Removal of C-Terminal Src Kinase from the Immune Synapse by a New Binding Protein (original) (raw)
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Journal of Biological Chemistry, 2001
In resting peripheral T cells, Csk is constitutively present in lipid rafts through an interaction with the Csk SH2-binding protein, PAG, also known as Cbp. Upon triggering of the T cell antigen receptor (TCR), PAG/Cbp is rapidly dephosphorylated leading to dissociation of Csk from lipid rafts. However, tyrosine phosphorylation of PAG/Cbp resumes after 3-5 min, at which time Csk reassociates with the rafts. Cells overexpressing a mutant Csk that lacks the catalytic domain, but displaces endogenous Csk from lipid rafts, have elevated basal levels of TCR--chain phosphorylation and spontaneous activation of an NFAT-AP1 reporter from the proximal interleukin-2 promoter as well as stronger and more sustained responses to TCR triggering than controls. We suggest that a transient release from Csk-mediated inhibition by displacement of Csk from lipid rafts is important for normal T cell activation.
Journal of Experimental Medicine, 2001
In T cells, cAMP-dependent protein kinase (PKA) type I colocalizes with the T cell receptor-CD3 complex (TCR/CD3) and inhibits T cell function via a previously unknown proximal target. Here we examine the mechanism for this PKA-mediated immunomodulation. cAMP treatment of Jurkat and normal T cells reduces Lck-mediated tyrosine phosphorylation of the TCR/CD3 chain after T cell activation, and decreases Lck activity. Phosphorylation of residue Y505 in Lck by COOH-terminal Src kinase (Csk), which negatively regulates Lck, is essential for the inhibitory effect of cAMP on chain phosphorylation. PKA phosphorylates Csk at S364 in vitro and in vivo leading to a two-to fourfold increase in Csk activity that is necessary for cAMP-mediated inhibition of TCR-induced interleukin 2 secretion. Both PKA type I and Csk are targeted to lipid rafts where proximal T cell activation occurs, and phosphorylation of raft-associated Lck by Csk is increased in cells treated with forskolin. We propose a mechanism whereby PKA through activation of Csk intersects signaling by Src kinases and inhibits T cell activation.
Journal of Biological Chemistry, 2003
Raft-associated Csk controls signaling through the T cell receptor (TCR) and was mainly anchored to Cbp/ PAG (phosphoprotein associated with glycosphingolipid-enriched membrane domains). Treatment of cells with the cAMP-elevating agent prostaglandin E 2 (PGE 2) augmented the level of Cbp/PAG phosphorylation with a concomitant increase in amounts of Csk bound to Cbp/ PAG. While TCR-triggering resulted in transient dissociation of Csk from Cbp/PAG/rafts allowing TCR-induced tyrosine phosphorylation to occur, pretreatment with PGE 2 reduced Csk dissociation upon TCR triggering. This correlated with lowered TCR-induced phosphorylation of CD3-chain and linker for activation of T cells. Moreover, competition of endogenous Csk from lipid rafts abolished PGE 2-mediated inhibition of TCRinduced-chain phosphorylation and activation of the nuclear factor of activated T cells (NFAT) activator protein 1 (AP-1). Finally, raft-associated Csk already activated via Cbp/PAG binding, gained additional increase in phosphotransferase activity upon protein kinase Amediated phosphorylation of Csk. We propose that cAMP regulates Csk via both spatial and enzymatic mechanisms, thereby inhibiting signaling through the TCR. Upon triggering of the T cell receptor (TCR) 1 activation of Lck leads to phosphorylation of the immunoreceptor tyrosine
Journal of Experimental Medicine, 2000
According to a recently proposed hypothesis, initiation of signal transduction via immunoreceptors depends on interactions of the engaged immunoreceptor with glycosphingolipid-enriched membrane microdomains (GEMs). In this study, we describe a novel GEM-associated transmembrane adaptor protein, termed phosphoprotein associated with GEMs (PAG). PAG comprises a short extracellular domain of 16 amino acids and a 397-amino acid cytoplasmic tail containing ten tyrosine residues that are likely phosphorylated by Src family kinases. In lymphoid cell lines and in resting peripheral blood α/β T cells, PAG is expressed as a constitutively tyrosine-phosphorylated protein and binds the major negative regulator of Src kinases, the tyrosine kinase Csk. After activation of peripheral blood α/β T cells, PAG becomes rapidly dephosphorylated and dissociates from Csk. Expression of PAG in COS cells results in recruitment of endogenous Csk, altered Src kinase activity, and impaired phosphorylation of S...
