Burkhart Schraven

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.

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.

Molecular Interaction between the Fyn-associated Protein SKAP55 and the SLP-76-associated Phosphoprotein SLAP-130

It has previously been reported that in resting T-lymphocytes the protein tyrosine kinase p59 constitutively co-precipitates with four phosphoproteins of 43, 55, 85, and 120 kDa, respectively. We have recently cloned the 55-kDa protein that was termed Src kinase-associated phosphoprotein of 55 kDa (SKAP55). Here we demonstrate that the recently characterized SH2-domain-containing leukocyte protein 76-associated phosphoprotein of 130 kDa (SLAP-130) is one of the components of the Fyn complex and that it also co-precipitates with SKAP55 in human T-cells. We establish that SKAP55 and SLAP-130 associate with each other when both molecules are co-expressed in COS cells. By co-transfection of truncated mutants of SKAP55 and SLAP-130 as well as by using the two-hybrid selection system, we further demonstrate that the association between SLAP-130 and SKAP55 is direct and involves the Src homology 3 domain of SKAP55 and the proline-rich sequence of SLAP-130.

SKAP-HOM, a novel adaptor protein homologous to the FYN-associated protein SKAP551

A recombinant GST‐Fyn‐SH2 domain was used to purify proteins from lysates of pervanadate treated EL4 cells. N‐terminal sequencing and molecular cloning of one of the purified polypeptides resulted in the identification of a novel adaptor protein that shares strong structural homology to the recently cloned Fyn‐associated adaptor protein SKAP55. This protein was termed SKAP‐HOM ( 55 ologue). Despite their striking homology, SKAP55 and SKAP‐HOM have distinct characteristics. Thus, unlike SKAP55, which is exclusively expressed in T lymphocytes, SKAP‐HOM expression is ubiquitous. Furthermore, while SKAP55 is constitutively tyrosine phosphorylated in resting human T cells, SKAP‐HOM is expressed as a non‐phosphorylated protein in the absence of external stimulus but becomes phosphorylated following T cell activation. In addition, SKAP‐HOM does not associate with p59fyn in T cells although it represents a specific substrate for the kinase in COS cells. Finally, we demonstrate that, as previously shown for SKAP55, SKAP‐HOM interacts with the recently identified polypeptide SLAP‐130.

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.

APO-1(CD95)-mediated apoptosis in Jurkat cells does not involve src kinases or CD45

Tyrosine phosphorylation has been reported to be an early event required for APO‐1/Fas(CD95) signalling in lymphocytes [Eischen, C.M., Dick, C.J. and Leibson, P.J. (1994) J. Immunol. 153, 1947–1954]. We have compared two mutant Jurkat cells, one largely deficient in expression of CD45 (J45.01) and a second one deficient in expression of p56lck (JCaM1.6) with wild type Jurkat cells for their ability to undergo APO‐1‐induced apoptosis. No significant difference was observed among the three cell lines. In the mutant Jurkat cells APO‐1 triggering did not result in increased tyrosine phosphorylation of cytosolic proteins. Furthermore, herbimycin A did not inhibit but rather augmented apoptosis at concentrations which effectively degraded the src related kinases lck and fyn. The data suggest that APO‐1‐mediated signalling is independent from src kinases and CD45.

Integration of receptor-mediated signals in T cells by transmembrane adaptor proteins

Abstract Our knowledge of the molecular events leading to T-cell activation has not yet fully explained how intracellular effector molecules are recruited to the plasma membrane after engagement of the TCR by antigen–MHC. The identification of transmembrane adaptor proteins might help to elucidate the mechanisms involved.

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.

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 α‐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.

Coupling the TCR to downstream signalling pathways: the role of cytoplasmic and transmembrane adaptor proteins.

Engagement of the T-cell receptor (TCR) complex initiates a cascade of intracellular events ultimately leading to T-cell proliferation and differentiation. One of the first detectable consequences of TCR triggering is the activation of cytoplasmic protein kinases which, through phosphorylation of specific substrates, couple the TCR to downstream signalling cascades. Although it is well established that activation of the Ras- and the calcium-dependent calcineurin pathway is required for the achievement of T-cell activation, the precise mechanism as to how the TCR is connected to these intracellular effector molecules is still unclear. Major progress has been made in this regard with the molecular characterization of novel cytoplasmic and transmembrane molecules called adaptor proteins which integrate TCR-mediated signals at the intracellular level thus allowing fine tuning of T-cell responses.