48 results on '"Rudd, C E"'
Search Results
2. Adaptors TRIM and LAX regulate transport and surface expression of co-receptor CTLA-4 on T-cells: W06.005
- Author
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Schneider, H., Banton, M. C., Inder, K. L., Valk, E., and Rudd, C. E.
- Published
- 2012
3. Specific Augmentation of Treg TCR Signaling and Antigen-Specific Proliferation In Vivo by Anti-CD45RB.: Abstract# 364
- Author
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Camirand, G., Wang, Y., Lu, Y., Rudd, C. E., and Rothstein, D. M.
- Published
- 2012
4. A role for adhesion and degranulation-promoting adapter protein in collagen-induced platelet activation mediated via integrin α2β1
- Author
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JARVIS, G. E., BIHAN, D., HAMAIA, S., PUGH, N., GHEVAERT, C. J. G., PEARCE, A. C., HUGHES, C. E., WATSON, S. P., WARE, J., RUDD, C. E., and FARNDALE, R. W.
- Published
- 2012
- Full Text
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5. A Raf-1-related p110 polypeptide associates with the CD4-p56lck complex in T cells
- Author
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Prasad, K V and Rudd, C E
- Subjects
animal structures ,T-Lymphocytes ,Molecular Sequence Data ,hemic and immune systems ,Protein Serine-Threonine Kinases ,Protein-Tyrosine Kinases ,Lymphocyte Activation ,Proto-Oncogene Proteins c-raf ,Lymphocyte Specific Protein Tyrosine Kinase p56(lck) ,Proto-Oncogene Proteins ,embryonic structures ,CD4 Antigens ,Humans ,Amino Acid Sequence ,Peptides ,Research Article ,HeLa Cells - Abstract
The CD4 and CD8 antigens on T cells have been shown to associate with the Src family member p56lck and a GTP-binding protein, p32. The identification of receptor interactions with intracellular mediators is essential in the elucidation of downstream signals mediated by engagement of these receptor complexes. In this study, we report the detection of an additional 110-kDa polypeptide (p110) associated with the CD4-p56lck complex in human peripheral blood T lymphocytes and leukemic T-cell lines. p110 bound preferentially to CD4-p56lck as an assembled complex and poorly, if at all, to the individual components. p110 was recognized directly by an antiserum to the C-terminal region of the serine/threonine kinase Raf-1 and is related to a p110 polypeptide detected in anti-Raf-1 immunoprecipitates. Despite its association with the CD4-p56lck complex, p110 was found to be phosphorylated predominantly on serine residues. Furthermore, phorbol ester treatment of cells resulted in a transient increase in the detection of p110 associated with CD4-p56lck, concomitant with the modulation of CD4-p56lck from the cell surface. This Raf-1-related p110 is therefore likely to play a role in signals generated from the CD4-p56lck complex. p110 may serve as a bridge between the CD4-p56lck complex and the serine/threonine kinase pathways of T-cell activation.
- Published
- 1992
6. The association between glycosylphosphatidylinositol-anchored proteins and heterotrimeric G protein alpha subunits in lymphocytes.
- Author
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Solomon, K R, primary, Rudd, C E, additional, and Finberg, R W, additional
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- 1996
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7. p56Lck and p59Fyn regulate CD28 binding to phosphatidylinositol 3-kinase, growth factor receptor-bound protein GRB-2, and T cell-specific protein-tyrosine kinase ITK: implications for T-cell costimulation.
- Author
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Raab, M, primary, Cai, Y C, additional, Bunnell, S C, additional, Heyeck, S D, additional, Berg, L J, additional, and Rudd, C E, additional
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- 1995
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8. CTLA-4 binding to the lipid kinase phosphatidylinositol 3-kinase in T cells.
- Author
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Schneider, H, primary, Prasad, K V, additional, Shoelson, S E, additional, and Rudd, C E, additional
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- 1995
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9. The T-cell antigen CD5 acts as a receptor and substrate for the protein-tyrosine kinase p56lck
- Author
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Raab, M, primary, Yamamoto, M, additional, and Rudd, C E, additional
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- 1994
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10. T-cell antigen CD28 interacts with the lipid kinase phosphatidylinositol 3-kinase by a cytoplasmic Tyr(P)-Met-Xaa-Met motif.
- Author
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Prasad, K V, primary, Cai, Y C, additional, Raab, M, additional, Duckworth, B, additional, Cantley, L, additional, Shoelson, S E, additional, and Rudd, C E, additional
- Published
- 1994
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11. Phosphatidylinositol (PI) 3-kinase and PI 4-kinase binding to the CD4-p56lck complex: the p56lck SH3 domain binds to PI 3-kinase but not PI 4-kinase
- Author
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Prasad, K V, primary, Kapeller, R, additional, Janssen, O, additional, Repke, H, additional, Duke-Cohan, J S, additional, Cantley, L C, additional, and Rudd, C E, additional
- Published
- 1993
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12. T cell receptor zeta/CD3-p59fyn(T)-associated p120/130 binds to the SH2 domain of p59fyn(T).
- Author
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da Silva, A J, primary, Janssen, O, additional, and Rudd, C E, additional
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- 1993
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13. Src-homology 3 domain of protein kinase p59fyn mediates binding to phosphatidylinositol 3-kinase in T cells.
- Author
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Prasad, K V, primary, Janssen, O, additional, Kapeller, R, additional, Raab, M, additional, Cantley, L C, additional, and Rudd, C E, additional
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- 1993
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14. A Raf-1-related p110 polypeptide associates with the CD4-p56lck complex in T cells.
- Author
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Prasad, K V, primary and Rudd, C E, additional
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- 1992
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15. CD5 acts as a tyrosine kinase substrate within a receptor complex comprising T-cell receptor zeta chain/CD3 and protein-tyrosine kinases p56lck and p59fyn.
- Author
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Burgess, K E, primary, Yamamoto, M, additional, Prasad, K V, additional, and Rudd, C E, additional
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- 1992
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16. Biosynthesis and post-translational modification of CD6, a T cell signal-transducing molecule.
- Author
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Swack, J A, primary, Mier, J W, additional, Romain, P L, additional, Hull, S R, additional, and Rudd, C E, additional
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- 1991
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17. Resting lymphocyte kinase (Rlk/Txk) targets lymphoid adaptor SLP-76 in the cooperative activation of interleukin-2 transcription in T-cells.
- Author
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Schneider, H, Guerette, B, Guntermann, C, and Rudd, C E
- Abstract
Rlk/Txk is a T-cell-specific member of the Btk/Tec family of tyrosine kinases, whereas SLP-76 is a lymphoid adaptor that is essential for pre-TcR and mature TcR signaling. Although Rlk deficient T-cells show partial defects in T-cell proliferation, Rlk can complement ITK-/- cells with multiple defects in TcR initiated early events and interleukin (IL)-2 production. A key question is the nature of the target of Rlk responsible for bridging the TcR with the activation of IL-2 transcription. In this study, we identify a pathway in which Rlk phosphorylates SLP-76 leading to the phosphorylation of PLCgamma1, activation of ERKs, and the synergistic up-regulation of TcR-driven IL-2 NFAT/AP-1 transcription. Rlk phosphorylated the N-terminal region of SLP-76, a region that has been previously shown to serve as a target for ZAP-70. Loss of N-terminal YESP/YEPP sites of SLP-76 or the Rlk kinase activity attenuated cooperativity between Rlk and SLP-76. These observations support a model where the TcR can utilize Rlk (as well as ZAP-70) in the phosphorylation of key sites in SLP-76 leading to the up-regulation of Th1 preferred cytokine IL-2.
- Published
- 2000
18. Novel isoform of lymphoid adaptor FYN-T-binding protein (FYB-130) interacts with SLP-76 and up-regulates interleukin 2 production.
