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«

The death factor CD95Ligand (CD95L, FasL) plays an essential role for the induction of apoptosis within the immune system. Thus, CD95L is involved in the termination of immune responses by activation induced cell death (AICD). It is an effector molecule for cytotoxic T cells and Natural Killer (NK) cells and is involved in the establishment of immune privileged tissues. Being expressed on many tumor cells, CD95L can act as a tumor defense molecule that counterattacks CD95-postive tumor infiltrating T lymphocytes. In addition, CD95L regulates activation of peripheral T cells as a costimulatory molecule and positive selection of thymocytes as an accessory molecule in the context of TCR-mediated activation. CD95L is a type II transmembrane protein and can be shedded by metalloproteases. Elevated levels of soluble CD95L are often observed in immunological and non-immunological diseases, although strict correlations with pathophysiology, disease progression or prognosis are only rarely found.

In cytotoxic T cells and NK cells, CD95L is stored in secretory lysosomes and only upon stimulation released into the immunological synapse. During AICD of CD4 T cells, CD95L is de novo synthetized and rapidly and transiently expressed on the cell surface. To date, it is absolutely unclear how the different expression patterns and functions of CD95L are regulated at the molecular and biochemical level.

We previously identified a number proteins that could be involved in the regulation of expression and reverse signal transduction of CD95L. In fact, numerous tested SH3 and WW domains in vitro strongly interact with CD95L via its proline-rich intracellular domain. The association is selective and can be specifically blocked by CD95L peptides. Moreover, many of the candidate CD95L binding proteins are enzymes and adapter proteins that play essential roles in TCR/CD3-mediated signal transduction.

In the following term, we will analyze the crosstalk between TCR- and CD95L-dependent signaling cascades that became evident from the observed inhibition of TCR/CD3-induced proliferation and tyrosine phosphorylation after pretreatment with FasFc fusion protein or anti-FasL monoclonal antibodies. Based on a report in the context of CD95L-related TNF and on our own preliminary observations, we will also address the question whether casein kinases are involved in the regulation of reverse signalling of TNF family members and especially CD95L.

Among other proteins, the cytosolic portion of CD95L precipitated PACSIN2 and FBP17 from T cell lysates. Both proteins are known to be important in the context of lysosomal transport and cytoskeletal organization in various cellular systems. Cotransfection and coprecipitation with CD95L confirm their association with CD95L and suggest that the interaction might be of cell biological relevance for the intracellular localization of CD95L. Thus, wild-type but not SH3-mutated FBP17 and PACSIN2 redistribute CD95L to the lysosomal compartment in non-hematopoietc cells. We will further investigate the exact function of FBP17 and PACSIN2 and other related proteins in lysosomal targeting and transport of CD95L in T cells and tumor cells.

Two of three SH3 domains of the adapter protein Nck interact with CD95L in vitro. In T cells, Nck plays a role in the inducible recruitment of cytoskeletal elements during receptor-triggered formation of the immunological synapse (IS). Again, cotransfection and coprecipitation with CD95L confirm this interaction. Moreover, analyses by confocal microscopy suggest that upon stimulation, e.g. with phorbolester and ionophore, Nck, FasL and CathepsinD as a marker for lysosomes translocate together to the IS. During the following term, we will investigate the nature of this translocation in different cell populations with a major focus on contact areas between T cells and target cells, T cells and B cells, and T cells and dendritic cells.

The notion that CD95L is expressed in a biphasic fashion in long-term T cell clones points to a yet unknown mechanism of shedding or internalization and degradation of the molecule. In collaboration with other groups within the SFB, we will be able to analyze the exact mechanisms of expression and shedding of FasL in different cell types.

Original papers

Lettau M, Pieper J, Gerneth A, Lengl-Janßen B, Voss M, Linkermann A, Schmidt H, Gelhaus C, Leippe M, Kabelitz D, Janssen O. (2010) The adapter protein Nck: Role of individual SH3 and SH2 binding modules for protein interactions in T lymphocytes.

Protein Science 19: 658-669

Lettau M, Pieper J, Gerneth A, Lengl-Janßen B, Voss M, Linkermann A, Schmidt H, Gelhaus C, Leippe M, Kabelitz D, Janssen O. (2010) The adapter protein Nck: role of individual SH3 and SH2 binding modules for protein interactions in T lymphocytes.

Protein Sci 19:658-669

Paulsen M, Valentin S, Mathew B, Adam-Klages S, Bertsch U, Lavrik I, Krammer PH, Kabelitz D, Janssen O. (2010) Modulation of CD4+ T cell activation by CD95 costimulation.

