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.

• Keywords
• Lck, RACK1, TCR triggering, cytoskeleton, lipid rafts, membrane redistribution, α-actinin,
• Publication type
• Journal Article MeSH

Lymphocyte membrane rafts contain molecules critical for immunoreceptor signaling. Here, we report identification of a new raft-associated adaptor protein LIME (Lck-interacting molecule) expressed predominantly in T lymphocytes. LIME becomes tyrosine phosphorylated after cross-linking of the CD4 or CD8 coreceptors. Phospho-LIME associates with the Src family kinase Lck and its negative regulator, Csk. Ectopic expression of LIME in Jurkat T cells results in an increase of Csk in lipid rafts, increased phosphorylation of Lck and higher Ca2+ response to CD3 stimulation. Thus, LIME appears to be involved in regulation of T cell activation by coreceptors.

• MeSH
• Adaptor Proteins, Vesicular Transport genetics   immunology   metabolism   MeSH
• CD4 Antigens immunology   MeSH
• CD8 Antigens immunology   MeSH
• CSK Tyrosine-Protein Kinase MeSH
• Databases, Protein MeSH
• Phosphorylation MeSH
• DNA, Complementary MeSH
• Humans MeSH
• Membrane Microdomains immunology   MeSH
• Molecular Sequence Data MeSH
• Amino Acid Sequence MeSH
• src-Family Kinases MeSH
• Lymphocyte Specific Protein Tyrosine Kinase p56(lck) metabolism   MeSH
• Protein-Tyrosine Kinases metabolism   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Adaptor Proteins, Vesicular Transport MeSH
• CD4 Antigens MeSH
• CD8 Antigens MeSH
• CSK Tyrosine-Protein Kinase MeSH
• CSK protein, human MeSH Browser
• DNA, Complementary MeSH
• Lck-interacting protein, mouse MeSH Browser
• src-Family Kinases MeSH
• Lymphocyte Specific Protein Tyrosine Kinase p56(lck) MeSH
• Protein-Tyrosine Kinases MeSH

A key molecule necessary for activation of T lymphocytes through their antigen-specific T cell receptor (TCR) is the transmembrane adaptor protein LAT (linker for activation of T cells). Upon TCR engagement, LAT becomes rapidly tyrosine phosphorylated and then serves as a scaffold organizing a multicomponent complex that is indispensable for induction of further downstream steps of the signaling cascade. Here we describe the identification and preliminary characterization of a novel transmembrane adaptor protein that is structurally and evolutionarily related to LAT and is expressed in B lymphocytes, natural killer (NK) cells, monocytes, and mast cells but not in resting T lymphocytes. This novel transmembrane adaptor protein, termed NTAL (non-T cell activation linker) is the product of a previously identified WBSCR5 gene of so far unknown function. NTAL becomes rapidly tyrosine-phosphorylated upon cross-linking of the B cell receptor (BCR) or of high-affinity Fcgamma- and Fc epsilon -receptors of myeloid cells and then associates with the cytoplasmic signaling molecules Grb2, Sos1, Gab1, and c-Cbl. NTAL expressed in the LAT-deficient T cell line J.CaM2.5 becomes tyrosine phosphorylated and rescues activation of Erk1/2 and minimal transient elevation of cytoplasmic calcium level upon TCR/CD3 cross-linking. Thus, NTAL appears to be a structural and possibly also functional homologue of LAT in non-T cells.

