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Ronald D Vale - One of the best experts on this subject based on the ideXlab platform.

  • a composition dependent molecular clutch between t cell Signaling clusters and actin
    bioRxiv, 2018
    Co-Authors: Jonathon A Ditlev, Anthony R Vega, Darius V Koster, Ashley M Lakoduk, Ronald D Vale, Satyajit Mayor, Khuloud Jaqaman, Michael K Rosen
    Abstract:

    During T cell activation, phase-separated Signaling protein clusters are moved toward the center of the immunological synapse by two distinct, concentric actin networks. How clusters move with actin is unknown. We observed that clusters lose the adaptor protein Nck as the clusters move across the boundary of the two actin networks. Biochemically reconstituted clusters with weak actin binders, Nck and its ligand N-WASP, promoted the strong association and movement of clusters with mobile actomyosin filaments. Clusters lacking these components were instead propelled by mechanical interactions. Basic elements of Nck and N-WASP coupled clusters to actin in vitro, and clusters constitutively containing basic elements moved aberrantly in cells. We propose that Nck and N-WASP act as a clutch between clusters and actin, and that changes in composition of these condensates enable cluster movement by the distinct dynamics of actin networks in different regions of the immunological synapse.

  • in vitro reconstitution of t cell receptor mediated segregation of the cd45 phosphatase
    Proceedings of the National Academy of Sciences of the United States of America, 2017
    Co-Authors: Ronald D Vale, Catherine B Carbone, Nadja Kern, Ricardo A Fernandes, Enfu Hui, Christopher K Garcia
    Abstract:

    Abstract T cell Signaling initiates upon the binding of peptide-loaded MHC (pMHC) on an antigen-presenting cell to the T cell receptor (TCR) on a T cell. TCR phosphorylation in response to pMHC binding is accompanied by segregation of the transmembrane phosphatase CD45 away from TCR–pMHC complexes. The kinetic segregation hypothesis proposes that CD45 exclusion shifts the local kinase–phosphatase balance to favor TCR phosphorylation. Spatial partitioning may arise from the size difference between the large CD45 extracellular domain and the smaller TCR–pMHC complex, although parsing potential contributions of extracellular protein size, actin activity, and lipid domains is difficult in living cells. Here, we reconstitute segregation of CD45 from bound receptor–ligand pairs using purified proteins on model membranes. Using a model receptor–ligand pair (FRB–FKBP), we first test physical and computational predictions for protein organization at membrane interfaces. We then show that the TCR–pMHC interaction causes partial exclusion of CD45. Comparing two developmentally regulated isoforms of CD45, the larger RABC variant is excluded more rapidly and efficiently (∼50%) than the smaller R0 isoform (∼20%), suggesting that CD45 isotypes could regulate Signaling thresholds in different T cell subtypes. Similar to the sensitivity of T cell Signaling, TCR–pMHC interactions with Kds of ≤15 µM were needed to exclude CD45. We further show that the coreceptor PD-1 with its ligand PD-L1, immunotherapy targets that inhibit T cell Signaling, also exclude CD45. These results demonstrate that the binding energies of physiological receptor–ligand pairs on the T cell are sufficient to create spatial organization at membrane–membrane interfaces.

  • in vitro reconstitution of t cell receptor mediated segregation of the cd45 phosphatase
    bioRxiv, 2017
    Co-Authors: Catherine B Carbone, Nadja Kern, Ricardo A Fernandes, Enfu Hui, Christopher K Garcia, Ronald D Vale
    Abstract:

    T cell Signaling initiates upon binding of peptide-major histocompatibility complex (pMHC) on an antigen-presenting cell (APC) to the T cell receptor (TCR) on a T cell. TCR phosphorylation in response to pMHC binding is accompanied by segregation of the transmembrane phosphatase CD45 away from TCR-pMHC complexes. The kinetic segregation hypothesis proposes that CD45 exclusion shifts the local kinase-phosphatase balance to favor TCR phosphorylation. Spatial partitioning may arise from the size difference between the large CD45 extracellular domain and the smaller TCR-pMHC complex, although parsing potential contributions of extracellular protein size, actin activity, and lipid domains is difficult in living cells. Here, we reconstitute segregation of CD45 from bound receptor-ligand pairs using purified proteins on model membranes. Using a model receptor-ligand pair (FRB-FKBP), we first test physical and computational predictions for protein organization at membrane interfaces. We then show that the TCR-pMHC interaction causes partial exclusion of CD45. Comparing two developmentally-regulated isoforms of CD45, the larger RABC variant is excluded more rapidly and efficiently (~50%) than the smaller R0 isoform (~20%), suggesting that CD45 isotypes could regulate Signaling thresholds in different T cell subtypes. Similar to the sensitivity of T cell Signaling, TCR-pMHC interactions with Kds of