- Author
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Veale, M, Raab, M, Li, Z, da Silva, A J, Kraeft, S K, Weremowicz, S, Morton, C C, and Rudd, C E
- Abstract
T-cell activation involves the participation of protein-tyrosine kinases p56(lck) and ZAP-70/SYK as well as lymphoid proteins such as SLP-76 and FYB/SLAP. FYB/SLAP has the hallmarks of an adaptor protein that binds to the SH2 domains of the Src kinase FYN-T and SLP-76. Whereas two forms of FYB at 120 and 130 kDa have been identified biochemically, a cDNA encoding only the lower molecular weight isoform has been cloned (termed FYB-120 or SLAP-130). In this study, we report the isolation of an alternative isoform of FYB with a molecular mass of 130 kDa (FYB-130) that has the same structure as FYB-120 except for an insertion of 46 amino acids toward the carboxyl-terminal region of the protein. FYB-120 and FYB-130 share an ability to bind to the SH2 domains of FYN-T and SLP-76, to act as substrates for p59(FYN-T), and to be expressed in the cytoplasm and nucleus of T-cells. Differences were noted between the isoforms in the efficiency of binding to SLP-76 and in the preferential expression of FYB-130 in mature T-cells. When co-expressed together with FYN-T and SLP-76, FYB-130 caused a significant increase in anti-CD3-driven NF-AT transcription. Finally, fluorescence in situ hybridization analysis localized the FYB gene to human chromosome 5 at position p13.1. FYB-130 therefore represents a novel variant of FYB protein that can up-regulate T-cell receptor-driven interleukin 2 production in mature T-cells.
- Published
- 1999
19. FYN-T-FYB-SLP-76 interactions define a T-cell receptor zeta/CD3-mediated tyrosine phosphorylation pathway that up-regulates interleukin 2 transcription in T-cells.
- Author
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Raab, M, Kang, H, da Silva, A, Zhu, X, and Rudd, C E
- Abstract
Protein-tyrosine kinases p56(Lck), SYK, and ZAP-70 and downstream adaptors LAT and SLP-76 have been implicated as essential components in T-cell activation. Another lymphoid-specific adaptor FYB/SLAP has also been identified as a predominant binding partner of SLP-76 and the Src kinase FYN-T, although its role in the activation process has been unclear. In this study, we demonstrate that FYN-T selectively phosphorylates FYB providing a template for the recruitment of FYN-T and SLP-76 SH2 domain binding. This interaction is unusual in its distinct cytoplasmic localization and its long term stable kinetics of phosphorylation. Furthermore, we demonstrate for the first time that the co-expression of all three components of the FYN-T-FYB-SLP-76 matrix can synergistically up-regulate T-cell receptor-driven interleukin 2 transcription activity. These findings document the existence of a T-cell receptor-regulated FYN-T-FYB pathway that interfaces with the adaptor SLP-76 and up-regulates lymphokine production in T-cells.
- Published
- 1999
20. The CD4 receptor is complexed in detergent lysates to a protein-tyrosine kinase (pp58) from human T lymphocytes.
- Author
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Rudd, C E, Trevillyan, J M, Dasgupta, J D, Wong, L L, and Schlossman, S F
- Abstract
The CD4 (T4) antigen is a cell-surface glycoprotein that is expressed predominantly on the surface of helper T cells and has been implicated in the regulation of T-cell activation and in the associative recognition of class II antigens of the major histocompatibility complex. In addition, the CD4 antigen appears to serve as a receptor for the human immunodeficiency virus (HIV). An important question has been whether the CD4 receptor is linked to an intracellular mediator that could regulate the activation of the CD4+ subset. In this paper, we provide preliminary evidence that the CD4 receptor is complexed in detergent lysates to a protein-tyrosine kinase (PTK) of 55-60 kDa, which is expressed specifically in T cells. The PTK is the human analogue of the murine pp56LSTRA (pp56lck) and has significant homology with c-src, c-yes, and other members of the src family. The identification of the PTK associated with CD4 receptor was made by use of an antiserum to a synthetic peptide that was deduced from the DNA sequence of PTK. Two-dimensional nonequilibrium pH gradient gel electrophoresis/NaDodSO4/PAGE revealed the kinase to focus as a heterogeneous collection of spots in the pH range of 4.0-5.0. Furthermore, in vitro phosphorylation revealed the phosphorylation of two additional polypeptides at 40 and 80 kDa, in addition to the autophosphorylation of the PTK at 55-60 kDa. The potential importance of the association between the CD4 receptor and the PTK of T cells is discussed in relation to T-cell activation and HIV infectivity.
- Published
- 1988
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21. The CD4 and CD8 antigens are coupled to a protein-tyrosine kinase (p56lck) that phosphorylates the CD3 complex.
- Author
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Barber, E K, Dasgupta, J D, Schlossman, S F, Trevillyan, J M, and Rudd, C E
- Abstract
Many mammalian receptors have been found to regulate cell growth by virtue of a protein-tyrosine kinase domain in their cytoplasmic tail. We recently described an association of the CD4 antigen with a T-cell-specific protein-tyrosine kinase (p56lck; formerly termed pp58lck; EC 2.7.1.112). This interaction represents a potential mechanism by which T-cell growth may be regulated and offers a model by which other members of the src family (products of c-src, c-yes, c-fgr, etc.) may interact with mammalian growth factor receptors. As in the case of the CD4 antigen, the CD8 antigen appears to serve as a receptor for nonpolymorphic regions of products of the major histocompatibility complex and has been implicated in the regulation of T-cell growth. In this study, we reveal that the human CD8 antigen is also associated with the T-cell-specific protein-tyrosine kinase (p56lck). The associated p56lck kinase was detected by use of both in vitro and in vivo labeling regimes using an antiserum to the C terminus of p56lck. Two-dimensional nonequilibrium pH-gradient gel electrophoresis and sodium dodecyl sulfate/polyacrylamide gel electrophoresis demonstrated the similarity of p56lck to the protein-tyrosine kinase associated with the CD4 antigen. The catalytic activity of p56lck was revealed by the autophosphorylation of the 55- to 60-kDa kinase and the occasional labeling of a 35-kDa protein. Last, we demonstrate directly that members of the CD3 complex, including the gamma, delta, and epsilon chains, as well as a putative zeta subunit, can be phosphorylated at tyrosine residues by the CD4/CD8.p56lck complex.
- Published
- 1989
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22. HLA-D region antigen-associated invariant polypeptides as revealed by two-dimensional gel analysis. Glycosylation and structural inter-relationships.
- Author
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Rudd, C E, Bodmer, J G, Bodmer, W F, and Crumpton, M J
- Abstract
Two-dimensional polyacrylamide gel analyses of immunoprecipitates of HLA-D region antigens prepared from [35S]methionine-labeled B lymphoblastoid cells revealed a number of invariant polypeptides (Ii and theta) that co-precipitate with the alpha and beta polypeptides of the class II (Ia) antigens. The invariant polypeptides comprised at least three Ii spots of Mr = 31,000 (Ii1-Ii3) and a series of six theta spots of Mr = 34,000 (theta 1-theta 6). The structural inter-relationships of these polypeptides have been investigated. Tryptic peptide fingerprints showed that Ii and theta have closely related amino acid sequences. In contrast, the fingerprints of the HLA-DR alpha and beta polypeptides clearly differed from those of theta and Ii as well as from each other. Analyses of immunoprecipitates prepared from cells cultured in the presence of tunicamycin revealed the presence of two N-linked oligosaccharides on each invariant polypeptide and suggested that the more acidic theta polypeptides (theta 1 and theta 2) differed from the other invariant polypeptides by the presence of sialic acid on one or both N-linked oligosaccharides. Removal of sialic acid by neuraminidase simplified the pattern of theta spots into three distinct Ii-related polypeptides. Endo-beta-N-acetylglycosaminidase H digestion indicated that the individual theta polypeptides represent stages in carbohydrate processing whereby Ii with two N-linked immature oligosaccharides are converted initially to theta 6-theta 3 with one immature and one complex, but nonsialylated, oligosaccharide and finally to theta 2-theta 1 with two complex oligosaccharides. Digestion of the theta polypeptides with N-acetylgalactosamine oligosaccharidase indicated that the theta spots are also derived by O-glycosylation from the Ii polypeptides. This assignment is supported by results obtained using monensin to block glycosylation within the Golgi. At least three spots persisted after complete removal of the N- and O-linked oligosaccharides, suggesting the presence of a family of invariant polypeptides differing in amino acid sequence.
