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Biomedical subjects

A S Shaw

Publications and source records attributed to A S Shaw.

At least 37 records · Page 2Linked to original sources
PubMed1999

Congenital nephrotic syndrome in mice lacking CD2-associated protein.

CD2-associated protein (CD2AP) is an 80-kilodalton protein that is critical for stabilizing contacts between T cells and antigen-presenting cells. In CD2AP-deficient mice, immune function was compromised, but the mice died at 6 to 7 weeks of age from renal failure. In the kidney, CD2AP was expressed primarily in glomerular epithelial cells. Knockout mice exhibited defects in epithelial cell foot processes, accompanied by mesangial cell hyperplasia and extracellular matrix deposition. Supporting a role for CD2AP in the specialized cell junction known as the slit diaphragm, CD2AP associated with nephrin, the primary component of the slit diaphragm.

Adaptor Proteins, Signal Transducing↗
PubMed1999

Proline residues in CD28 and the Src homology (SH)3 domain of Lck are required for T cell costimulation.

The Src family tyrosine kinases Lck and Fyn are critical for signaling via the T cell receptor. However, the exact mechanism of their activation is unknown. Recent crystal structures of Src kinases suggest that an important mechanism of kinase activation is via engagement of the Src homology (SH)3 domain by proline-containing sequences. To test this hypothesis, we identified several T cell membrane proteins that contain potential SH3 ligands. Here we demonstrate that Lck and Fyn can be activated by proline motifs in the CD28 and CD2 proteins, respectively. Supporting a role for Lck in CD28 signaling, we demonstrate that CD28 signaling in both transformed and primary T cells requires Lck as well as proline residues in CD28. These data suggest that Lck plays an essential role in CD28 costimulation.

Alanine↗
PubMed1999

The immunological synapse: a molecular machine controlling T cell activation.

The specialized junction between a T lymphocyte and an antigen-presenting cell, the immunological synapse, consists of a central cluster of T cell receptors surrounded by a ring of adhesion molecules. Immunological synapse formation is now shown to be an active and dynamic mechanism that allows T cells to distinguish potential antigenic ligands. Initially, T cell receptor ligands were engaged in an outermost ring of the nascent synapse. Transport of these complexes into the central cluster was dependent on T cell receptor-ligand interaction kinetics. Finally, formation of a stable central cluster at the heart of the synapse was a determinative event for T cell proliferation.

Animals↗
PubMed1998

The 14-3-3 proteins positively regulate rapamycin-sensitive signaling.

BACKGROUND: The kinase Tor is the target of the immunosuppressive drug rapamycin and is a member of the phosphatidylinositol kinase (PIK)-related kinase family. It plays an essential role in progression through the G1 phase of the cell cycle. The molecular details of Tor signaling remain obscure, however. RESULTS: We isolated two Saccharomyces cerevisiae genes, BMH1 and BMH2, as multicopy suppressors of the growth-inhibitory phenotype caused by rapamycin in budding yeast. BMH1 and BMH2 encode homologs of the 14-3-3 signal transduction proteins. Deletion of one or both BMH genes caused hypersensitivity to rapamycin in a manner that was dependent on gene dosage. In addition, alterations in the phosphopeptide-binding pocket of the 14-3-3 proteins had dramatically different effects on their ability to relieve the growth-arresting rapamycin phenotype. Mutations that prevented 14-3-3 from binding to a phosphoserine motif abolished its ability to confer rapamycin resistance. In contrast, substitution of two residues in 14-3-3 that surround these phosphoserine-binding sites conferred a dominant rapamycin-resistant phenotype. CONCLUSIONS: Our studies reveal 14-3-3 as an important component in rapamycin-sensitive signaling and provide significant new insights into the structure and function of 14-3-3 proteins.

14-3-3 Proteins↗
PubMed1998

A novel adaptor protein orchestrates receptor patterning and cytoskeletal polarity in T-cell contacts.

Recognition of antigen by T cells requires the formation of a specialized junction between the T cell and the antigen-presenting cell. This junction is generated by the recruitment and the exclusion of specific proteins from the contact area. The mechanisms that regulate these events are unknown. Here we demonstrate that ligand engagement of the adhesion molecule, CD2, initiates a process of protein segregation, CD2 clustering, and cytoskeletal polarization. Although protein segregation was not dependent on the cytoplasmic domain of CD2, CD2 clustering and cytoskeletal polarization required an interaction of the CD2 cytoplasmic domain with a novel SH3-containing protein. This novel protein, called CD2AP, is likely to facilitate receptor patterning in the contact area by linking specific adhesion receptors to the cytoskeleton.