  • single molecule microscopy reveals plasma membrane microdomains created by protein protein networks that exclude or trap Signaling molecules in t cells
    Cell, 2005
    Co-Authors: Adam D Douglass, Ronald D Vale
    Abstract:

    Membrane subdomains have been implicated in T cell Signaling, although their properties and mechanisms of formation remain controversial. Here, we have used single-molecule and scanning confocal imaging to characterize the behavior of GFP-tagged Signaling proteins in Jurkat T cells. We show that the coreceptor CD2, the adaptor protein LAT, and tyrosine kinase Lck cocluster in discrete microdomains in the plasma membrane of Signaling T cells. These microdomains require protein-protein interactions mediated through phosphorylation of LAT and are not maintained by interactions with actin or lipid rafts. Using a two color imaging approach that allows tracking of single molecules relative to the CD2/LAT/Lck clusters, we demonstrate that these microdomains exclude and limit the free diffusion of molecules in the membrane but also can trap and immobilize specific proteins. Our data suggest that diffusional trapping through protein-protein interactions creates microdomains that concentrate or exclude cell surface proteins to facilitate T cell Signaling.

Arthur Weiss - One of the best experts on this subject based on the ideXlab platform.

  • fine tuning of substrate preferences of the src family kinase lck revealed through a high throughput specificity screen
    eLife, 2018
    Co-Authors: Arthur Weiss, Neel H Shah, Mark Lobel, John Kuriyan
    Abstract:

    The specificity of tyrosine kinases is attributed predominantly to localization effects dictated by non-catalytic domains. We developed a method to profile the specificities of tyrosine kinases by combining bacterial surface-display of peptide libraries with next-generation sequencing. Using this, we showed that the tyrosine kinase ZAP-70, which is critical for T cell Signaling, discriminates substrates through an electrostatic selection mechanism encoded within its catalytic domain (Shah et al., 2016). Here, we expand this high-throughput platform to analyze the intrinsic specificity of any tyrosine kinase domain against thousands of peptides derived from human tyrosine phosphorylation sites. Using this approach, we find a difference in the electrostatic recognition of substrates between the closely related Src-family kinases Lck and c-Src. This divergence likely reflects the specialization of Lck to act in concert with ZAP-70 in T cell Signaling. These results point to the importance of direct recognition at the kinase active site in fine-tuning specificity.

  • an extracatalytic function of cd45 in b cells is mediated by cd22
    Proceedings of the National Academy of Sciences of the United States of America, 2015
    Co-Authors: Sarah Coughlin, Arthur Weiss, Mark Noviski, James L Mueller, Ammarina Chuwonpad, William C Raschke, Julie Zikherman
    Abstract:

    The receptor-like tyrosine phosphatase CD45 regulates antigen receptor Signaling by dephosphorylating the C-terminal inhibitory tyrosine of the src family kinases. However, despite its abundance, the function of the large, alternatively spliced extracellular domain of CD45 has remained elusive. We used normally spliced CD45 transgenes either incorporating a phosphatase-inactivating point mutation or lacking the cytoplasmic domain to uncouple the enzymatic and noncatalytic functions of CD45 in lymphocytes. Although these transgenes did not alter T-Cell Signaling or development irrespective of endogenous CD45 expression, both partially rescued the phenotype of CD45-deficient B cells. We identify a noncatalytic role for CD45 in regulating tonic, but not antigen-mediated, B-cell antigen receptor (BCR) Signaling through modulation of the function of the inhibitory coreceptor CD22. This finding has important implications for understanding how naive B cells maintain tonic BCR Signaling while restraining inappropriate antigen-dependent activation to preserve clonal "ignorance."

  • investigation of novel zap 70 functionality in t cell Signaling pathways using computational modeling
    Biophysical Journal, 2014
    Co-Authors: Maria P Frushicheva, Arthur Weiss, Arup K Chakraborty
    Abstract:

    Aberrant regulation of cellular processes in immune systems can result in human disease. Therefore, we need a better understanding of the mechanistic principles of biochemical Signaling pathways that regulate immune responses. This study investigates a novel functionality of the tyrosine kinase zeta-associated protein Zap-70 in T cell Signaling pathways, using the quantitative phosphoproteomics data obtained from our experimental collaborators. The Zap-70 alterations in cellular Signaling pathways (loss of its function or expression) can cause an unusual form of severe combined immune deficiency (SCID) that often leads to fatal outcomes. Therefore, the analysis of Signaling events using computational modeling and modern proteomics technique (e.g., stable isotopic labeling of amino acids in cell culture (SILAC)) provides a network map of possible molecular targets guiding disease diagnosis. Additionally, this network map presents information about the placement of newly observed phosphorylation sites in T cell Signaling pathways across a time course after receptor stimulation. In this study, we tested several computational models of Zap-70 T cell Signaling pathways to experiment with different hypotheses. Specifically, we calculated the phosphorylation levels of tyrosine residues of N- and C- terminals of immunoreceptor tyrosine-based activation motif (ITAM) for different expression levels of Zap-70 in T cells. We performed fully stochastic Signaling simulations using stochastic simulation complier (SSC) developed in our lab and modeling of ordinary differential equations. Subsequently, the sensitivity analysis of deterministic simulations was performed for identifications of key proteins and biochemical reactions in Signaling networks that regulate stochastic transitions leading to pathological cellular function. The calculated ITAM phosphorylation levels are well correlated with the corresponding experimental ones. Finally, using our computational modeling, we formulated novel testable hypotheses that can guide future experiments.

