Sonderforschungsbereich 415:
"Specifity and Pathophysiology of Signal Transduction Pathways"

Project A9: »Regulation of Expression and Signal Transduction of the CD95 Ligand on T-Lymphocytes and Tumor Cells«

Summary

Repeated stimulation of T-lymphocytes induces CD95/CD95L-mediated apoptosis in sensitive cells. Regulation of this 'activation-induced cell death' (AICD) is associated with an induction CD95L surface expression. Signaling pathways leading to cell death upon binding of the death factor CD95L to its death receptor CD95 have been analyzed in great detail over the past decade. In contrast, the regulation of induction and stabilization of expression of CD95L is still unclear. Furthermore, beside its function as a death factor, CD95L has been ascribed a significant function as a modulator of T cell activation. The aim of our project is to analyze transport and retrograde signaling capacity of the CD95L biochemically and functionally. As a working hypothesis, we postulate that the reverse signals, the cross talk to other signaling molecules and the transport and expression are regulated in a cell-type specific fashion via SH3- or WW-mediated interactions. SH3 domains and WW domains are interaction modules that specifically bind to proline-containing recognition motifs as also present in the long and highly conserved cytosolic region of the CD95L.

The project was started as an integral part of the Collaborative Research Center in 2000. Since then, we have identified a large set of putative interactors for CD95L by mainly two approaches:

1. We generated a large collection of SH3 modules expressed as GST fusion proteins and used them for pull down assays from lysates of CD95L transfectants. CD95L was found to bind to SH3 domains of Src-related kinases, Grb2-related adapter proteins, NCF-1 (neutrophil cytosol factor 1 = p47-phox), Nck and the p85-subunit of PI 3-Kinase.

2. We generated GST fusion proteins that contained different portions of the CD95L cytoplasmic tail and used these for pull down assays from T-cell lysates. Precipitated proteins were separated by 2D gel electrophoresis and analyzed by peptide mass fingerprinting. Here, we found Grb2 again, several proteins associated with the cytoskeleton, and PACSIN2 and FBP17, two proteins that belong to family of SH3-containing putative transporter proteins. Moreover, upon expression in 293T cells, we were able to co-precipitate the latter two proteins with CD95L and we could demonstrate that binding strictly depends on an intact SH3 domain.

The next series of experiments are designed to get insight into the functional relevance of the identified protein-protein interactions.

1. The different interactions have to be verified by co-transfection studies in different cell types. The main goal is to identify the exact molecules that target and transport CD95L to secretory lysosomes and the cell surface.

2. To get a hint on mechanisms of reverse signaling, T cell clones, PHA Blasts and CD95L transfected (CD95-resistant) Jurkat variants will be used to analyze a TCR/CD95L cross talk. Readout will be changes in the TCR/CD3 signaling capacity, growth, cytokine production etc. Similar experiments might be conducted with other CD95L expressing (tumor) cells.

3. In collaboration with Martin Zörnig and colleagues at the Georg-Speyer-Haus in Frankfurt, we try to generate mutant mice that lack different parts of the cytosolic region of CD95L. If successful, this will ultimately demonstrate the role of CD95L as a T-cell effector and regulator molecule.

Original papers

Kreuz, S., Siegmund, D., Rumpf, J.-J., Samel, D., Leverkus, M., Janssen, O., Hacker, G., Dittrich-Breiholz, O., Kracht, M., Scheurich, P., Wajant, H. (2004) NFkB activation by Fas is mediated through FADD, Caspase-8 and RIP and is inhibited by FLIP.

EMBO J. submitted

Oberg, H.H., Sipos, B., Kalthoff, H., Janssen, O., Kabelitz, D. (2004) Expression of the human TDAG51 gene is regulated by MAP kinase via PKC and/or Ras and does not correlate with AICD.

