Removal of C-Terminal Src Kinase from the Immune Synapse by a New Binding Protein
| Autor: | Andres Alonso, Tomas Mustelin, Souad Rahmouni, Marianne J. B. van Stipdonk, Chiara Soncini, Torkel Vang, Michel Moutschen, Stephen P. Schoenberger, Scott Williams |
|---|---|
| Rok vydání: | 2005 |
| Předmět: |
CD3 Complex
T-Lymphocytes Receptors Antigen T-Cell Protein tyrosine phosphatase Biology Ligands Lymphocyte Activation SH2 domain CSK Tyrosine-Protein Kinase src Homology Domains Jurkat Cells chemistry.chemical_compound Membrane Microdomains Proto-Oncogene Proteins Immunoreceptor tyrosine-based activation motif Humans Phosphorylation RNA Small Interfering Poly-ADP-Ribose Binding Proteins Molecular Biology Adaptor Proteins Signal Transducing Tyrosine-protein kinase CSK Phosphotransferases T-cell receptor DNA Helicases Membrane Proteins Tyrosine phosphorylation Cell Biology Protein-Tyrosine Kinases Phosphoproteins Cell biology RNA Recognition Motif Proteins src-Family Kinases Protein kinase domain chemistry Lymphocyte Specific Protein Tyrosine Kinase p56(lck) Tyrosine Carrier Proteins RNA Helicases Signal Transduction Proto-oncogene tyrosine-protein kinase Src |
| Zdroj: | Molecular and Cellular Biology. 25:2227-2241 |
| ISSN: | 1098-5549 |
| DOI: | 10.1128/mcb.25.6.2227-2241.2005 |
| Popis: | The molecular mechanisms of T-cell-antigen receptor (TCR) signal transduction and T-cell activation have been intensely studied during the past decade. It has become evident that several protein tyrosine kinases (PTKs) and protein tyrosine phosphatases (PTPs) play crucial roles (reviewed in references 33 and 38). The earliest known biochemical response to TCR ligation is an increased phosphorylation of a number of cellular proteins on tyrosine residues (21, 25). Pharmacological agents that prevent this phosphorylation block T-cell activation altogether (37, 26), whereas inhibitors of PTPs mimic TCR ligation and cause T-cell activation (44, 52) and prevent reversion of activated T cells to a resting phenotype (22). Biochemical and genetic evidence indicates that the Src-family PTK Lck plays a crucial receptor-proximal role in TCR signaling (31), even in T cells that lack CD4 or CD8 (55). Although the molecular mechanism for the TCR-Lck connection is unclear, it seems that Lck responds to TCR stimulation with a rapid increase in its phosphorylation of tyrosines within the immunoreceptor tyrosine-based activation motifs (ITAMs) of the CD3 and ζ subunits of the TCR. Once phosphorylated, these motifs serve to recruit a second type of cytoplasmic PTK, ZAP-70 (8, 23), which is subsequently activated by direct phosphorylation at Y493 in its activation loop by Lck (7). Due to the presence of 10 ITAMs in the TCR complex, up to 10 ZAP-70 molecules may cluster on the fully phosphorylated receptor. Once activated by Lck, ZAP-70 autophosphorylates, presumably in trans, to create docking sites for SH2 domain-containing signaling proteins (41). The Src family PTKs are also responsible for recruitment and activation of the cytoplasmic Tec-related kinases Itk/Emt and Txk/Rlk (16, 19), which are directly involved in phosphorylation and activation of phospholipase Cγ1 (29, 48). It also appears that Src family PTKs have numerous other substrates, including cytoskeletal proteins, adapters, and other signaling molecules. Given the central role of Src family PTKs, particularly Lck in T-cell activation, it seems obvious that these kinases must be extraordinarily tightly regulated to ensure that T cells respond appropriately to antigen. Indeed, Lck is regulated at all available levels from transcription and translation to multiple posttranslational modifications and controlled subcellular location. Perhaps the best studied regulation is the phosphorylation of an inhibitory tyrosine in the C terminus of Lck, Y505 (reviewed in reference 32). Mutation of this residue results in a constitutively active form of Lck, which can transform fibroblasts (2, 30). In T cells, Y505 is phosphorylated by the Csk PTK (4) and dephosphorylated by the CD45 PTP (34, 36, 45). It has been estimated that ca. 50% of Lck molecules are Y505 phosphorylated under physiological conditions in T cells (53), with a relatively slow turnover (43). In agreement with the notion that the balance between CD45 and Csk is important (35), most CD45-negative T cells fail to respond to TCR stimulation (6, 28, 49), whereas increased CD45 expression, e.g., in memory T cells (50), correlates with increased sensitivity to TCR ligation. Conversely, overexpression of Csk very efficiently reduces TCR signaling (9, 58), whereas a dominant-negative Csk augments it (56). In addition, a two- to threefold activation of Csk is used as a physiological mechanism for immunosuppression by cyclic AMP-inducing stimuli (58). Csk is a 50-kDa cytoplasmic PTK comprised of Src homology 3 (SH3) and SH2 domains and a catalytic kinase domain (39, 47), but it differs from other nonreceptor PTKs in that it lacks N-terminal membrane docking motifs, tyrosine phosphorylation sites, and C-terminal regulatory sequences. Csk has a highly specialized and unique function as a general negative regulator of all Src family kinases (32, 40). Csk is expressed in all examined cell types but is particularly abundant in hematopoietic cells. An important advance in our understanding of Csk regulation was the recent discovery of a transmembrane molecule, termed PAG (5) or Cbp (27), which specifically binds Csk through its SH2 domain. PAG/Cbp resides in lipid rafts and is phosphorylated on tyrosine in resting T cells (5, 56), thus anchoring a portion of Csk in the subcellular compartment that is enriched in Src family kinases. Upon TCR triggering, PAG/Cbp is rapidly dephosphorylated by an unknown PTP, resulting in dissociation of Csk (56). This apparently allows lipid raft-located Lck and Fyn to remain active longer and to phosphorylate ITAMs and other molecules. After ca. 10 min (in primary T cells), however, PAG/Cbp is rephosphorylated and Csk begins to return to the lipid rafts. This coincides with the downturn of tyrosine phosphorylation. The importance of this mechanism is perhaps best illustrated by the consequences of expression of a Csk-SH3-SH2 protein (lacking kinase domain), which will compete with endogenous Csk for binding to PAG/Cbp (56). This truncated protein caused a striking increase in basal and induced levels of tyrosine phosphorylation, which also lasted longer than in controls. The protein also augmented NFAT/AP-1 reporter gene activation (56). Here we address the question of where Csk goes when it dissociates from PAG/Cbp and leaves the lipid rafts. We have identified another ligand for Csk, termed G3BP, which appears to be anchored at some distance from the immune synapse. The time course of Csk binding to G3BP is similar to the time course of Csk dissociation from PAG/Cbp upon TCR stimulation. In agreement with the notion that G3BP may sequester a portion of Csk away from the immune synapse, we found that expression of G3BP reduced Lck phosphorylation at Y505 and improved T-cell activation. |
| Databáze: | OpenAIRE |
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