Adaptor Proteins, Signal Transducing↗
PubMed1998

Fidelity of T cell activation through multistep T cell receptor zeta phosphorylation.

The T cell receptor (TCR) alphabeta heterodimer interacts with its ligands with high specificity, but surprisingly low affinity. The role of the zeta component of the murine TCR in contributing to the fidelity of antigen recognition was examined. With sequence-specific phosphotyrosine antibodies, it was found that zeta undergoes a series of ordered phosphorylation events upon TCR engagement. Completion of phosphorylation steps is dependent on the nature of the TCR ligand. Thus, the phosphorylation steps establish thresholds for T cell activation. This study documents the sophisticated molecular events that follow the engagement of a low-affinity receptor.

Animals↗
PubMed1998

14-3-3 proteins are required for maintenance of Raf-1 phosphorylation and kinase activity.

By binding to serine-phosphorylated proteins, 14-3-3 proteins function as effectors of serine phosphorylation. The exact mechanism of their action is, however, still largely unknown. Here we demonstrate a requirement for 14-3-3 for Raf-1 kinase activity and phosphorylation. Expression of dominant negative forms of 14-3-3 resulted in the loss of a critical Raf-1 phosphorylation, while overexpression of 14-3-3 resulted in enhanced phosphorylation of this site. 14-3-3 levels, therefore, regulate the stoichiometry of Raf-1 phosphorylation and its potential activity in the cell. Phosphorylation of Raf-1, however, was insufficient by itself for kinase activity. Removal of 14-3-3 from phosphorylated Raf abrogated kinase activity, whereas addition of 14-3-3 restored it. This supports a paradigm in which the effects of phosphorylation on serine as well as tyrosine residues are mediated by inducible protein-protein interactions.

14-3-3 Proteins↗
PubMed1997

Mitotic and G2 checkpoint control: regulation of 14-3-3 protein binding by phosphorylation of Cdc25C on serine-216.

Human Cdc25C is a dual-specificity protein phosphatase that controls entry into mitosis by dephosphorylating the protein kinase Cdc2. Throughout interphase, but not in mitosis, Cdc25C was phosphorylated on serine-216 and bound to members of the highly conserved and ubiquitously expressed family of 14-3-3 proteins. A mutation preventing phosphorylation of serine-216 abrogated 14-3-3 binding. Conditional overexpression of this mutant perturbed mitotic timing and allowed cells to escape the G2 checkpoint arrest induced by either unreplicated DNA or radiation-induced damage. Chk1, a fission yeast kinase involved in the DNA damage checkpoint response, phosphorylated Cdc25C in vitro on serine-216. These results indicate that serine-216 phosphorylation and 14-3-3 binding negatively regulate Cdc25C and identify Cdc25C as a potential target of checkpoint control in human cells.

14-3-3 Proteins↗
PubMed1997

Phenotypic characterization and analysis of allogeneic T cell responses in ZAP-70 dominant negative transgenic mice.

Antigen stimulation of T cells results in a series of biochemical events including the interaction of both SH2 domains of ZAP-70 with phosphorylated ITAMS on the T cell receptor. In order to study the physiological relevance of decreasing native ZAP-70-SH2 interaction in vivo, we generated transgenic mice expressing a T cell-specific, dominant negative form of ZAP-70 consisting of only the tandem SH2 domains (ZAP-NC). Phenotypically, these animals had a comparable distribution of lymphocyte subsets in the thymus and spleen compared with the wild-type (WT) controls. However, examination of peripheral blood revealed a slow but progressive decrease in the number of lymphocytes, particularly CD4+ cells, with age (17% reduction by 3 months, 58% reduction by 6 months). Allogeneic responses were then evaluated in vitro as well as in vivo using a subcutaneous sponge matrix implant. Although spleen cells cultured for 4 days in vitro with alloantigen developed normal functional responses, allogeneic responses generated in vivo within a subcutaneous sponge matrix were impaired. This was characterized by a depression in cytotoxic T lymphocyte (CTL) activity, a 82% reduction in the frequency of helper T cells, and a 78% reduction in the capacity of sponge-infiltrating lymphocytes to produce IL-2 in response to secondary antigen stimulation. These results indicate that although overt lymphocyte development and in vitro function were unremarkable, expression of a truncated ZAP-70 affected the in vivo survival of peripheral lymphocytes and altered the in vivo generation of functional activity to alloantigen.

