The initiation of T-cell signaling is critically dependent on the function of the member of Src family tyrosine kinases, Lck. Upon T-cell antigen receptor (TCR) triggering, Lck kinase activity induces the nucleation of signal-transducing hubs that regulate the formation of complex signaling network and cytoskeletal rearrangement. In addition, the delivery of Lck function requires rapid and targeted membrane redistribution, but the mechanism underpinning this process is largely unknown. To gain insight into this process, we considered previously described proteins that could assist in this process via their capacity to interact with kinases and regulate their intracellular translocations. An adaptor protein, receptor for activated C kinase 1 (RACK1), was chosen as a viable option, and its capacity to bind Lck and aid the process of activation-induced redistribution of Lck was assessed. Our microscopic observation showed that T-cell activation induces a rapid, concomitant, and transient co-redistribution of Lck and RACK1 into the forming immunological synapse. Consistent with this observation, the formation of transient RACK1-Lck complexes were detectable in primary CD4+ T-cells with their maximum levels peaking 10 s after TCR-CD4 co-aggregation. Moreover, RACK1 preferentially binds to a pool of kinase active pY394Lck, which co-purifies with high molecular weight cellular fractions. The formation of RACK1-Lck complexes depends on functional SH2 and SH3 domains of Lck and includes several other signaling and cytoskeletal elements that transiently bind the complex. Notably, the F-actin-crosslinking protein, α-actinin-1, binds to RACK1 only in the presence of kinase active Lck suggesting that the formation of RACK1-pY394Lck-α-actinin-1 complex serves as a signal module coupling actin cytoskeleton bundling with productive TCR/CD4 triggering. In addition, the treatment of CD4+ T-cells with nocodazole, which disrupts the microtubular network, also blocked the formation of RACK1-Lck complexes. Importantly, activation-induced Lck redistribution was diminished in primary CD4+ T-cells by an adenoviral-mediated knockdown of RACK1. These results demonstrate that in T cells, RACK1, as an essential component of the multiprotein complex which upon TCR engagement, links the binding of kinase active Lck to elements of the cytoskeletal network and affects the subcellular redistribution of Lck.

• Klíčová slova
• Lck, RACK1, TCR triggering, cytoskeleton, lipid rafts, membrane redistribution, α-actinin,
• Publikační typ
• časopisecké články MeSH

In spite of a comprehensive understanding of the schematics of T cell receptor (TCR) signaling, the mechanisms regulating compartmentalization of signaling molecules, their transient interactions, and rearrangement of membrane structures initiated upon TCR engagement remain an outstanding problem. These gaps in our knowledge are exemplified by recent data demonstrating that TCR triggering is largely dependent on a preactivated pool of Lck concentrated in T cells in a specific type of membrane microdomains. Our current model posits that in resting T cells all critical components of TCR triggering machinery including TCR/CD3, Lck, Fyn, CD45, PAG, and LAT are associated with distinct types of lipid-based microdomains which represent the smallest structural and functional units of membrane confinement able to negatively control enzymatic activities and substrate availability that is required for the initiation of TCR signaling. In addition, the microdomains based segregation spatially limits the interaction of components of TCR triggering machinery prior to the onset of TCR signaling and allows their rapid communication and signal amplification after TCR engagement, via the process of their coalescence. Microdomains mediated compartmentalization thus represents an essential membrane organizing principle in resting T cells. The integration of these structural and functional aspects of signaling into a unified model of TCR triggering will require a deeper understanding of membrane biology, novel interdisciplinary approaches and the generation of specific reagents. We believe that the fully integrated model of TCR signaling must be based on membrane structural network which provides a proper environment for regulatory processes controlling TCR triggering.

• Klíčová slova
• Fyn, Lck, TCR triggering, compartmentalization, heavy and light DRMs, membrane microdomains, spatio-temporal regulation,
• Publikační typ
• časopisecké články MeSH

Formation of the immunological synapse between an antigen-presenting cell (APC) and a T cell leads to signal generation in both cells involved. In T cells, the lipid raft-associated transmembrane adaptor protein LAT plays a central role. Its phosphorylation is a crucial step in signal propagation, including the calcium response and mitogen-activated protein kinase activation, and largely depends on its association with the SLP76 adaptor protein. Here we report the discovery of a new palmitoylated transmembrane adaptor protein, termed SCIMP. SCIMP is expressed in B cells and other professional APCs and is localized in the immunological synapse due to its association with tetraspanin-enriched microdomains. In B cells, it is constitutively associated with Lyn kinase and becomes tyrosine phosphorylated after major histocompatibility complex type II (MHC-II) stimulation. When phosphorylated, SCIMP binds to the SLP65 adaptor protein and also to the inhibitory kinase Csk. While the association with SLP65 initiates the downstream signaling cascades, Csk binding functions as a negative regulatory loop. The results suggest that SCIMP is involved in signal transduction after MHC-II stimulation and therefore serves as a regulator of antigen presentation and other APC functions.

