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Biomedical subjects

B Schraven

Publications and source records attributed to B Schraven.

At least 19 recordsLinked to original sources
PubMed2001

Complex formation and cooperation of protein kinase C theta and Akt1/protein kinase B alpha in the NF-kappa B transactivation cascade in Jurkat T cells.

Protein kinase C theta (PKC theta) is known to induce NF-kappa B, an essential transcriptional element in T cell receptor/CD28-mediated interleukin-2 production but also T cell survival. Here we provide evidence that PKC theta is physically and functionally coupled to Akt1 in this signaling pathway. First, T cell receptor/CD3 ligation was sufficient to induce activation as well as plasma membrane recruitment of PKC theta. Second, PKC theta selectively cooperated with Akt1, known to act downstream of CD28 co-receptor signaling, in activating a NF-kappa B reporter in T cells. Third, Akt1 function was shown to be required for PKC theta-mediated NF-kappa B transactivation. Fourth, PKC theta co-immunoprecipitated with Akt1; however, neither Akt1 nor PKC theta served as a prominent substrate for each other in vitro as well as in intact T cells. Finally, plasma membrane targeting of PKC theta and Akt1 exerted synergistic transactivation of the I-kappa B kinase beta/inhibitor of NF-kappa B/NF-kappa B signaling cascade independent of T cell activation. Taken together, these findings suggest a direct cross-talk between PKC theta and Akt1 in Jurkat T cells.

Base Sequence↗
PubMed2001

Release from tonic inhibition of T cell activation through transient displacement of C-terminal Src kinase (Csk) from lipid rafts.

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-zeta-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.

Antigens, Polyomavirus Transforming↗
PubMed2001

The transmembrane adaptor protein TRIM regulates T cell receptor (TCR) expression and TCR-mediated signaling via an association with the TCR zeta chain.

T cell receptor (TCR)-interacting molecule (TRIM) is a recently identified transmembrane adaptor protein, which is exclusively expressed in T cells. Here we demonstrate that in mature T cells, TRIM preferentially interacts with the TCR via the TCR-zeta chains and to a lesser extent via the CD3-straightepsilon/gamma heterodimer. Transient or stable overexpression of TRIM in Jurkat T cells results in enhancement of TCR expression on the cell surface and elevated induction of Ca(2+) mobilization after T cell activation. TRIM-mediated upregulation of TCR expression results from inhibition of spontaneous TCR internalization and stabilization of TCR complexes on the cell surface. Collectively, our data identify TRIM as a novel integral component of the TCR complex and suggest that one function of TRIM might be to modulate the strength of signals transduced through the TCR through regulation of TCR expression on the cell surface.

Adaptor Proteins, Signal Transducing↗
PubMed2001

Structural and functional dissection of the cytoplasmic domain of the transmembrane adaptor protein SIT (SHP2-interacting transmembrane adaptor protein).

SIT (SHP2-interacting transmembrane adaptor protein) is a recently identified transmembrane adaptor protein, which is expressed in lymphocytes. Its structural properties, in particular the presence of five potential tyrosine phosphorylation sites, suggest involvement of SIT in TCR-mediated recruitment of SH2 domain-containing intracellular signaling molecules to the plasma membrane. Indeed, it has recently been demonstrated that SIT inducibly interacts with the SH2-containing protein tyrosine phosphatase 2 (SHP2) via an immunoreceptor tyrosine-based inhibition motif (ITIM). Moreover, SIT is capable to inhibit TCR-mediated signals proximal of activation of protein kinase C. However, inhibition of T cell activation by SIT occurs independently of SHP2 binding. The present study was performed to further characterize the molecular interaction between SIT and intracellular effector molecules and to identify the protein(s) mediating its inhibitory function. We demonstrate that SIT not only interacts with SHP2 but also with the adaptor protein Grb2 via two consensus YxN motifs. However, mutation of both Grb2-binding sites also does not influence the inhibitory function of SIT. In contrast, mutation of the tyrosine-based signaling motif Y(168) ASV completely abrogates the ability of SIT to inhibit T cell activation. Co-precipitation experiments revealed that the tyrosine kinase p50(csk) could represent the negative regulatory effector molecule that binds to this motif.

Adaptor Proteins, Signal Transducing↗
PubMed2001

Archived polyacrylamide gels as a resource for proteome characterization by mass spectrometry.