Protein tyrosine phosphorylation in T cell signaling
Frontiers in Bioscience, 2002
Introduction 2.1. Inositol phospholipid hydrolysis and calcium mobilization 2.2. TCR-induced tyrosine phosphorylation 3. Src-related kinases in T cells 3.1. Lck-a co-receptor associated PTK 3.2. Fyn-a Src-family PTK associated with the TCR complex. 3.3. c-Yes, the third and least understood Src-related kinase in T cells 3.4. Structure and Regulation of Src family PTKs 3.4.1. Lipid modification and lipid raft association of Lck and Fyn 3.4.2. Unique N-Terminal Region 3.4.3. SH3 Domain 3.4.4. SH2 Domain 3.4.5. Kinase Domain 3.5. Regulation of Src-family PTKs 3.5.1. Positive regulation by tyrosine phosphorylation in the kinase domain 3.5.2. Dephosphorylation of the activation loop tyrosine. 3.5.3. Regulatory C-terminus 3.5.4. Suppression of Src family kinases by Csk 3.5.5. Csk is opposed by CD45 3.5.6. Additional phosphorylation events 3.5.7. Src family PTKs and viruses 4. Syk-family PTKs in T cells 4.1. Regulatory mechanisms for Zap-70 4.2. Regulation of Syk 4.3. Both Zap-70 and Syk act as docking proteins 5. Structure and function of the Tec-family PTKs in T cells 5.1. Other tyrosine kinases in T cells 6. Protein tyrosine phosphatases in T cell activation 6.1. PTPases present in T lymphocytes 6.2. Role of CD45 in T cell activation 6.3. HePTP-in control of MAP kinases 6.4. SHP1-mediator of inhibitory signaling 6.5. SHP2 in T cell activation 6.6. PEP-a partner of Csk in suppression of TCR signaling 6.7. PTP-MEG2-a regulator of secretory vesicles 6.8. ERM-PTPases-gatekeepers at the membrane-cytoskeleton interface 6.9. PTEN-in control of lymphocyte life and death 6.10. LMPTP-a small and peculiar enzyme 7. Initiation of the TCR signaling cascade 7.1. ITAM phosphorylation by Src family PTKs 7.2. Which PTPase dephosphorylates the ITAMs? 7.3. Consequences of ITAM phosphorylation 7.4. Multiple ITAMs-redundancy, amplification or sensitivity? 7.5. Partial TCR-zeta phosphorylation and T cell anergy 7.6. The role of CD4 and CD8 in the initiation of T cell activation 7.7. Role of lipid rafts in TCR signaling 7.8. Nonredundant functions of Fyn (T) 7.9. Does Syk participate in T cell activation? 8. Substrates for kinases and phosphatases: enzymes, adapters and downstream signaling pathways 8.1.
Molecular and Cellular Biology, 2005
Ras GTPases are on/off switches regulating numerous cellular responses by signaling to various effector molecules. In T lymphocytes, Ras can be activated by two Ras exchange factors, SOS and RasGRP1, which are recruited through the adapters Grb2 and LAT and via the second-messenger diacylglycerol (DAG), respectively. Mitogen-activated protein (MAP) kinase phosphorylation patterns induced by active Ras can vary and contribute to distinct cellular responses. The different consequences of Ras activation by either guanine exchange factor are unknown. DAG also recruits and activates the kinase protein kinase C (PKC) turning on the Erk MAP kinase pathway, but the biochemical mechanism responsible is unclear.
Proceedings of the National Academy of Sciences, 1991
Stimulation of the T-cell antigen receptor (TCR), which itself is not a protein-tyrosine kinase (PTK), activates a PTK and phospholipase C (PLC). Using the human T-cell leukemic line Jurkat and normal peripheral blood lymphocytes, we demonstrate that stimulation of the TCR specifically induces the recovery of PLC activity in eluates from anti-phosphotyrosine immunoprecipitates. Stimulation of the human muscarinic receptor, subtype 1, when expressed in Jurkat activates PLC through a guanine nucleotide binding protein but does not induce the recovery of PLC activity in eluates from anti-phosphotyrosine immunoprecipitates. Western blot analysis reveals that PLC-y1 is tyrosine-phosphorylated in response to TCR stimulation. Nearly all of the PLC activity recovered in eluates from anti-phosphotyrosine immunoprecipitates was depleted by anti-PLC-yl antibodies. Stimulation of the TCR on mutants derived from Jurkat that are defective in TCR-induced PLC activation results in markedly reduced, if any, PLC activity recovered in phosphotyrosine immunoprecipitates and in no detectable PLC-yl tyrosine phosphorylation. Thus, the TCR functions like PTK growth factor receptors, but through an indirect interaction, to induce tyrosine phosphorylation of PLC-y1. Since other studies have implicated two members of the src family of PTKs in TCRmediated signal transduction, our findings suggest that the induction of tyrosine phosphorylation of PLC--1 by a mechanism involving a src-like kinase may be the means by which the TCR regulates PLC activity in T cells.