• MeSH
• adaptorové proteiny signální transdukční chemie   metabolismus   MeSH
• aktivace lymfocytů MeSH
• antigen prezentující buňky imunologie   MeSH
• B-lymfocyty imunologie   metabolismus   MeSH
• C-terminální Src kinasa MeSH
• fosfoproteiny metabolismus   MeSH
• HEK293 buňky MeSH
• imunologické synapse chemie   MeSH
• lidé MeSH
• malá interferující RNA MeSH
• membránové mikrodomény chemie   metabolismus   MeSH
• membránové proteiny chemie   genetika   imunologie   metabolismus   MeSH
• MHC antigeny II. třídy imunologie   metabolismus   MeSH
• mitogenem aktivované proteinkinasy metabolismus   MeSH
• molekulární sekvence - údaje MeSH
• prezentace antigenu MeSH
• RNA interference MeSH
• sekvence aminokyselin MeSH
• signální transdukce MeSH
• skupina kinas odvozených od src-genu metabolismus   MeSH
• src homologní domény MeSH
• T-lymfocyty imunologie   MeSH
• tyrosinkinasy chemie   metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• adaptorové proteiny signální transdukční MeSH
• B cell linker protein MeSH Prohlížeč
• C-terminální Src kinasa MeSH
• CSK protein, human MeSH Prohlížeč
• fosfoproteiny MeSH
• lyn protein-tyrosine kinase MeSH Prohlížeč
• malá interferující RNA MeSH
• membránové proteiny MeSH
• MHC antigeny II. třídy MeSH
• mitogenem aktivované proteinkinasy MeSH
• SCIMP protein, human MeSH Prohlížeč
• skupina kinas odvozených od src-genu MeSH
• SLP-76 signal Transducing adaptor proteins MeSH Prohlížeč
• tyrosinkinasy MeSH

Transmembrane adaptor proteins (TRAPs) are important organizers and regulators of immunoreceptor-mediated signaling. A bioinformatic search revealed several potential novel TRAPs, including a highly conserved protein, proline rich 7 (PRR7), previously described as a component of the PSD-95/N-methyl-d-aspartate receptor protein complex in postsynaptic densities (PSD) of rat neurons. Our data demonstrate that PRR7 is weakly expressed in other tissues but is readily up-regulated in activated human peripheral blood lymphocytes. Transient overexpression of PRR7 in Jurkat T cell line led to gradual apoptotic death dependent on the WW domain binding motif surrounding Tyr-166 in the intracellular part of PRR7. To circumvent the pro-apoptotic effect of PRR7, we generated Jurkat clones with inducible expression of PRR7 (J-iPRR7). In these cells acute induction of PRR7 expression had a dual effect. It resulted in up-regulation of the transcription factor c-Jun and the activation marker CD69 as well as enhanced production of IL-2 after phorbol 12-myristate 13-acetate (PMA) and ionomycin treatment. On the other hand, expression of PRR7 inhibited general tyrosine phosphorylation and calcium influx after T cell receptor cross-linking by antibodies. Moreover, we found PRR7 constitutively tyrosine-phosphorylated and associated with Src. Collectively, these data indicate that PRR7 is a potential regulator of signaling and apoptosis in activated T cells.

• MeSH
• adaptorové proteiny signální transdukční biosyntéza   genetika   imunologie   MeSH
• aminokyselinové motivy MeSH
• apoptóza účinky léků   fyziologie   MeSH
• Caco-2 buňky MeSH
• CD antigeny biosyntéza   genetika   imunologie   MeSH
• diferenciační antigeny T-lymfocytů biosyntéza   genetika   imunologie   MeSH
• fosforylace účinky léků   fyziologie   MeSH
• HEK293 buňky MeSH
• interleukin-2 biosyntéza   genetika   imunologie   MeSH
• ionofory farmakologie   MeSH
• ionomycin farmakologie   MeSH
• Jurkat buňky MeSH
• karcinogeny farmakologie   MeSH
• krysa rodu Rattus MeSH
• lektiny typu C biosyntéza   genetika   imunologie   MeSH
• lidé MeSH
• protoonkogenní proteiny c-jun genetika   imunologie   metabolismus   MeSH
• receptory antigenů T-buněk genetika   imunologie   metabolismus   MeSH
• regulace genové exprese fyziologie   MeSH
• T-lymfocyty imunologie   metabolismus   MeSH
• terciární struktura proteinů MeSH
• tetradekanoylforbolacetát farmakologie   MeSH
• U937 buňky MeSH
• vápníková signalizace účinky léků   fyziologie   MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• adaptorové proteiny signální transdukční MeSH
• CD antigeny MeSH
• CD69 antigen MeSH Prohlížeč
• diferenciační antigeny T-lymfocytů MeSH
• IL2 protein, human MeSH Prohlížeč
• interleukin-2 MeSH
• ionofory MeSH
• ionomycin MeSH
• karcinogeny MeSH
• lektiny typu C MeSH
• protoonkogenní proteiny c-jun MeSH
• receptory antigenů T-buněk MeSH
• tetradekanoylforbolacetát MeSH