Animals↗
PubMed1996

Interaction of 14-3-3 with signaling proteins is mediated by the recognition of phosphoserine.

The highly conserved and ubiquitously expressed 14-3-3 family of proteins bind to a variety of proteins involved in signal transduction and cell cycle regulation. The nature and specificity of 14-3-3 binding is, however, not known. Here we show that 14-3-3 is a specific phosphoserine-binding protein. Using a panel of phosphorylated peptides based on Raf-1, we have defined the 14-3-3 binding motif and show that most of the known 14-3-3 binding proteins contain the motif. Peptides containing the motif could disrupt 14-3-3 complexes and inhibit maturation of Xenopus laevis oocytes. These results suggest that the interactions of 14-3-3 with signaling proteins are critical for the activation of signaling proteins. Our findings also suggest novel roles for serine/threonine phosphorylation in the assembly of protein-protein complexes.

14-3-3 Proteins↗
PubMed1996

Identification of an interferon-gamma receptor alpha chain sequence required for JAK-1 binding.

We have shown previously that a four-amino acid block residing at positions 266-269 (LPKS) in the intracellular domain of the human interferon-gamma (IFN-gamma) receptor alpha chain is critical for IFN-gamma-dependent tyrosine kinase activation and biologic response induction. Herein we show that this sequence is required for the constitutive attachment of the tyrosine kinase JAK-1. Using a vaccinia expression system, a receptor alpha chain-specific monoclonal antibody coprecipitated JAK-1 from cells coexpressing JAK-1 and either (a) wild type IFN-gamma receptor alpha chain, (b) a receptor alpha chain truncation mutant containing only the first 59 intracellular domain amino acids, or (c) a receptor mutant containing alanine substitutions for the functionally irrelevant residues 272-275. In contrast, JAK-1 was not coprecipitated when coexpressed with a receptor alpha chain mutant containing alanine substitutions for the functionally critical residues 266-269 (LPKS). Mutagenesis of the LPKS sequence revealed that Pro-267 is the only residue obligatorily required for receptor function. In addition, Pro-267 is required for JAK-1 binding. These results thus identify a site in the IFN-gamma receptor alpha chain required for constitutive JAK-1 association and establish that this association is critical for IFN-gamma signal transduction.

Amino Acid Sequence↗
PubMed1996

Regulation of antigen receptor signal transduction by protein tyrosine kinases.

The past two years have seen further clarification of the early events occurring in antigen receptor signal transduction that are mediated by the immunoreceptor tyrosine-based activation motif (ITAM). The ITAM was shown to be a specific binding site for the ZAP-70/Syk protein tyrosine kinases and the structure of this complex was solved. In addition, possible mechanisms of activation and functions for these kinases were reported. Lastly, genetic studies established the critical importance of these kinases in antigen-receptor signaling and lymphocyte development.

Animals↗
PubMed1996

Multiple features of the p59fyn src homology 4 domain define a motif for immune-receptor tyrosine-based activation motif (ITAM) binding and for plasma membrane localization.

The src family tyrosine kinase p59fyn binds to a signaling motif contained in subunits of the TCR known as the immune-receptor tyrosine-based activation motif (ITAM). This is a specific property of p59fyn because two related src family kinases, p60src and p56lck, do not bind to ITAMs. In this study, we identify the residues of p59fyn that are required for binding to ITAMs. We previously demonstrated that the first 10 residues of p59fyn direct its association with the ITAM. Because this region of src family kinases also directs their fatty acylation and membrane association (Resh, M.D. 1993, Biochim. Biophys. Acta 1155:307-322; Resh, M.D. 1994. Cell. 76:411-413), we determined whether fatty acylation and membrane association of p59fyn correlates with its ability to bind ITAMs. Four residues (Gly2, Cys3, Lys7, and Lys9) were required for efficient binding of p59fyn to the TCR. Interestingly, the same four residues are present in p56lyn, the other src family tyrosine kinase known to bind to the ITAM, suggesting that this set of residues constitutes an ITAM recognition motif. These residues were also required for efficient fatty acylation (myristoylation at Gly2 and palmitoylation at Cys3), and plasma membrane targeting of p59fyn. Thus, the signals that direct p59fyn fatty acylation and plasma membrane targeting also direct its specific ability to bind to TCR proteins.