Cell Death Differ, in press

Qian, J., Linkermann, A., Schulze-Osthoff, K., Wajant, H., Kabelitz, D. Janssen, O. (2004) FasL-induced interference with TCR/CD3 stimulation of human peripheral blood lymphocytes

Cell Death Differ, submitted

Ghadimi, M.P., Sanzenbacher, R., Thiede, B., Wenzel, J., Jing, Q., Plomann, M., Borkhardt, A., Kabelitz, D., Janssen, O. (2002) Identification of interaction partners of the cytosolic polyproline region of CD95 ligand (CD178).

FEBS Lett 519: 50-58

Glatzel, A., Entschladen, F., Zollner, T.M., Kraiczy, P., Brade, V., Kaufmann, R., Janßen, O., Lengl-Janßen, B., Wesch,D., Kabelitz, D. (2002) The responsiveness of human V-delta-1 gamma-delta T-lymphocytes to Borrelia burgdorferi is largely restricted to synovial fluid cells from Lyme arthritis patients.

J Infect Dis 186: 1043-1046

Glatzel, A., Wesch, D., Schiemann, F., Brandt, E., Janssen, O., Kabelitz, D. (2002) Patterns of chemokine receptor expression on peripheral blood gamma-delta T lymphocytes: strong expression of CCR5 is a selective feature of V-delta-2/Vdelta9 gamma-delta T cells.

J Immunol 168: 4920-4929

Glauner, H., Siegmund, D., Motejadded, H., Scheurich, P., Henkler, F., Janssen, O. , Wajant, H. (2002) Intracellular localization and transcriptional regulation of Tumor Necrosis Factor (TNF) Receptor-associated Factor 4 (TRAF4).

Eur J Biochem.; 269: 4819-4829

Hinz, T., Flindt, S., Marx, A., Janssen, O., Kabelitz, D. (2001) Inhibition of protein synthesis by the T cell receptor-inducible human TDAG51 gene product.

Cell Signal 13: 345-352.

Hinz, T., Flindt, S., Marx, A., Janssen, O., Kabelitz, D. (2001) The human homologue of the T cell death associated gene 51 inhibits protein biosynthesis in vitro and in mammalian cells.

Cell Signal, 13, 345-352

Stepczynskaya, A., Lauber, K., Engels, I. H., Janssen, O., Kabelitz, D., Wesselborg, S., Schulze-Osthoff, K. (2001) Staurosporine and conventional anticancer drugs induce overlapping, yet distinct pathways of apoptosis and caspase activation.

Oncogene 20: 1193-1202.

Weidmann, E., Boehrer, S., Chow, K.U., Engels, K., Harder, S., Hinz, T., Janssen, O., Kriener, S., Rummel, M.J., Siebert, R., Kabelitz, D., Hansmann, M.L., Mitrou, P.S. (2001) Treatment of aggressive and progressing indolent peripheral T- and NK-cell neoplasias by combination of Fludarabine, Cyclophosphamide and Doxorubicine - Introduction of a protocol by the 'Study Group Peripheral T- and NK-cell neoplasias'.

Onkologie 24: 162-164.

Wenzel, J., Sanzenbacher, R., Ghadimi, M., Lewitzky, M., Zhou, Q., Kaplan, D., Kabelitz, D., Feller, S.M., Janssen, O. (2001) Multiple interactions of the cytosolic polyproline region of the CD95 ligand: hints for the reverse signal transduction capacity of a death factor.

FEBS Letters 509: 255-262.

Reviews

Janssen, O., Qian, J., Linkermann, A., Kabelitz, D. (2003) CD95 ligand - death factor and costimulatory molecule?

Cell Death Diff,. 10, 1215-1225

Linkermann, A., Qian, J., Janssen, O. (2003) Slowly getting a clue on CD95L biology.

Biochem Pharmacol. 66, 1417-1426

Linkermann, A., Qian, J., Kabelitz, D., Janssen, O. (2003) FasL as a death regulator and signal transducer.

Signal Transduction 3, 33-46

Other Publications

Linkermann, A., Qian, J., Janssen, O. (2004) FasL retrograde signalling.

in: Wajant H (Ed.) Fas signalling. Landes Bioscience, in press