• MeSH
• Adaptor Proteins, Signal Transducing * MeSH
• Lymphocyte Activation MeSH
• B-Lymphocytes immunology   metabolism   MeSH
• Cell Line MeSH
• Killer Cells, Natural immunology   metabolism   MeSH
• Phosphoproteins chemistry   genetics   isolation & purification   metabolism   MeSH
• Phosphorylation MeSH
• Humans MeSH
• Lymphoid Tissue cytology   metabolism   MeSH
• Membrane Microdomains chemistry   metabolism   MeSH
• Membrane Proteins chemistry   genetics   isolation & purification   metabolism   MeSH
• Molecular Sequence Data MeSH
• Monocytes immunology   metabolism   MeSH
• Mice, Inbred C57BL MeSH
• Mice, Knockout MeSH
• Mice MeSH
• Proteins * MeSH
• Receptors, Antigen, B-Cell metabolism   MeSH
• Receptors, Fc metabolism   MeSH
• Receptors, IgE metabolism   MeSH
• Receptors, IgG metabolism   MeSH
• Amino Acid Sequence MeSH
• Signal Transduction * MeSH
• T-Lymphocytes immunology   metabolism   MeSH
• Carrier Proteins chemistry   genetics   isolation & purification   metabolism   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Adaptor Proteins, Signal Transducing * MeSH
• Phosphoproteins MeSH
• LAT protein, human MeSH Browser
• Lat protein, mouse MeSH Browser
• LAT2 protein, human MeSH Browser
• LAT2 protein, mouse MeSH Browser
• Membrane Proteins MeSH
• Proteins * MeSH
• Receptors, Antigen, B-Cell MeSH
• Receptors, Fc MeSH
• Receptors, IgE MeSH
• Receptors, IgG MeSH
• Carrier Proteins MeSH

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 alpha/beta 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 alpha/beta 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 Src-specific substrates. Moreover, overexpression of PAG in Jurkat cells downregulates T cell receptor-mediated activation of the transcription factor nuclear factor of activated T cells. These findings collectively suggest that in the absence of external stimuli, the PAG-Csk complex transmits negative regulatory signals and thus may help to keep resting T cells in a quiescent state.

• MeSH
• Adaptor Proteins, Signal Transducing MeSH
• Lymphocyte Activation * MeSH
• CD3 Complex metabolism   MeSH
• CSK Tyrosine-Protein Kinase MeSH
• Phosphoproteins genetics   metabolism   MeSH
• Glycosphingolipids metabolism   MeSH
• Cloning, Molecular MeSH
• DNA, Complementary genetics   MeSH
• Humans MeSH
• Membrane Proteins genetics   metabolism   MeSH
• Molecular Sequence Data MeSH
• Receptors, Antigen, T-Cell metabolism   MeSH
• Amino Acid Sequence MeSH
• Signal Transduction MeSH
• src-Family Kinases MeSH
• T-Lymphocytes immunology   MeSH
• Protein-Tyrosine Kinases metabolism   MeSH
• Protein Binding MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Adaptor Proteins, Signal Transducing MeSH
• CD3 Complex MeSH
• CSK Tyrosine-Protein Kinase MeSH
• CSK protein, human MeSH Browser
• Phosphoproteins MeSH
• Glycosphingolipids MeSH
• DNA, Complementary MeSH
• Membrane Proteins MeSH
• PAG1 protein, human MeSH Browser
• Receptors, Antigen, T-Cell MeSH
• src-Family Kinases MeSH
• Protein-Tyrosine Kinases MeSH

The glycosyl-phosphatidylinositol (GPI)-anchored glycoprotein Thy-1 is one of the most abundant molecules expressed on the surface of rat mast cells and rat basophilic leukemia (RBL) cells. The finding that Thy-1 from detergent-solubilized RBL-2H3 cells forms complexes with src-related protein-tyrosine kinase p56/p53lyn suggested that this kinase may play a key role in Thy-1-mediated mast-cell activation. The molecular mechanism of this activation is, however, unknown. Here we show that in RBL-2H3-derived cells extracted by the standard procedure with several non-ionic detergents, the majority of Thy-1 and p56/p53lyn were not released into postnuclear supernatant but remained associated with the detergent-resistant cytoskeletal/nuclear fraction. Pretreatment of the cells with the cholesterol-complexing agents, saponin or digitonin, resulted in complete solubilization of Thy-1 and p56/p53lyn in non-ionic detergents and dissociation of the complexes; this implies that cholesterol plays a crucial role in stabilization of the complexes. This conclusion was supported by double immunofluorescence colocalization experiments which also allowed us to estimate the size of the insoluble complexes to be about 0.1 micron. Sequential treatment with saponin and Nonidet P-40 was used to fractionate tyrosine-phosphorylated proteins during Thy-1-mediated activation of RBL-2H3 cells. Among the soluble cytoplasmic proteins the most dramatic change in tyrosine phosphorylation was found in pp72, whereas pp40 and pp33 were found mainly in the membrane fraction. Our data suggest that surface aggregation of GPI-anchored Thy-1 molecules leads to aggregation of p56/p53lyn kinase located in the same membrane microdomain, followed by transphosphorylation of both soluble and membrane-bound substrates.