  • zap 70 an essential kinase in t cell Signaling
    Cold Spring Harbor Perspectives in Biology, 2010
    Co-Authors: Haopeng Wang, Theresa A Kadlecek, Byron B Auyeung, Hanna Sjolin E Goodfellow, Lih Yun Hsu, Tanya S Freedman, Arthur Weiss
    Abstract:

    ZAP-70 is a cytoplasmic protein tyrosine kinase that plays a critical role in the events involved in initiating T-Cell responses by the antigen receptor. Here we review the structure of ZAP-70, its regulation, its role in development and in disease. We also describe a model experimental system in which ZAP-70 function can be interrupted by a small chemical inhibitor.

Michael L Dustin - One of the best experts on this subject based on the ideXlab platform.

  • spatiotemporal regulation of t cell costimulation by tcr cd28 microclusters and protein kinase c theta translocation
    Immunity, 2008
    Co-Authors: Tadashi Yokosuka, Michael L Dustin, Wakana Kobayashi, Kumiko Sakatasogawa, Masako Takamatsu, Akiko Hashimototane, Makio Tokunaga, Takashi Saito
    Abstract:

    T cell activation is mediated by microclusters (MCs) containing T cell receptors (TCRs), kinases, and adaptors. Although TCR MCs translocate to form a central supramolecular activation cluster (cSMAC) of the immunological synapse at the interface of a T cell and an antigen-presenting cell, the role of MC translocation in T cell Signaling remains unclear. Here, we found that the accumulation of MCs at cSMAC was important for T cell costimulation. Costimulatory receptor CD28 was initially recruited coordinately with TCR to MCs, and its signals were mediated through the assembly with the kinase PKCtheta. The accumulation of MCs at the cSMAC was accompanied by the segregation of CD28 from the TCR, which resulted in the translocation of both CD28 and PKCtheta to a spatially unique subregion of cSMAC. Thus, costimulation is mediated by the generation of a unique costimulatory compartment in the cSMAC via the dynamic regulation of MC translocation.

  • spatiotemporal regulation of t cell costimulation by tcr cd28 microclusters and protein kinase c theta translocation
    Immunity, 2008
    Co-Authors: Tadashi Yokosuka, Michael L Dustin, Wakana Kobayashi, Kumiko Sakatasogawa, Masako Takamatsu, Akiko Hashimototane, Makio Tokunaga, Takashi Saito
    Abstract:

    Summary T cell activation is mediated by microclusters (MCs) containing T cell receptors (TCRs), kinases, and adaptors. Although TCR MCs translocate to form a central supramolecular activation cluster (cSMAC) of the immunological synapse at the interface of a T cell and an antigen-presenting cell, the role of MC translocation in T cell Signaling remains unclear. Here, we found that the accumulation of MCs at cSMAC was important for T cell costimulation. Costimulatory receptor CD28 was initially recruited coordinately with TCR to MCs, and its signals were mediated through the assembly with the kinase PKCθ. The accumulation of MCs at the cSMAC was accompanied by the segregation of CD28 from the TCR, which resulted in the translocation of both CD28 and PKCθ to a spatially unique subregion of cSMAC. Thus, costimulation is mediated by the generation of a unique costimulatory compartment in the cSMAC via the dynamic regulation of MC translocation.

  • the immunological synapse and the actin cytoskeleton molecular hardware for t cell Signaling
    Nature Immunology, 2000
    Co-Authors: Michael L Dustin, John A Cooper
    Abstract:

    . The integrin LFA-1 and its major immunoglobulin superfamily ligand ICAM-1 can interact at ∼40 nm and can thereby initiate adhesive interactions when appropri- ately activated 33,34 . These large adhesion molecules initiate active mech- anisms to promote the interaction of smaller adhesion molecules, bring- ing the actin cytoskeleton to center stage. The actin cytoskeleton plays a dual role in regulation of LFA-1. The resting leukocyte has two surface domains: low flat surfaces and microvilli. Integrins such as LFA-1 are located on the low flat surface along with other large glycoproteins like CD43 35

Shigemi M Kinoshita - One of the best experts on this subject based on the ideXlab platform.