Mass spectrometry was applied to identify protein spots excised from an archived two-dimensional polyacrylamide gel that had been dried and stored for eight years at room temperature. All proteins were successfully identified. Detailed characterization of protein digests by matrix-assisted laser desorption/ionization (MALDI) peptide mapping, nanoelectrospray tandem mass spectrometry and MALDI-quadrupole time-of-flight mass spectrometry revealed no evidence of protein degradation or modifications that could hamper identification of proteins in a sequence database. The experiment with a model protein demonstrated that the pattern of tryptic peptides and the yield of individual peptides were not noticeably changed in the in-gel digest of the archived protein spot compared to the digest of the spot excised from a fresh gel. Thus, the characterization of "archived proteomes" has the potential to advance proteomic research without repeating "wet" biochemistry experiments, that had been perfected in the laboratory years ago.

Acrylic Resins↗
PubMed2001

Adapters in lymphocyte signalling.

Adapter proteins are well recognised as important molecular switches connecting immunoreceptors with intracellular signalling pathways. However, recent data suggest that homeostasis within the lymphatic system also depends on the coordinated activities of negative regulatory adapter proteins. These prevent activation of lymphocytes in the absence of externally applied signals and regulate termination/limitation of ongoing immune responses via different mechanisms.

Animals↗
PubMed2000

Interaction of linker for activation of T cells with multiple adapter proteins in platelets activated by the glycoprotein VI-selective ligand, convulxin.

The snake venom toxin convulxin activates platelets through the collagen receptor glycoprotein VI (GPVI)/Fc receptor gamma-chain (FcR gamma-chain) complex leading to tyrosine phosphorylation and activation of the tyrosine Syk and phospholipase Cgamma2 (PLCgamma2). In the present study, we demonstrate that convulxin is a considerably more powerful agonist than collagen or the GPVI-selective collagen-related peptide (CRP). Confirmation that the response to convulxin is mediated solely via Syk was provided by studies on Syk-deficient platelets. The increase in phosphorylation of the FcR gamma-chain is associated with marked increases in tyrosine phosphorylation of downstream proteins including Syk, linker for activation of T cells (LAT), SLP-76, and PLCgamma2. The transmembrane adapter LAT coprecipitates with SLP-76 and PLCgamma2, as well as with a number of other adapter proteins, some of which have not been previously described in platelets, including Cbl, Grb2, Gads, and SKAP-HOM. Gads is constitutively associated with SLP-76 and is probably the protein bridging its association with LAT. There was no detectable association between Grb2 and SLP-76 in control or stimulated cells, suggesting that the interaction of LAT with Grb2 is present in a separate complex to that of LAT-Gads-SLP-76. These results show that the trimeric convulxin stimulates a much greater phosphorylation of the FcR gamma-chain and subsequent downstream responses relative to CRP and collagen, presumably because of its ability to cause a greater degree of cross-linking of GPVI. The adapter LAT appears to play a critical role in recruiting a number of other adapter proteins to the surface membrane in response to activation of GPVI, presumably at sites of glycolipid-enriched microdomains, enabling an organized signaling cascade that leads to platelet activation.

Adaptor Proteins, Signal Transducing↗
PubMed2000

Phosphoprotein associated with glycosphingolipid-enriched microdomains (PAG), a novel ubiquitously expressed transmembrane adaptor protein, binds the protein tyrosine kinase csk and is involved in regulation of T cell activation.

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.

Adaptor Proteins, Signal Transducing↗
PubMed2000

The Yersinia tyrosine phosphatase YopH targets a novel adhesion-regulated signalling complex in macrophages.

The Yersinia protein tyrosine phosphatase (PTP) YopH is translocated into eukaryotic cells by a type III secretion system that requires bacterial-host cell contact. YopH is composed of two modular effector domains: a substrate-binding domain located in the N-terminal region (residues 1-130) and a PTP catalytic domain located in the C-terminal region (residues 206-468). Previous studies have shown that YopH selectively targets tyrosine-phosphorylated proteins of approximate molecular weight 120 kDa (p120) and 55 kDa (p55) in murine macrophages. It has been demonstrated that p120 actually represents two tyrosine-phosphorylated target proteins, Cas and Fyb. We used the substrate-binding domain of YopH to affinity purify tyrosine-phosphorylated target proteins from lysates of J774A.1 macrophages. Protein microsequencing identified p55 as murine SKAP-HOM. Direct interaction between SKAP-HOM and a catalytically inactive form of YopH was demonstrated in vitro and in macrophages. In addition, we obtained evidence that SKAP-HOM is tyrosine phosphorylated in response to macrophage cell adhesion and that it forms a signalling complex with Fyb. We suggest that dephosphorylation of SKAP-HOM and Fyb by YopH allows yersiniae to interfere with a novel adhesion-regulated signal transduction pathway in macrophages.

Amino Acid Sequence↗
PubMed2000

Passenger lymphocyte syndrome with severe hemolytic anemia due to an anti-Jk(a) after allogeneic PBPC transplantation.