• MeSH
• Thy-1 Antigens immunology   MeSH
• Cell Membrane immunology   MeSH
• Cholesterol physiology   MeSH
• Detergents MeSH
• Electrophoresis, Polyacrylamide Gel MeSH
• Fluorescent Antibody Technique MeSH
• Rats MeSH
• Humans MeSH
• Mast Cells immunology   MeSH
• Molecular Sequence Data MeSH
• Mice MeSH
• Precipitin Tests MeSH
• Base Sequence MeSH
• src-Family Kinases metabolism   MeSH
• Animals MeSH
• Check Tag
• Rats MeSH
• Humans MeSH
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Thy-1 Antigens MeSH
• Cholesterol MeSH
• Detergents MeSH
• lyn protein-tyrosine kinase MeSH Browser
• src-Family Kinases MeSH

The T-lymphocyte co-receptors of MHC glycoproteins CD4 and CD8 are known to be associated with the protein tyrosine kinase Lck via cysteine-containing sequences in the cytoplasmic domains of CD4 and CD8 and in the N-terminal domain of Lck. Here we demonstrate that a fraction of CD4 and CD8 molecules are associated with very large, detergent-resistant complexes containing several glycosylphosphatidylinositol-anchored proteins, (glyco)lipids, and protein tyrosine kinases Lck and Fyn but apparently no other major transmembrane proteins. Association of Lck and Fyn with these large complexes is, in contrast to simple CD4/CD8-Lck complexes, not sensitive to alkylation with iodoacetamide. These large complexes therefore represent an alternative way of association of CD4 and CD8 with the protein tyrosine kinases, which may play a role in signaling through these receptors.

• MeSH
• CD4 Antigens analysis   MeSH
• CD8 Antigens analysis   MeSH
• Cell Membrane chemistry   MeSH
• Glycosylphosphatidylinositols MeSH
• Humans MeSH
• Lymphocytes chemistry   MeSH
• Neoplasm Proteins * MeSH
• Lymphocyte Specific Protein Tyrosine Kinase p56(lck) MeSH
• Protein-Tyrosine Kinases analysis   classification   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• CD4 Antigens MeSH
• CD8 Antigens MeSH
• FRK protein, human MeSH Browser
• Glycosylphosphatidylinositols MeSH
• Neoplasm Proteins * MeSH
• Lymphocyte Specific Protein Tyrosine Kinase p56(lck) MeSH
• Protein-Tyrosine Kinases MeSH

Four of the tetraspans family antigens expressed in B cells, CD37, CD53, TAPA-1, and R2/C33, as well as at least two other molecules, CD19 and CD21, coprecipitate with DR antigens from mild detergent lysates of human B-cell lines and tonsillar B cells. Coprecipitation and preclearing experiments indicate the existence of large multicomponent complexes containing jointly the seven components, although some "incomplete" complexes lacking some of the components may also exist. The complexes contain only a relatively small fraction of the total cellular pool of relevant molecules. The existence of these "tetraspans-DR complexes" may be related to the previously reported antiproliferative and signaling effects of antibodies against most of their components.