- Published
- 1985
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23. Growth factor receptor-bound protein 2 SH2/SH3 domain binding to CD28 and its role in co-signaling.
- Author
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Kim, H H, Tharayil, M, and Rudd, C E
- Abstract
The co-stimulatory antigen CD28 has been shown to bind to several intracellular proteins including phosphatidylinositol 3-kinase, growth factor receptor-bound protein 2 (Grb2), and ITK. Paradoxically, Grb2 and phosphatidylinositol 3-kinase binding has been mapped to a similar pYMNM motif within the CD28 cytoplasmic tail. Given the importance of CD28 co-signaling to T cell function, questions exist regarding the mechanism by which Grb2 binds to CD28, and whether the interaction plays a role in co-stimulation. To biochemically characterize Grb2/CD28 binding, we initially utilized glutathione S-transferase-Grb2 fusion proteins carrying inactivating mutations within the SH2 and SH3 domains of Grb2, and assessed their ability to bind to CD28. In vitro binding experiments indicated that the Grb2 SH2 domain is critical for the association, while the SH3 domain plays an additional role in facilitating optimal binding. Enhanced binding via the SH3 domains was not observed when the C-terminal PXXP motif within CD28 was disrupted, thereby indicating that both SH2 and SH3 domains contribute to CD28 binding. Mutations that alter Grb2 binding were found to block the CD28-dependent interleukin-2 production. Further, tyrosine phosphorylation of Vav and the costimulation-dependent activation of Jun N-terminal kinase was blocked in cells defective in CD28/Grb2 binding. These results provide evidence for an alternate CD28-mediated signaling process involving Grb2 binding to the co-receptor.
- Published
- 1998
24. The Fes protein-tyrosine kinase phosphorylates a subset of macrophage proteins that are involved in cell adhesion and cell-cell signaling.
- Author
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Jücker, M, McKenna, K, da Silva, A J, Rudd, C E, and Feldman, R A
- Abstract
The c-fps/fes proto-oncogene encodes a 92-kDa protein-tyrosine kinase that is expressed at high levels in macrophages. We have previously shown that overexpression of c-fps/fes in a CSF-1-dependent macrophage cell line (BAC1.2F5) partially released these cells from their factor dependence and that this correlated with the tyrosine phosphorylation of a subset of proteins in a tissue-specific manner. We have now identified one of the macrophage substrates of Fes as the crk-associated substrate (Cas) and a second substrate as a 130-kDa protein that has been previously described as a T cell activation-dependent substrate and is unrelated to Cas. Both of these proteins, which have optimal consensus sequences for phosphorylation by Fes, were tightly associated with this kinase through its SH2 domain, suggesting that they were direct substrates of Fes. Remarkably, when the Fes SH2 domain was used as an affinity reagent to identify potential substrates of endogenous Fes in control BAC1.2F5 cells, the phosphotyrosyl proteins that were recognized were the same as those that were specifically phosphorylated when Fes was overexpressed in the same cells. We conclude that the substrates we identified may be structurally related or identical to the physiological targets of this kinase in macrophages. The known functions of Cas and p130 suggest that Fes kinase may play a role in signaling triggered by cell adhesion and cell-cell interactions during immune responses of macrophages.
- Published
- 1997
25. The subdivision of the T4 (CD4) subset on the basis of the differential expression of L-C/T200 antigens.
- Author
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Rudd, C E, Morimoto, C, Wong, L L, and Schlossman, S F
- Abstract
The T4 (CD4) subset of T lymphocytes has been subdivided into two major subsets, a suppressor/inducer subset (T4+,2H4+) and a helper subset (T4+,2H4-) on the basis of the differential expression of the L-C/T200 (CD45) antigens. The 2H4 antigen itself comprises at least three distinct polypeptides at 125,200, and 220 X 10(3) Mr, of which the 200 and 220 X 10(3) Mr polypeptides constitute the highest Mr isoforms of a pool of five distinct L-C/T200 antigens. The T4+,2H4+ subset expresses at least four of these isoforms at 180, 190, 200, and 220 X 10(3) on the cell surface, while the T4+,2H4- subset expresses only the 180 and 190 X 10(3) Mr forms. Pulse-chase analysis and endoglycosidase treatment revealed that the 125 X 10(3) Mr chain of the 2H4 antigen is nonglycosylated, while the 200 and 220 X 10(3) polypeptides are structurally related and derived by N- and O-linked glycosylation from two nascent subunits at 150 and 160 X 10(3) Mr. The function of the T4+,2H4+ subset could be blocked only by an antibody reactive with the L-C/T200 isoforms enriched with O-linked oligosaccharides at 200 and 220 X 10(3) Mr.
- Published
- 1987
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26. Lymphokine regulation of CD45R expression on human T cell clones.
- Author
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Brod, S A, Rudd, C E, Purvee, M, and Hafler, D A
- Abstract
Whether the expression of higher molecular weight isoforms of the T-200 complex represents different lineages of T cells and/or a sequential stage of the differential pathway of T cells has been unclear. Understanding T cell expression of higher molecular weight isoforms of the T-200 complex (CD45R) may be important because of their association with regulation of immune responses. By direct single cell cloning, we observed a number of long-term T cell clones that expressed CD45RA (2H4). CD45RA expression could be further regulated by ionomycin or the cytokines IL-1 and IL-6, but not IL-2, IL-4, or IFN-gamma. These results indicate that CD45RA expression may define T cell lineages of activated T cells partially controlled by the cytokines IL-1 and IL-6. Further, these results may associate regulatory actions of IL-1 and IL-6 with their ability to increase CD45RA expression in subpopulations of human T cells.
- Published
- 1989
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27. Lithium salts as a treatment for COVID-19: Pre-clinical outcomes.
- Author
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Soriano-Torres O, Noa Romero E, González Sosa NL, Enríquez Puertas JM, Fragas Quintero A, García Montero M, Martín Alfonso D, Infante Hernández Y, Lastre M, Rodríguez-Pérez L, Borrego Y, González VE, Vega IG, Ramos Pupo R, Reyes LM, Zumeta Dubé MT, Hernández A 1st, García de la Rosa I, Minguez Suárez A, Alarcón Camejo LA, Rodríguez M, Oliva Hernández R, Rudd CE, and Pérez O
- Subjects
- Animals, Antiviral Agents pharmacology, Antiviral Agents therapeutic use, Cricetinae, Humans, Lithium, SARS-CoV-2, Salts, COVID-19 Drug Treatment
- Abstract
Introduction: Identifying effective drugs for Coronavirus disease 2019 (COVID-19) is urgently needed. An efficient approach is to evaluate whether existing approved drugs have anti-SARS-CoV-2 effects. The antiviral properties of lithium salts have been studied for many years. Their anti-inflammatory and immune-potentiating effects result from the inhibition of glycogen synthase kinase-3., Aims: To obtain pre-clinical evidence on the safety and therapeutic effects of lithium salts in the treatment of COVID-19., Results: Six different concentrations of lithium, ranging 2-12 mmol/L, were evaluated. Lithium inhibited the replication of SARS-CoV-2 virus in a dose-dependent manner with an IC
50 value of 4 mmol/L. Lithium-treated wells showed a significantly higher percentage of monolayer conservation than viral control, particularly at concentrations higher than 6 mmol/L, verified through microscopic observation, the neutral red assay, and the determination of N protein in the supernatants of treated wells. Hamsters treated with lithium showed less intense disease with fewer signs. No lithium-related mortality or overt signs of toxicity were observed during the experiment. A trend of decreasing viral load in nasopharyngeal swabs and lungs was observed in treated hamsters compared to controls., Conclusions: These results provide pre-clinical evidence of the antiviral and immunotherapeutic effects of lithium against SARS-CoV-2, which supports an advance to clinical trials on COVID-19's patients., (Copyright © 2022 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)- Published
- 2022
- Full Text
- View/download PDF
28. Cytotoxic T lymphocyte antigen 4 and CD28 modulate cell surface raft expression in their regulation of T cell function.