BACKGROUND: After allogeneic peripheral blood progenitor cell (PBPC) transplantation, a patient developed a severe hemolytic transfusion reaction due to passenger lymphocyte syndrome. CASE REPORT: A 50-year-old woman with secondary acute myeloid leukemia transforming from a myelodysplastic syndrome received an ABO-compatible PBPC graft from her HLA-identical sister. For prophylaxis of GVHD, the patient was treated with cyclosporine and methotrexate. Eighteen days after the transplant, the patient experienced a severe hemolytic transfusion reaction due to an alloantibody (anti-Jk(a)) produced by donor lymphocytes. RESULTS: The patient was typed as group A, Jk(a+) before transplantation; the donor was typed as group A, Jk(a-). On Day 18 after transplantation, the immunohematologic screening revealed a positive DAT (C3d 3+) and an alloanti-Jk(a). Hemolysis in the patient at that time was indicated by a drop in the Hb and an increase in the LDH level (maximum, 592 IU/L on Day 23). CONCLUSION: The course of hemolysis and the time of appearance of an alloantibody in this patient meet the criteria for passenger lymphocyte syndrome. In most cases, this syndrome is triggered by ABO system antibodies. This is the first reported case of passenger lymphocyte syndrome after PBPC transplantation that was due to an alloantibody that did not belong to the ABO system.

Acute Disease↗
PubMed2000

Networks in signal transduction: the role of adaptor proteins in platelet activation.

In multicellular organisms, the translation of externally applied signals into appropriate cellular responses is mediated by a multitude of complex intracellular signalling cascades. The accurate function of these signalling pathways is based on the sound interaction of proteins of different categories such as transmembrane receptors, protein kinases, protein phosphatases and g-proteins in three-dimensional signalling complexes. During the past 10 years it has became evident that a new class of proteins termed adaptor proteins is indispensable for the assembly of these intracellular signalling scaffolds. The primary function of adaptor proteins is to mediate protein-protein and protein-lipid interactions and thus to integrate receptor-mediated signals at the intracellular level and to couple signalling receptors to cytosolic signalling pathways. In order to perform this task, adapter proteins are equipped with particular protein-protein and/or protein-lipid interaction modules allowing them to communicate with other signalling proteins. While the essential function of adaptor proteins is clearly established in a variety of cell types (e.g. immune cells), the current knowledge about their role in platelet activation is still in the beginning. Numerous adaptor proteins have been shown to be expressed in platelets and many of them seem to be involved in the assembly of signalling complexes after engagement of platelet receptors such as the collagen receptor glycoprotein VI (GPVI), thrombin receptors, integrin receptors and the GP Ib receptor. This review will focus on the functional role of the most extensively studied adaptor proteins during platelet activation.

Animals↗
PubMed1999

SHPS-1 is a scaffold for assembling distinct adhesion-regulated multi-protein complexes in macrophages.

Inhibitory immunoreceptors downregulate signaling by recruiting Src homology 2 (SH2) domain-containing tyrosine and/or lipid phosphatases to activating receptor complexes [1]. There are indications that some inhibitory receptors might also perform other functions [2] [3]. In adherent macrophages, two inhibitory receptors, SHPS-1 and PIR-B, are the major proteins binding to the tyrosine phosphatase SHP-1. SHPS-1 also associates with two tyrosine-phosphorylated proteins (pp55 and pp130) and a protein tyrosine kinase [4]. Here, we have identified pp55 and pp130 as the adaptor molecules SKAP55hom/R (Src-kinase-associated protein of 55 kDa homologue) and FYB/SLAP-130 (Fyn-binding protein/SLP-76-associated protein of 130 kDa), respectively, and the tyrosine kinase activity as PYK2. Two distinct SHPS-1 complexes were formed, one containing SKAP55hom/R and FYB/SLAP-130, and the other containing PYK2. Recruitment of FYB/SLAP-130 to SHPS-1 required SKAP55hom/R, whereas PYK2 associated with SHPS-1 independently. Formation of both complexes was independent of SHP-1 and tyrosine phosphorylation of SHPS-1. Finally, tyrosine phosphorylation of members of the SHPS-1 complexes was regulated by integrin-mediated adhesion. Thus, SHPS-1 provides a scaffold for the assembly of multi-protein complexes that might both transmit adhesion-regulated signals and help terminate such signals through SHP-1-directed dephosphorylation. Other inhibitory immunoreceptors might have similar scaffold-like functions.

Animals↗
PubMed1999

Tumor therapy with bispecific antibody: the targeting and triggering steps can be separated employing a CD2-based strategy.