• MeSH
• Tetraspanin 25 MeSH
• Tetraspanin 28 MeSH
• Antigens, Neoplasm * MeSH
• Antigens, Surface isolation & purification   metabolism   MeSH
• B-Lymphocytes immunology   MeSH
• Cell Line MeSH
• Antigens, CD isolation & purification   metabolism   MeSH
• Antigens, Differentiation, T-Lymphocyte isolation & purification   metabolism   MeSH
• Antigens, Differentiation isolation & purification   metabolism   MeSH
• Glycoproteins isolation & purification   metabolism   MeSH
• HLA-DR Antigens isolation & purification   metabolism   MeSH
• Humans MeSH
• Membrane Glycoproteins * MeSH
• Membrane Proteins * MeSH
• Antibodies, Monoclonal MeSH
• Precipitin Tests MeSH
• Kangai-1 Protein MeSH
• Proto-Oncogene Proteins * MeSH
• Tetraspanins MeSH
• Blotting, Western MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Tetraspanin 25 MeSH
• Tetraspanin 28 MeSH
• Antigens, Neoplasm * MeSH
• Antigens, Surface MeSH
• Antigens, CD MeSH
• CD37 protein, human MeSH Browser
• CD53 protein, human MeSH Browser
• CD81 protein, human MeSH Browser
• CD82 protein, human MeSH Browser
• Antigens, Differentiation, T-Lymphocyte MeSH
• Antigens, Differentiation MeSH
• Glycoproteins MeSH
• HLA-DR Antigens MeSH
• Membrane Glycoproteins * MeSH
• Membrane Proteins * MeSH
• Antibodies, Monoclonal MeSH
• Kangai-1 Protein MeSH
• Proto-Oncogene Proteins * MeSH
• Tetraspanins MeSH

Thy-1 is a surface glycoprotein that is attached to the plasma membrane by a glycosyl-phosphatidyl-inositol anchor. Crosslinking of Thy-1 in rat mast cells and basophilic leukemia cells (RBL-2H3) induces cell activation including histamine release and tyrosine phosphorylation of several proteins. Here we show that glycosyl-phosphatidylinositol-linked Thy-1 forms noncovalent complexes with src-related protein-tyrosine kinase p53/p56lyn and other protein-tyrosine kinases and/or their substrates. These complexes are resistant to solubilization by a nonionic detergent, sedimentable at 200,000 x g, and very large ( > 10 MDa) as determined by gel chromatography. Activation of RBL-2H3 cells by crosslinking of the high-affinity IgE receptors resulted in decreased recovery of the complexes. The combined data indicate the existence of large detergent-resistant domains in the surface membrane of mast cells that may play an important role in their activation.

• MeSH
• Leukemia, Basophilic, Acute MeSH
• Antigens, Surface isolation & purification   metabolism   MeSH
• Thy-1 Antigens MeSH
• CSK Tyrosine-Protein Kinase MeSH
• Electrophoresis, Polyacrylamide Gel MeSH
• Phosphoric Diester Hydrolases MeSH
• Chromatography, Gel MeSH
• Immunoblotting MeSH
• Rats MeSH
• Phosphatidylinositol Diacylglycerol-Lyase MeSH
• Macromolecular Substances MeSH
• Membrane Glycoproteins isolation & purification   metabolism   MeSH
• Molecular Weight MeSH
• Antibodies, Monoclonal MeSH
• Tumor Cells, Cultured MeSH
• Proto-Oncogene Proteins pp60(c-src) isolation & purification   metabolism   MeSH
• Receptors, IgE metabolism   MeSH
• src-Family Kinases * MeSH
• Protein-Tyrosine Kinases isolation & purification   metabolism   MeSH
• Animals MeSH
• Check Tag
• Rats MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, U.S. Gov't, P.H.S. MeSH
• Names of Substances
• Antigens, Surface MeSH
• Thy-1 Antigens MeSH
• CSK Tyrosine-Protein Kinase MeSH
• Phosphoric Diester Hydrolases MeSH
• Phosphatidylinositol Diacylglycerol-Lyase MeSH
• lyn protein-tyrosine kinase MeSH Browser
• Macromolecular Substances MeSH
• Membrane Glycoproteins MeSH
• Antibodies, Monoclonal MeSH
• Proto-Oncogene Proteins pp60(c-src) MeSH
• Receptors, IgE MeSH
• src-Family Kinases * MeSH
• Protein-Tyrosine Kinases MeSH