- Author
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Martin M, Schneider H, Azouz A, and Rudd CE
- Subjects
- Abatacept, Antigens, CD, CD3 Complex metabolism, CTLA-4 Antigen, Carrier Proteins metabolism, Cells, Cultured, G(M1) Ganglioside metabolism, Humans, Phosphoproteins metabolism, Receptors, Antigen, T-Cell metabolism, Signal Transduction, T-Lymphocytes, Cytotoxic cytology, Adaptor Proteins, Signal Transducing, Antigens, Differentiation metabolism, CD28 Antigens metabolism, Immunoconjugates, Membrane Microdomains metabolism, Membrane Proteins, T-Lymphocytes, Cytotoxic metabolism
- Abstract
Coreceptors CD28 and cytotoxic T lymphocyte antigen (CTLA)-4 have opposing effects on TcR/CD3 activation of T cells. While CD28 enhances and CTLA-4 inhibits activation, the underlying molecular basis of these effects has yet to be established. In this context, ganglioside and cholesterol enriched membrane microdomains (rafts, GEMs) serve as centers of signaling in T cells. Although CD28 can promote TcR/raft colocalization, evidence is lacking on whether the surface expression of membrane rafts can be targeted by CTLA-4 in its modulation of T cell responses. In this study, we demonstrate that both CD28 and CTLA-4 profoundly alter the surface expression of membrane rafts during T cell activation. While CD28 increased expression and the number of peripheral T cells induced to express surface rafts in response to TcR ligation, CTLA-4 potently inhibited both TcR and TcR x CD28 induced raft expression on the surface of T cells. Consistent with this, CD28 increased the presence of the linker of activated T cells (LAT) in purified membrane rafts, while CTLA-4 coligation effectively blocked this increase. Further, the reversal of the CTLA-4 block with CD3/CD28 ligation was accompanied by an increase in surface raft expression and associated LAT. Our observations demonstrate for the first time that CTLA-4 targets the release of rafts to the surface of T cells, and provides a mechanism for the opposing effects of CD28 and CTLA-4 on costimulation.
- Published
- 2001
- Full Text
- View/download PDF
29. CD28 signaling via VAV/SLP-76 adaptors: regulation of cytokine transcription independent of TCR ligation.
- Author
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Raab M, Pfister S, and Rudd CE
- Subjects
- Actins metabolism, Adaptor Proteins, Signal Transducing, Amino Acid Motifs, Animals, B7-1 Antigen genetics, B7-1 Antigen immunology, Biopolymers, CHO Cells, COS Cells, Cell Membrane metabolism, Chlorocebus aethiops, Cricetinae, Cricetulus, DNA-Binding Proteins metabolism, Humans, Interleukin-2 genetics, Interleukin-4 genetics, Jurkat Cells immunology, Ligands, Macromolecular Substances, NFATC Transcription Factors, Phosphoproteins chemistry, Protein Binding, Proto-Oncogene Proteins chemistry, Proto-Oncogene Proteins c-vav, Recombinant Fusion Proteins physiology, Transcription Factors metabolism, rac1 GTP-Binding Protein physiology, CD28 Antigens physiology, Cell Cycle Proteins, Gene Expression Regulation physiology, Interleukin-2 biosynthesis, Interleukin-4 biosynthesis, Lymphocyte Activation physiology, Nuclear Proteins, Phosphoproteins physiology, Proto-Oncogene Proteins physiology, Receptors, Antigen, T-Cell immunology, Signal Transduction physiology, T-Lymphocytes immunology, Transcription, Genetic physiology
- Abstract
Since CD28 provides cosignals in T cell responses, a key question is whether the coreceptor operates exclusively via TCRzeta/CD3 or also operates as an independent signaling unit. In this study, we show that CD28 can cooperate with VAV/SLP-76 adaptors to upregulate interleukin 2/4 transcription independently of TCR ligation. CD28 signaling is dependent on VAV/SLP-76 complex formation and induces membrane localization of these complexes. CD28-VAV/SLP-76 also functions in nonlymphoid cells to promote nuclear entry of NFAT, indicating that these adaptors are the only lymphoid components needed for this pathway. Further downstream, CD28-VAV/SLP-76 synergizes with Rac1 and causes F-actin remodelling proximal to receptor. Autonomous CD28 signaling may account for the distinct nature of the second signal and in trans amplification of T cell responses.
- Published
- 2001
- Full Text
- View/download PDF
30. Adaptor FYB (Fyn-binding protein) regulates integrin-mediated adhesion and mediator release: differential involvement of the FYB SH3 domain.
- Author
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Geng L, Pfister S, Kraeft SK, and Rudd CE
- Subjects
- Carrier Proteins genetics, Cloning, Molecular, Fibronectins immunology, Humans, Ionomycin pharmacology, Microscopy, Confocal, Tetradecanoylphorbol Acetate pharmacology, Transfection, src Homology Domains, Adaptor Proteins, Signal Transducing, Carrier Proteins immunology, Cell Adhesion immunology, Integrins immunology
- Abstract
Aggregation of the high-affinity IgE receptor (FcepsilonRI) on mast cells activates a tyrosine phosphorylation cascade that is required for adhesion and degranulation events leading to the release of histamine and other inflammatory mediators. The full range of intracellular mediators that regulate this process is unknown. Recent studies have identified a group of immune cell-specific adaptor proteins that include linker for activation of T-cell (LAT), SH2-domain-containing leukocyte protein (SLP-76), and Fyn-T-binding protein (FYB)/SLP-76-associated protein (SLAP). In this study, we demonstrate that FYB can up-regulate integrin-mediated adhesion to fibronectin and mediator release in RBL-2H3 mast cells. The regulation of these two events could be distinguished from each other by the requirement of the FYB SH3 domain in beta-hexosaminidase release, but not adhesion, and the up-regulation of mediator release by FYB in nonadherent cells. FcepsilonRI aggregation increased FYB tyrosine phosphorylation, whereas confocal immunofluorescence microscopy showed that FYB colocalizes with F-actin in membrane ruffles and plaques. Our findings identify FYB as a regulator of integrin-mediated adhesion and degranulation events, which, in the case of mast cells, has potential applications to inflammatory and allergic responses.