  • cop9 signalosome component jab1 csn5 is necessary for t cell Signaling through lfa 1 and hiv 1 replication
    PLOS ONE, 2012
    Co-Authors: Peter O Krutzik, Shigemi M Kinoshita, Garry P Nolan
    Abstract:

    To determine critical host factors involved in HIV-1 replication, a dominant effector genetics approach was developed to reveal Signaling pathways on which HIV-1 depends for replication. A large library of short peptide aptamers was expressed via retroviral delivery in T cells. Peptides that interfered with T cell activation-dependent processes that might support HIV-1 replication were identified. One of the selected peptides altered Signaling, lead to a difference in T cell activation status, and inhibited HIV-1 replication. The target of the peptide was JAB1/CSN5, a component of the signalosome complex. JAB1 expression overcame the inhibition of HIV-1 replication in the presence of peptide and also promoted HIV-1 replication in activated primary CD4+ T cells. This peptide blocked physiological release of JAB1 from the accessory T cell surface protein LFA-1, downstream AP-1 dependent events, NFAT activation, and HIV-1 replication. Thus, genetic selection for intracellular aptamer inhibitors of host cell processes proximal to signals at the immunological synapse of T cells can define unique mechanisms important to HIV-1 replication.

George C Tsokos - One of the best experts on this subject based on the ideXlab platform.

  • spleen tyrosine kinase syk regulates systemic lupus erythematosus sle t cell Signaling
    PLOS ONE, 2013
    Co-Authors: Alexandros P Grammatikos, Debjani Ghosh, Amy Devlin, Vasileios C Kyttaris, George C Tsokos
    Abstract:

    Engagement of the CD3/T cell receptor complex in systemic lupus erythematosus (SLE) T cells involves Syk rather than the zeta-associated protein. Because Syk is being considered as a therapeutic target we asked whether Syk is central to the multiple aberrantly modulated molecules in SLE T cells. Using a gene expression array, we demonstrate that forced expression of Syk in normal T cells reproduces most of the aberrantly expressed molecules whereas silencing of Syk in SLE T cells normalizes the expression of most abnormally expressed molecules. Protein along with gene expression modulation for select molecules was confirmed. Specifically, levels of cytokine IL-21, cell surface receptor CD44, and intracellular molecules PP2A and OAS2 increased following Syk overexpression in normal T cells and decreased after Syk silencing in SLE T cells. Our results demonstrate that levels of Syk affect the expression of a number of enzymes, cytokines and receptors that play a key role in the development of disease pathogenesis in SLE and provide support for therapeutic targeting in SLE patients.

  • fc epsilon receptor type i gamma chain replaces the deficient t cell receptor zeta chain in t cells of patients with systemic lupus erythematosus
    Arthritis & Rheumatism, 2001
    Co-Authors: Edith J Enyedy, Madhusoodana P Nambiar, Stamatisnick C Liossis, Gregory J Dennis, Gary M Kammer, George C Tsokos
    Abstract:

    Objective T cells from the majority of patients with systemic lupus erythematosus (SLE) express significantly lower levels of T cell receptor ζ chain, a critical Signaling molecule. However, TCR/CD3 triggering of SLE T cells shows increased phosphorylation of downstream Signaling intermediates and increased [Ca2+]i response, suggesting the presence of alternative Signaling mechanisms. We investigated whether Fcϵ receptor type I γ chain (FcϵRIγ) could substitute for TCR ζ chain and contribute to T cell Signaling in SLE. Methods T cells were purified from the peripheral blood of 21 patients with SLE and 5 healthy volunteers. The expression of FcϵRIγ was investigated using immunoblotting, reverse transcriptase–polymerase chain reaction, and flow cytometry methods. Involvement of the FcϵRIγ in T cell Signaling was studied by immunoprecipitation and/or immunoblotting after TCR/CD3 stimulation. Results Western blotting and densitometric analysis showed that the expression of FcϵRIγ in SLE T cells was 4.3-fold higher than in normal T cells (P < 0.001). Flow cytometric analyses of T lymphocyte subsets revealed that the proportions of FcϵRIγ+,CD3+, FcϵRIγ+,CD4+, and FcϵRIγ+, CD8+ cells were significantly greater in SLE patients than in healthy controls (P < 0.001). Immunoprecipitation of SLE T cell lysates with an anti-FcϵRIγ antibody showed that FcϵRIγ associates with the tyrosine kinase Syk and the CD3ϵ chain, suggesting that FcϵRIγ is functionally involved in TCR Signaling. Conclusion These results demonstrate that the FcϵRI γ chain is expressed at high levels in a large proportion of SLE T cells. The increased expression of FcϵRI γ chain in SLE T cells may account in part for the aberrant antigen receptor–initiated Signaling and contribute to the diverse cellular abnormalities found in this disease.