For tumor therapy with unprimed effector cells, we developed a novel combination of a CD2 x tumor Ag bispecific targeting Ab and an anti-CD2 triggering Ab. These Ab constructs were derived from two novel CD2 mAbs, termed M1 and M2 that, together, but not individually activate T cells. Unlike many other CD2 Abs, M1 and M2 do not interfere with TCR/CD3 triggering nor do they inhibit binding of CD2 to its ligand CD58, thus preserving the physiological functions of these important effector cell molecules. M2 was chemically conjugated with an Ab recognizing the epidermal growth factor-receptor (EGF-R). Incubation of unprimed peripheral blood mononuclear cells with the bispecific F(ab')2 construct (M2xEGF-R) in the presence of trigger Ab M1 led to efficient selective lysis of EGF-R-positive targets by CTL and NK cells. Importantly, the need for trigger Ab M1 for effector cell stimulation allowed to separate targeting from triggering steps in vitro and should thus enable to focus immune responses to sites of target Ag expression in vivo.

Animals↗
PubMed1999

SHP2-interacting transmembrane adaptor protein (SIT), a novel disulfide-linked dimer regulating human T cell activation.

T lymphocytes express several low molecular weight transmembrane adaptor proteins that recruit src homology (SH)2 domain-containing intracellular molecules to the cell membrane via tyrosine-based signaling motifs. We describe here a novel molecule of this group termed SIT (SHP2 interacting transmembrane adaptor protein). SIT is a disulfide-linked homodimeric glycoprotein that is expressed in lymphocytes. After tyrosine phosphorylation by src and possibly syk protein tyrosine kinases SIT recruits the SH2 domain-containing tyrosine phosphatase SHP2 via an immunoreceptor tyrosine-based inhibition motif. Overexpression of SIT in Jurkat cells downmodulates T cell receptor- and phytohemagglutinin-mediated activation of the nuclear factor of activated T cells (NF-AT) by interfering with signaling processes that are probably located upstream of activation of phospholipase C. However, binding of SHP2 to SIT is not required for inhibition of NF-AT induction, suggesting that SIT not only regulates NF-AT activity but also controls NF-AT unrelated pathways of T cell activation involving SHP2.

Adaptor Proteins, Signal Transducing↗
PubMed1999

Molecular alterations of the Fyn-complex occur as late events of human T cell activation.

Two-dimensional gel electrophoresis of anti-p59fyn immunoprecipitates obtained from non-transformed resting human T lymphocytes resulted in the identification of an oligomeric protein complex which is constitutively formed between Fyn and several additional phosphoproteins (pp43, pp72, pp85, the protein tyrosine kinase Pyk2, as well as the two recently cloned adaptor proteins, SKAP55 and SLAP-130). With the exception of pp85, these proteins seem to preferentially interact with Fyn since they are not detectable in Lck immunoprecipitates prepared under the same experimental conditions. Among the individual members of the Fyn-complex pp85, SKAP55 and pp43 are constitutively phosphorylated on tyrosine residue(s) in vivo and likely interact with Fyn via its src homology 2 (SH2)-domain. In contrast to non-transformed T lymphocytes, continuously proliferating transformed human T cell lines express an altered Fyn-complex. Thus, despite normal expression and tyrosine phosphorylation, SKAP55 does not associate with Fyn in Jurkat cells and in other human T cell lines. Instead two novel proteins interact with Fyn among which one has previously been identified as alpha-tubulin. Importantly, almost identical alterations of the Fyn-complex as observed in Jurkat cells are induced in non-transformed T lymphocytes following mitogenic stimulation. These data suggest that Fyn and its associated proteins could be involved in the control of human T cell proliferation. Moreover, the analogous constitutive alterations in transformed T cell lines could indicate that deregulation of the Fyn-complex might be functionally associated with the malignant phenotype of these cells.

Adaptor Proteins, Signal Transducing↗
PubMed1999

Biochemical and functional analysis of mice deficient in expression of the CD45-associated phosphoprotein LPAP.

The role of the CD45-associated phosphoprotein (LPAP / CD45-AP) during an immune response remains unclear. To understand better the function of LPAP we generated LPAP-deficient mice by disrupting exon 2 of the LPAP gene. LPAP-null mice were healthy and did not show gross abnormalities compared to their wild-type littermates. However, immunofluorescence analysis of T and B lymphocytes revealed a reduced expression of CD45, which did not affect a particular subpopulation. In contrast to a recent report (Matsuda et al., J. Exp. Med. 1998. 187: 1863 - 1870) we neither observed significant alterations of the assembly of the CD45 / lck-complex nor of polyclonal T-cell responses. However, lymphnodes from LPAP-null mice showed increased cellularity, which could indicate that expression of LPAP might be required to prevent expansion of lymphocytes in particular lymphatic organs rather than potentiating immune responses.

Animals↗