- Published
- 2001
- Full Text
- View/download PDF
31. Positive regulation of T cell activation and integrin adhesion by the adapter Fyb/Slap.
- Author
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Griffiths EK, Krawczyk C, Kong YY, Raab M, Hyduk SJ, Bouchard D, Chan VS, Kozieradzki I, Oliveira-Dos-Santos AJ, Wakeham A, Ohashi PS, Cybulsky MI, Rudd CE, and Penninger JM
- Subjects
- Actins metabolism, Animals, Antigens, CD metabolism, Antigens, Differentiation, T-Lymphocyte metabolism, B-Lymphocytes immunology, CD3 Complex metabolism, Carrier Proteins genetics, Cell Adhesion, Cell Adhesion Molecules metabolism, Chimera, Gene Targeting, Humans, Immunization, Immunoglobulin G biosynthesis, Intercellular Adhesion Molecule-1 metabolism, Interferon-gamma biosynthesis, Interleukin-2 biosynthesis, Interleukin-2 pharmacology, Lectins, C-Type, Lymphocyte Function-Associated Antigen-1 metabolism, Mice, Phosphoproteins genetics, Receptors, Antigen, T-Cell immunology, Receptors, Antigen, T-Cell metabolism, Receptors, Interleukin-2 metabolism, Recombinant Proteins metabolism, Signal Transduction, T-Lymphocytes immunology, T-Lymphocytes metabolism, Adaptor Proteins, Signal Transducing, Carrier Proteins physiology, Integrins metabolism, Lymphocyte Activation, Phosphoproteins physiology, T-Lymphocytes physiology
- Abstract
The molecular adapter Fyb/Slap regulates signaling downstream of the T cell receptor (TCR), but whether it plays a positive or negative role is controversial. We demonstrate that Fyb/Slap-deficient T cells exhibit defective proliferation and cytokine production in response to TCR stimulation. Fyb/Slap is also required in vivo for T cell-dependent immune responses. Functionally, Fyb/Slap has no apparent role in the activation of known TCR signaling pathways, F-actin polymerization, or TCR clustering. Rather, Fyb/Slap regulates TCR-induced integrin clustering and adhesion. Thus, Fyb/Slap is the first molecular adapter to be identified that couples TCR stimulation to the avidity modulation of integrins governing T cell adhesion.
- Published
- 2001
- Full Text
- View/download PDF
32. CTLA-4 blockade of antigen-induced cell death.
- Author
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da Rocha Dias S and Rudd CE
- Subjects
- Abatacept, Amino Acid Motifs, Animals, Antigens, CD, Antigens, Differentiation genetics, Apoptosis drug effects, CTLA-4 Antigen, Fas Ligand Protein, Gene Expression Regulation, Hybridomas, Interleukin-2 biosynthesis, Interleukin-2 genetics, Interleukin-2 metabolism, Lymphocyte Activation, Membrane Glycoproteins biosynthesis, Membrane Glycoproteins genetics, Mice, Muromonab-CD3 pharmacology, Point Mutation, Receptor-CD3 Complex, Antigen, T-Cell immunology, Signal Transduction, T-Lymphocytes, Cytotoxic drug effects, T-Lymphocytes, Cytotoxic metabolism, Transfection, Antigens immunology, Antigens, Differentiation physiology, Apoptosis physiology, Immunoconjugates, T-Lymphocytes, Cytotoxic immunology
- Abstract
While cytotoxic T lymphocyte antigen-4 (CTLA-4) negatively regulates T-cell receptor (TCR)-driven interleukin (IL)-2 production and proliferation, little is known regarding whether the coreceptor has the capacity to inhibit other events, such as Fas ligand (FasL) expression and antigen-induced cell death (AICD). In this study, it is shown that CTLA-4 expressed in a T-cell hybridoma can elicit a potent block of FasL expression and AICD. Inhibition occurred independently of CTLA-4 blockage of IL-2 production and was partially reversed by a single mutation in the cytoplasmic YVKM motif. These findings indicate that CTLA-4 can block TCR signaling prior to bifurcation of signals leading to IL-2 production and apoptosis.
- Published
- 2001
- Full Text
- View/download PDF
33. The CD28-related molecule ICOS is required for effective T cell-dependent immune responses.
- Author
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Coyle AJ, Lehar S, Lloyd C, Tian J, Delaney T, Manning S, Nguyen T, Burwell T, Schneider H, Gonzalo JA, Gosselin M, Owen LR, Rudd CE, and Gutierrez-Ramos JC
- Subjects
- Abatacept, Animals, Antigens, CD, Antigens, Differentiation immunology, Antigens, Differentiation, T-Lymphocyte genetics, CTLA-4 Antigen, Cloning, Molecular, Cytokines biosynthesis, Gene Expression, Humans, Immunoglobulin G biosynthesis, Immunoglobulin M biosynthesis, Inducible T-Cell Co-Stimulator Protein, Jurkat Cells, Male, Mice, Mice, Inbred BALB C, Mice, Transgenic, Signal Transduction, Antigens, Differentiation, T-Lymphocyte immunology, CD28 Antigens immunology, Immunoconjugates, Th1 Cells immunology, Th2 Cells immunology
- Abstract
While CD28 is critical for expansion of naive T cells, recent evidence suggests that the activation of effector T cells is largely independent of CD28/B7. We suggest that ICOS, the third member of the CD28/CTLA-4 family, plays an important role in production of IL-2, IL-4, IL-5, and IFNgamma from recently activated T cells and contributes to T cell-dependent B help in vivo. Inhibition of ICOS attenuates lung mucosal inflammation induced by Th2 but not Th1 effector populations. Our data indicate a critical function for the third member of the CD28 family in T cell-dependent immune responses.
- Published
- 2000
- Full Text
- View/download PDF
34. SH3 domain recognition of a proline-independent tyrosine-based RKxxYxxY motif in immune cell adaptor SKAP55.
- Author
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Kang H, Freund C, Duke-Cohan JS, Musacchio A, Wagner G, and Rudd CE
- Subjects
- Animals, Arginine, Gene Expression Regulation, Histocompatibility Antigens Class I metabolism, Histocompatibility Antigens Class II metabolism, Humans, Interleukin-2 biosynthesis, Jurkat Cells, Lysine, Mice, Models, Molecular, Nuclear Magnetic Resonance, Biomolecular, Proline, Protein Binding, Receptors, Antigen, T-Cell metabolism, Signal Transduction, Spleen cytology, Surface Plasmon Resonance, Amino Acids, Diamino, Histocompatibility Antigens metabolism, Phosphoproteins metabolism, Tyrosine, src Homology Domains
- Abstract
Src-homology 3 (SH3) domains recognize PXXP core motif preceded or followed by positively charged residue(s). Whether SH3 domains recognize motifs other than proline-based sequences is unclear. In this study, we report SH3 domain binding to a novel proline-independent motif in immune cell adaptor SKAP55, which is comprised of two N-terminal lysine and arginine residues followed by two tyrosines (i.e. RKxxYxxY). Domains capable of binding to class I proline motifs bound to the motif, while the class II domains failed to bind. Peptide precipitation, alanine scanning and in vivo co-expression studies demonstrated a requirement for the arginine, lysine and tandem tyrosines of the motif. Two-dimensional NMR analysis of the peptide bound FYN-SH3 domain showed overlap with the binding site of a proline-rich peptide on the charged surface of the SH3 domain, while resonance signals for other residues (W119, W120, Y137) were not perturbed by the RKGDYASY based peptide. Expression of the RKGDYASY peptide potently inhibited TcRzeta/CD3-mediated NF-AT transcription in T cells. Our findings extend the repertoire of SH3 domain binding motifs to include a tyrosine-based motif and demonstrate a regulatory role for this motif in receptor signaling.
- Published
- 2000
- Full Text
- View/download PDF
35. Lymphocyte signaling: Cbl sets the threshold for autoimmunity.
- Author
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Rudd CE and Schneider H
- Subjects
- Animals, CD28 Antigens immunology, Mice, Proto-Oncogene Proteins c-cbl, Receptors, Antigen, T-Cell immunology, Ubiquitin-Protein Ligases, Adaptor Proteins, Signal Transducing, Autoimmunity immunology, B-Lymphocytes immunology, Carrier Proteins immunology, Ligases immunology, Phosphoproteins immunology, Proto-Oncogene Proteins immunology, Signal Transduction immunology, T-Lymphocytes immunology
- Abstract
Cbl, a negative regulator of immune signaling, has recently been shown to act as a ubiquitin-protein ligase. Further, two new papers describing Cbl-b-deficient mice suggest that Cbl-b sets the threshold of signaling in T and B cells and prevents the development of autoimmunity.