Acylation↗
PubMed1996

Ligand-induced assembly and activation of the gamma interferon receptor in intact cells.

Functionally active gamma interferon (IFN-gamma) receptors consist of an alpha subunit required for ligand binding and signal transduction and a beta subunit required primarily for signaling. Although the receptor alpha chain has been well characterized, little is known about the specific role of the receptor beta chain in IFN-gamma signaling. Expression of the wild-type human IFN-gamma receptor beta chain in murine L cells that stably express the human IFN-gamma receptor alpha chain (L.hgR) produced a murine cell line (L.hgR.myc beta) that responded to human IFN-gamma. Mutagenesis of the receptor beta-chain intracellular domain revealed that only two closely spaced, membrane-proximal sequences (P263PSIP267 and I270EEYL274) are required for function. Coprecipitation studies showed that these sequences are necessary for the specific and constitutive association of the receptor beta chain with the JAK-2 tyrosine kinase. These experiments also revealed that the IFN-gamma receptor alpha and beta chains are not preassociated on the surface of unstimulated cells but rather are induced to associate in an IFN-gamma-dependent fashion. A chimeric protein in which the intracellular domain of the beta chain was replaced by JAK-2 complemented human IFN-gamma signaling and biologic responsiveness in L.hgR. In contrast, a c-src-containing beta-chain chimera did not. These results indicate that the sole obligate role of the IFN-gamma receptor beta chain in signaling is to recruit JAK-2 into the ligand-assembled receptor complex.

Amino Acid Sequence↗
PubMed1995

Comparison of p56lck and p59fyn protein expression in thymocyte subsets, peripheral T cells, NK cells, and lymphoid cell lines.

The expression of the src-family kinases, p56lck and p59fyn, is critical for thymocyte development and TCR-mediated signal transduction, and may be important for signaling through other lymphoid receptors as well. Overexpression studies have demonstrated that the levels of p56lck and p59fyn expression can affect T cell development and signaling through the TCR. Therefore, it is likely that their exact expression levels play an important role in modulating signaling in thymocytes, mature T cells, and other lymphocytes. Here, we used quantitative immunoblotting to measure p56lck and p59fyn protein expression levels in thymocyte subsets, peripheral T cells, NK cells, and lymphoid cell lines. p59fyn expression levels were similar to p56lck in most cells that were examined demonstrating that p59fyn is abundantly expressed in T cells. In addition, we found that p56lck protein expression is equivalent in CD4 and CD8 double-negative, double-positive, and single-positive thymocytes. In contrast, p59fyn expression levels were significantly lower in double-positive thymocytes than in the other thymocyte subpopulations. Finally, we demonstrate that p56lck and p59fyn expression varies greatly in a number of cell lines used to study T cell activation and that IL-2 treatment can dynamically regulate p56lck and p59fyn expression in some cells.

Animals↗
PubMed1995

Analysis of the interaction of ZAP-70 and syk protein-tyrosine kinases with the T-cell antigen receptor by plasmon resonance.

Tyrosine phosphorylation of a 17-amino acid immunoreceptor tyrosine-based activation motif (ITAM), conserved in each of the signaling subunits of the T-cell antigen receptor (TCR), mediates the recruitment of ZAP-70 and syk protein-tyrosine kinases (PTKs) to the activated receptor. The interaction between the two tandemly arranged Src-homology 2 (SH2) domains of this family of PTKs and each of the phosphotyrosine-containing ITAMs was examined by real-time measurements of kinetic parameters. The association rate and equilibrium binding constants for the ZAP-70 and syk SH2 domains were determined for the CD3 epsilon ITAM. Both PTKs bound with ka and Kd values of 5 x 10(6) M-1.sec-1 and approximately 25 nM, respectively. Bindings to the other TCR ITAMs (zeta 1, zeta 2, gamma, and delta ITAMs) were comparable, although the zeta 3 ITAM bound approximately 2.5-fold less well. Studies of the affinity of a single functional SH2 domain of ZAP-70 provided evidence for the cooperative nature of binding of the dual SH2 domains. Mutation of either single SH2 domain decreased the Kd by > 100-fold. Finally, the critical features of the ITAM for syk binding were found to be similar to those required for ZAP-70 binding. These data provide insight into the mechanism by which the multisubunit TCR interacts with downstream effector molecules.

Amino Acid Sequence↗