling cascades triggered by both cytokines in Kit225 T-cells unveiling subtle differences that may contribute to their functional dichotomy. In the present study, we aimed to decipher the receptor complex assembly in IL-2- and IL-15-activated T-lymphocytes that is fine tune orchestrated by site-specific phosphorylation events. Comparing the cytokine-induced interactome of the interleukin receptor beta and gamma subunits shared by the two cytokines, we defined the components of the early IL-2 and IL-15 receptor-associated complex discovering novel constituents such as FAM59A and SOCS2. Additionally, phosphopeptide-directed analysis allowed us to detect several cytokine-dependent and –independent phosphorylation events within the activated receptor complex including novel phosphorylated sites located in the cytoplasmic region of IL-2R?. We proved that the distinct phosphorylations induced by the cytokines serve for recruiting different types of effectors to the initial receptor/ligand complex. Whereas IL-2R? pS431 binds to clathrins, which are involved in the receptor internalization and subsequent signal attenuation, IL-2R? pY325 and pY357 attract phosphatases, adaptor proteins, kinases as well as the newly identified member of the interleukin receptor complex SOCS2. Overall our study sheds new light into the initial molecular mechanisms triggered by IL-2 and IL-15 and constitutes a further step towards a better understanding of the early signaling aspects of the two closely-related cytokines in T-lymphocytes.Fri Mar 31 22:32:00 BST 2017MSV00008085027463037