- Published
- 2000
- Full Text
- View/download PDF
36. SHPS-1 is a scaffold for assembling distinct adhesion-regulated multi-protein complexes in macrophages.
- Author
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Timms JF, Swanson KD, Marie-Cardine A, Raab M, Rudd CE, Schraven B, and Neel BG
- Subjects
- Animals, COS Cells, Cell Adhesion, Cell Adhesion Molecules analysis, Cell Adhesion Molecules metabolism, Focal Adhesion Kinase 1, Focal Adhesion Kinase 2, Focal Adhesion Protein-Tyrosine Kinases, Immunoblotting, Macrophages drug effects, Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase pharmacology, Mice, Mice, Mutant Strains, Nuclear Proteins analysis, Nuclear Proteins metabolism, Phosphoproteins analysis, Phosphoproteins metabolism, Protein-Tyrosine Kinases analysis, Protein-Tyrosine Kinases metabolism, Sulfones analysis, Sulfones metabolism, Uridine analogs & derivatives, Uridine analysis, Uridine metabolism, Antigens, Differentiation, Bone Marrow Cells chemistry, Macrophages chemistry, Membrane Glycoproteins metabolism, Neural Cell Adhesion Molecule L1, Neural Cell Adhesion Molecules metabolism, Protein Folding, Receptors, Immunologic
- Abstract
Inhibitory immunoreceptors downregulate signaling by recruiting Src homology 2 (SH2) domain-containing tyrosine and/or lipid phosphatases to activating receptor complexes [1]. There are indications that some inhibitory receptors might also perform other functions [2] [3]. In adherent macrophages, two inhibitory receptors, SHPS-1 and PIR-B, are the major proteins binding to the tyrosine phosphatase SHP-1. SHPS-1 also associates with two tyrosine-phosphorylated proteins (pp55 and pp130) and a protein tyrosine kinase [4]. Here, we have identified pp55 and pp130 as the adaptor molecules SKAP55hom/R (Src-kinase-associated protein of 55 kDa homologue) and FYB/SLAP-130 (Fyn-binding protein/SLP-76-associated protein of 130 kDa), respectively, and the tyrosine kinase activity as PYK2. Two distinct SHPS-1 complexes were formed, one containing SKAP55hom/R and FYB/SLAP-130, and the other containing PYK2. Recruitment of FYB/SLAP-130 to SHPS-1 required SKAP55hom/R, whereas PYK2 associated with SHPS-1 independently. Formation of both complexes was independent of SHP-1 and tyrosine phosphorylation of SHPS-1. Finally, tyrosine phosphorylation of members of the SHPS-1 complexes was regulated by integrin-mediated adhesion. Thus, SHPS-1 provides a scaffold for the assembly of multi-protein complexes that might both transmit adhesion-regulated signals and help terminate such signals through SHP-1-directed dephosphorylation. Other inhibitory immunoreceptors might have similar scaffold-like functions.
- Published
- 1999
- Full Text
- View/download PDF
37. Adaptors and molecular scaffolds in immune cell signaling.
- Author
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Rudd CE
- Subjects
- B-Lymphocytes immunology, Enzyme Precursors immunology, Intracellular Signaling Peptides and Proteins, Protein-Tyrosine Kinases immunology, Receptors, Antigen, T-Cell immunology, Syk Kinase, ZAP-70 Protein-Tyrosine Kinase, Signal Transduction, T-Lymphocytes immunology
- Published
- 1999
- Full Text
- View/download PDF
38. Lymphocyte signaling: adapting new adaptors.
- Author
-
Rudd CE
- Subjects
- Adaptation, Physiological, Carrier Proteins metabolism, Membrane Proteins metabolism, Protein-Tyrosine Kinases metabolism, ZAP-70 Protein-Tyrosine Kinase, Adaptor Proteins, Signal Transducing, B-Lymphocytes metabolism, Phosphoproteins metabolism, Receptors, Antigen, B-Cell metabolism, Receptors, Antigen, T-Cell metabolism, Signal Transduction, T-Lymphocytes metabolism
- Abstract
New developments in lymphocyte signaling have revealed insights into the proximal phosphorylation events associated with the T-cell receptor and into the importance of the T-cell adaptor protein Slp-76 and its B-cell homolog in the transduction of the signal from the antigen receptor.
- Published
- 1998
- Full Text
- View/download PDF
39. FYB (FYN binding protein) serves as a binding partner for lymphoid protein and FYN kinase substrate SKAP55 and a SKAP55-related protein in T cells.
- Author
-
Liu J, Kang H, Raab M, da Silva AJ, Kraeft SK, and Rudd CE
- Subjects
- Amino Acid Sequence, Animals, COS Cells, Intracellular Signaling Peptides and Proteins, Mice, Microscopy, Fluorescence, Molecular Sequence Data, Phosphoproteins chemistry, Phosphorylation, Proteins chemistry, Proto-Oncogene Proteins c-fyn, Sequence Homology, Amino Acid, Substrate Specificity, T-Lymphocytes enzymology, Adaptor Proteins, Signal Transducing, Carrier Proteins metabolism, Phosphoproteins metabolism, Protein-Tyrosine Kinases metabolism, Proteins metabolism, Proto-Oncogene Proteins metabolism, T-Lymphocytes metabolism
- Abstract
TcRzeta/CD3 ligation initiates a signaling cascade involving CD4/CD8-p56(lck), p59(fyn), and ZAP-70, as well as lymphoid downstream proteins VAV, SLP-76, and FYB/SLAP. A current question concerns the nature of the downstream binding partner(s) of FYB in T cells. In this study, using a two-hybrid screen with FYB as bait, we have identified eight clones, four of which correspond to the recently published lymphoid protein SKAP55, and two which correspond to a related protein with some 44% homology to SKAP55 (termed SKAP55-related protein, SKAP55R). The SKAP55 clones showed only minor differences (two substitutions and one residue deletion) from SKAP55. SKAP55R has the same overall structure as SKAP55 except for the presence of a unique N terminus with a well-defined coiled-coil domain. Both SKAP55 and SKAP55R were found to bind FYB through their SH3 domains and to act as substrates for the FYN kinase in T cells. Furthermore, immunofluorescence confocal microscopy showed that FYB and SKAP55 colocalize in the perinuclear region of cells. SKAP55 also colocalizes with another FYB binding protein, SLP-76. Taken together, these observations demonstrate that FYB is part of an interactive matrix with SKAP55 and a SKAP55-related protein.
- Published
- 1998
- Full Text
- View/download PDF
40. Cloning of a novel T-cell protein FYB that binds FYN and SH2-domain-containing leukocyte protein 76 and modulates interleukin 2 production.
- Author
-
da Silva AJ, Li Z, de Vera C, Canto E, Findell P, and Rudd CE
- Subjects
- Amino Acid Sequence, Carrier Proteins metabolism, Cloning, Molecular, Humans, Interleukin-2 genetics, Jurkat Cells, Membrane Proteins metabolism, Molecular Sequence Data, Phosphoproteins genetics, Protein Binding, Protein-Tyrosine Kinases genetics, Protein-Tyrosine Kinases metabolism, Proteins metabolism, Proto-Oncogene Proteins metabolism, Proto-Oncogene Proteins c-fyn, Receptors, Antigen, T-Cell metabolism, Signal Transduction, Adaptor Proteins, Signal Transducing, Carrier Proteins genetics, Interleukin-2 metabolism, Phosphoproteins metabolism, Proteins genetics, Proto-Oncogene Proteins genetics, T-Lymphocytes metabolism
- Abstract
T cell receptor zeta (TcRzeta)/CD3 ligation initiates a signaling cascade that involves src kinases p56(lck) and zeta-associated protein 70, leading to the phosphorylation of substrates such as TcRzeta, Vav, SH2-domain-containing leukocyte protein 76 (SLP-76), cbl, and p120/130. FYN binding protein (FYB or p120/130) associates with p59(fyn), the TcRzeta/CD3 complex, and becomes tyrosine-phosphorylated in response to receptor ligation. In this study, we report the cDNA cloning of human and murine FYB and show that it is restricted in expression to T cells and myeloid cells and possesses an overall unique hydrophilic sequence with several tyrosine-based motifs, proline-based type I and type II SH3 domain binding motifs, several putative lysine/glutamic acid-rich nuclear localization motifs, and a SH3-like domain. In addition to binding the src kinase p59(fyn), FYB binds specifically to the hematopoietic signaling protein SLP-76, an interaction mediated by the SLP-76 SH2 domain. In keeping with this, expression of FYB augmented interleukin 2 secretion from a T cell hybridoma, DC27.10, in response to TcRzeta/CD3 ligation. FYB is therefore a novel hematopoietic protein that acts as a component of the FYN and SLP-76 signaling cascades in T cells.
- Published
- 1997
- Full Text
- View/download PDF
41. Regulation of Vav-SLP-76 binding by ZAP-70 and its relevance to TCR zeta/CD3 induction of interleukin-2.
- Author
-
Raab M, da Silva AJ, Findell PR, and Rudd CE
- Subjects
- Adaptor Proteins, Signal Transducing, Amino Acid Sequence, Animals, Cell Line, Humans, Jurkat Cells, Mice, Molecular Sequence Data, Protein Binding drug effects, Proto-Oncogene Proteins c-vav, T-Lymphocytes drug effects, T-Lymphocytes metabolism, ZAP-70 Protein-Tyrosine Kinase, Interleukin-2 biosynthesis, Interleukin-2 metabolism, Membrane Proteins immunology, Oncogene Proteins drug effects, Oncogene Proteins metabolism, Phosphoproteins drug effects, Phosphoproteins metabolism, Protein-Tyrosine Kinases pharmacology, Receptor-CD3 Complex, Antigen, T-Cell immunology, Receptors, Antigen, T-Cell immunology
- Abstract
T cell activation stimulates p56(lck), p59(fyn), ZAP-70, Vav-SLP-76 binding, and IL-2 transcription. Major questions concern the tyrosine-kinase and relevant site(s) needed for Vav-SLP-76 complex formation and its role in IL-2 production. Here, we show that of the three kinases, only ZAP-70 phosphorylates SLP-76 at specific sites that allow Vav SH2 domain binding. Therefore, while p56(lck) regulates proximal events, ZAP-70 acts downstream on targets such as SLP-76. We also show by in vitro and in vivo analysis that two SLP-76 pYESP motifs (Y113 and Y128) mediate binding, the first being more efficient. A third pYEPP motif (Y145) failed to bind. Finally, TCR zeta CD3 ligation of T cell hybridoma DC27.10 induces IL-2 production without detectable Vav-SLP-76 binding. Therefore, despite effects of Vav-SLP-76 on IL-2 expression, Vav-SLP-76 binding per se is not essential for IL-2 production in all T cells.
- Published
- 1997
- Full Text
- View/download PDF
42. Upstream-downstream: CD28 cosignaling pathways and T cell function.
- Author
-
Rudd CE
- Subjects
- Amino Acid Sequence, Animals, Humans, Molecular Sequence Data, CD28 Antigens physiology, Signal Transduction immunology, T-Lymphocytes immunology
- Published
- 1996
- Full Text
- View/download PDF
43. Selective CD28pYMNM mutations implicate phosphatidylinositol 3-kinase in CD86-CD28-mediated costimulation.
- Author
-
Cai YC, Cefai D, Schneider H, Raab M, Nabavi N, and Rudd CE
- Subjects
- Animals, B7-2 Antigen, CD28 Antigens genetics, CHO Cells, Cricetinae, Humans, Interleukin-2 biosynthesis, Mice, Mutation, Phosphatidylinositol 3-Kinases, Signal Transduction, Antigens, CD immunology, CD28 Antigens immunology, Lymphocyte Activation immunology, Membrane Glycoproteins immunology, Phosphotransferases (Alcohol Group Acceptor) immunology, T-Lymphocytes immunology
- Abstract
CD28 costimulatory signals are required for lymphokine production and T cell proliferation. CD28 signaling recruits the intracellular proteins PI 3-kinase, ITK, and GRB-2/SOS. PI 3-kinase and GRB-2/SOS bind the CD28 cytoplasmic pYMNM motif via SH2 domains. We generated CD28 pYMNM mutants and found that Y191 mutation (Y191CD28F) disrupted both PI 3-kinase and GRB-2 binding, while M194 mutation (M194CD28C) disrupted only PI 3-kinase binding. Both mutants still bound ITK. We have assessed the ability of these selective mutants to support IL-2 production upon TCR zeta/CD3 ligation in the presence of CHO-CD86 (B7-2) cells. Both Y191CD28F and M194CD28C mutants failed to generate IL-2. These data directly implicate PI 3-kinase in CD28-mediated costimulation leading to IL-2 secretion. Wortmannin, an inhibitor of PI 3-kinase, induced cell apoptosis and as such was unsuitable for use in this study.
- Published
- 1995
- Full Text
- View/download PDF
44. Identification of two SH3-binding motifs in the regulatory subunit of phosphatidylinositol 3-kinase.
- Author
-
Kapeller R, Prasad KV, Janssen O, Hou W, Schaffhausen BS, Rudd CE, and Cantley LC
- Subjects
- Amino Acid Sequence, Binding Sites, Cell Line, Cloning, Molecular, Glutathione Transferase isolation & purification, Humans, Macromolecular Substances, Molecular Sequence Data, Phosphatidylinositol 3-Kinases, Phosphotransferases (Alcohol Group Acceptor) isolation & purification, Phosphotransferases (Alcohol Group Acceptor) metabolism, Precursor Cell Lymphoblastic Leukemia-Lymphoma, Proline, Recombinant Fusion Proteins chemistry, Recombinant Fusion Proteins isolation & purification, Recombinant Fusion Proteins metabolism, Sequence Homology, Amino Acid, Tumor Cells, Cultured, Phosphotransferases (Alcohol Group Acceptor) chemistry
- Abstract
Src homology 3 (SH3) domains have been recently shown to bind to proline-rich sequences contained in 3BP1, 3BP2, and SOS. In a recent study we demonstrated that phosphatidylinositol 3-kinase (PI 3-kinase) associates with the Fyn SH3 domain. Here we show that p85, the regulatory subunit of PI 3-kinase, binds directly to the SH3 domains of Abl, Lck, Fyn, and p85 itself. An examination of p85 amino acid sequence revealed two proline-rich sequences in its N-terminal region similar to those present in 3BP1, 3BP2, and SOS. To test whether these sequences mediate the association of p85 with SH3 domains two peptides with amino acid composition corresponding to the p85 alpha proline-rich sequences were synthesized and used in competition assays. Both peptides worked equally well in inhibiting the binding of PI 3-kinase activity and p85 alpha to Fyn SH3 domain, whereas a control peptide had no effect. These results indicate that, as in 3BP1 and SOS, the proline-rich sequences in p85 mediate its interaction with SH3 domains. These results also suggest that the SH3 domain of p85 may "self-associate" with the proline-rich motifs of the same subunit as part of the PI 3-kinase regulatory mechanism.
- Published
- 1994
45. Regulation of CD4-p56lck-associated phosphatidylinositol 3-kinase (PI 3-kinase) and phosphatidylinositol 4-kinase (PI 4-kinase).
- Author
-
Prasad KV, Kapeller R, Janssen O, Duke-Cohan JS, Repke H, Cantley LC, and Rudd CE
- Subjects
- 1-Phosphatidylinositol 4-Kinase, Cell Line, Cross-Linking Reagents, HIV Envelope Protein gp120 metabolism, HIV-1 metabolism, Humans, Lymphocyte Specific Protein Tyrosine Kinase p56(lck), Phosphatidylinositol 3-Kinases, Precursor Cell Lymphoblastic Leukemia-Lymphoma, T-Lymphocytes immunology, Tumor Cells, Cultured, CD4 Antigens metabolism, Phosphotransferases (Alcohol Group Acceptor) metabolism, Protein-Tyrosine Kinases metabolism, T-Lymphocytes metabolism
- Abstract
CD4 serves as a receptor for MHC class II antigens and as a receptor for the human immunodeficiency virus (HIV-1) viral coat protein gp120. It is coupled to the protein-tyrosine kinase p56lck, an interaction necessary for an optimal response of certain T cells to antigen. Although anti-CD4 crosslinking may increase lck activity, the effects of HIV-1 gp120 have been controversial. Activated protein-tyrosine kinases are known to associate with certain intracellular proteins possessing src-homology regions (SH-2 domains) such as phosphatidylinositol 3-kinase (PI 3-kinase). In this paper, we demonstrate that the CD4:p56lck complex associates with significant amounts of phosphatidylinositol (PI) kinase activity. High pressure liquid chromatographic (HPLC) analysis of the reaction products demonstrated the presence of phosphatidylinositol 3-phosphate (PI 3-P) and phosphatidylinositol 4-phosphate (PI 4-P), thus indicating that PI 3 and PI 4 kinases associate with CD4-p56lck. The p85 subunit of PI 3-kinase was also detected in anti-CD4 immunoprecipitates by immunoblotting with anti-p85 antiserum. Significantly, p56lck binding to CD4 appears to be necessary for the detection of lipid kinase activity associated with p56lck. Also, anti-HIV gp120 and anti-CD4 crosslinking induced a 10-15-fold increase in levels of both PI 3- and PI 4-kinase activity in anti-CD4 precipitates. Stimulation of CD4-p56lck-linked PI kinases by crosslinked HIV-1 gp120 may play a role in HIV-1-induced immune defects.
- Published
- 1993
- Full Text
- View/download PDF
46. A 72-kilodalton fyn-related polypeptide (p72fyn-R) binds to the antigen-receptor/CD3 (TcR/CD3) complex.
- Author
-
da Silva AJ and Rudd CE
- Subjects
- Alkaline Phosphatase metabolism, Animals, Cricetinae, Electrophoresis, Gel, Two-Dimensional, Mice, Peptide Mapping, Proto-Oncogene Proteins c-fyn, Tumor Cells, Cultured, Proto-Oncogene Proteins metabolism, Receptor-CD3 Complex, Antigen, T-Cell metabolism
- Abstract
Protein-tyrosine kinases play crucial roles in the activation and transformation of T lymphocytes. In this study, we have identified a variant of the fyn kinase at 70-72 kDa (termed p72fyn-R) that can preferentially associate with the TcR/CD3 complex in certain T cells. Phosphoamine acid analysis revealed that the CD3-associated p72fyn-R is labeled on both tyrosine and serine/threonine residues. TcR/CD3-associated p72fyn-R could be specifically reprecipitated using anti-fyn antisera to both the N and C terminus of p59fyn. In addition, two-dimensional phosphotryptic peptide map patterns of TcR/CD3-associated p72fyn and anti-fyn-precipitable p72 were identical. By contrast, a comparison of p72fyn-R and p62fyn showed similarities and differences. p72fyn-R possesses a peptide corresponding to the autophosphorylation site that migrates in the same position as found for p59/62fyn. However, p72fyn-R possessed at least four novel phosphorylated sites labeled on serine and threonine residues that are absent in the p62fyn pattern. Phosphatase digestion experiments indicated that p72fyn-R is more resistant to dephosphorylation than p59/62fyn. Two-dimensional phosphotryptic analysis indicated that the novel serine/threonine phosphorylation sites were responsible for the resistance to phosphatase digestion. Although the exact nature of the relationship between p72fyn-R and p59/62fyn remains undetermined, these data indicate that TcR/CD3 may utilize novel variants of src-related kinases in the generation of signals which regulate T-cell growth.
- Published
- 1993
47. A 32-kD GTP-binding protein associated with the CD4-p56lck and CD8-p56lck T cell receptor complexes.
- Author
-
Telfer JC and Rudd CE
- Subjects
- Amino Acid Sequence, Cell Line, Humans, Lymphocyte Specific Protein Tyrosine Kinase p56(lck), Molecular Sequence Data, Phosphoproteins metabolism, Precipitin Tests, Protein Binding, CD4 Antigens metabolism, CD8 Antigens metabolism, GTP-Binding Proteins metabolism, Protein-Tyrosine Kinases metabolism, Receptors, Antigen, T-Cell metabolism
- Abstract
The guanosine triphosphate (GTP)-binding proteins include signal-transducing heterotrimeric G proteins (for example, Gs, Gi), smaller GTP-binding proteins that function in protein sorting, and the oncogenic protein p21ras. The T cell receptor complexes CD4-p56lck and CD8-p56lck were found to include a 32- to 33-kilodalton phosphoprotein (p32) that was recognized by an antiserum to a consensus GTP-binding region in G proteins. Immunoprecipitated CD4 and CD8 complexes bound GTP and hydrolyzed it to guanosine diphosphate (GDP). The p32 protein was covalently linked to [alpha-32P]GTP by ultraviolet photoaffinity labeling. These results demonstrate an interaction between T cell receptor complexes and an intracellular GTP-binding protein.
- Published
- 1991
- Full Text
- View/download PDF
48. A novel epitope of the LFA-1 antigen which can distinguish killer effector and suppressor cells in human CD8 cells.
- Author
-
Morimoto C, Rudd CE, Letvin NL, and Schlossman SF
- Subjects
- Animals, Antibodies, Monoclonal, Antigens, Surface immunology, Electrophoresis, Polyacrylamide Gel, Humans, Immunosorbent Techniques, Lymphocyte Culture Test, Mixed, Lymphocyte Function-Associated Antigen-1, Mice, Mice, Inbred BALB C, Molecular Weight, T-Lymphocytes, Cytotoxic immunology, Antigens, Differentiation, T-Lymphocyte immunology, Antigens, Surface analysis, Epitopes analysis, Killer Cells, Natural immunology, T-Lymphocytes, Regulatory immunology
- Abstract
The CD4 subset of cells displays helper/inducer activity and recognizes class II antigens of the major histocompatibility complex (MHC), while the CD8 subset recognizes class I MHC antigens and exhibits cytotoxic or suppressor function. Considerable functional as well as corresponding phenotypic heterogeneity exists within the two major T cell subsets. Although the CD8+ population contains pre-cytotoxic, cytotoxic, pre-suppressor and suppressor effector T cells, these distinctions still rest largely on the use of functional assays. Attempts have been made to define the CD8+ precursor of the killer cell with new monoclonal antibodies. But more precise phenotypic distinctions between the functional subpopulations within CD8+ cells will be needed. We have now developed a monoclonal antibody, anti-S6F1 which can distinguish killer effector and suppressor effector cells in CD8 lymphocyte populations. The cell-surface structure defined by this antibody comprises two glycoproteins with relative molecular mass (Mr) 180K and 95K respectively. Also sequential immunoprecipitation studies and two dimensional gel electrophoresis indicate that anti-S6F1 recognizes a novel epitope on the LFA-1 antigen.
- Published
- 1987
- Full Text
- View/download PDF
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