Cell Death Differ, in revision

Paulsen M, Valentin S, Mathew B, Adam-Klages S, Bertsch U, Lavrik I, Krammer PH, Kabelitz D, Janssen O. (2010) Modulation of CD4+ T cell activation by CD95 costimulation.

Cell Death Differ, in revision

Lettau M, Pieper J, Janssen O. (2009) Nck adapter proteins: Functional versatility in T cells.

Cell Commun Signal 7:1

Lettau M, Paulsen M, Kabelitz D, Janssen O. (2009) FasL expression and reverse signalling.

Results Probl Cell Diff 49:49-61

Lettau M, Paulsen M, Kabelitz D, Janssen O. (2009) FasL expression and reverse signalling.

Results Probl Cell Diff 49:49-61

Linkermann A, Gelhaus G, Lettau M, Qian J, Kabelitz D, Janssen O. (2009) Identification of interaction partners for individual SH3 domains of members of Fas ligand associated members of the PCH protein family in T lymphocytes.

Biochim Biophys Acta 1794:168-176

Linkermann A, Gelhaus C, Lettau M, Qian J, Kabelitz D, Janssen O. (2009) Identification of interaction partners for individual SH3 domains of members of Fas ligand associated members of the PCH protein family in T lymphocytes.

Biochim Biophys Acta 1794:168-176

Mirghomizadeh F, Bullwinkel J, Orinska Z, Janssen O, Petersen A, Singh PB, Bulfone-Paus S. (2009) Transcriptional regulation of mouse mast cell protease-2 by IL-15.

J Biol Chem 284:32635-32641

Mirghomizadeh F, Bullwinkel J, Orinska Z, Janssen O, Petersen A, Singh PB, Bulfone-Paus S. (2009) Transcriptional regulation of mouse mast cell protease-2 by IL-15.

J Biol Chem 284:32635-32641

Ohnesorge S, Oberg , Peters C, Janssen O, Kabelitz D, Wesch D. (2009) Differential polyI:C) responses of human Vg9Vd2 T cells stimulated with pyrophosphates versus aminobisphosphonates.

The Open Immunology Journal 2:135-142

Paulsen M, Mathew B, Qian J, Lettau M, Kabelitz D, Janssen O. (2009) FasL crosslinking inhibits activation of human peripheral blood T cells.

Int Immunol 21:587-598

Paulsen M, Mathew B, Qian J, Lettau M, Kabelitz D, Janssen O. (2009) FasL crosslinking inhibits activation of human peripheral blood T cells.

Int Immunol 21:587-598

Schmidt H, Gelhaus C, Lucius R, Nebendahl M, Leippe M, Janssen O. (2009) Enrichment and analysis of secretory lysosomes from lymphocyte populations.

BMC Immunology 10:41

Voss M, Lettau M, Janssen O. (2009) Identification of SH3 domain interaction partners of human FasL (CD178) by phage display screening.

BMC Immunology 10:53

Lettau M, Paulsen M, Kabelitz D, Janssen O. (2008) Storage, expression and function of fas ligand, the key death factor of immune cells.

Curr Med Chem 15:1684-1696,

Lettau M, Paulsen M, Kabelitz D, Janssen O (2008) Storage, expression and function of fas ligand, the key death factor of immune cells.

Curr Med Chem 15:1684-1696

Paulsen M, Ussat S, Jakob M, Scherer G, Lepenies I, Schütze S, Kabelitz D, Adam-Klages S. (2008) Interaction with XIAP prevents full caspase-3/7 activation in proliferating human T lymphocytes.

Eur J Immunol 38:1979-1987

Schmidt H, Gelhaus C, Nebendahl M, Lettau M, Watzl C, Kabelitz D, Leippe M, Janssen O. (2008) 2D-DIGE analyses of enriched secretory lysosomes reveal heterogeneous profiles of functionally relevant proteins in leukemic and activated human NK cells.

Proteomics 8:2911-2925

Schmidt H, Gelhaus C, Nebendahl M, Lettau M, Watzl C, Kabelitz D, Leippe M, Janssen O. (2008) 2D-DIGE analyses of enriched secretory lysosomes reveal heterogeneous profiles of functionally relevant proteins in leukemic and activated human NK cells.

Proteomics 8:2911-2925

Voss M, Lettau M, Paulsen M, Janssen O. (2008) Posttranslational regulation of Fas ligand function.

Cell Commun Signal 6:11

Ammerpohl O, Trauzold A, Schniewind B, Griep U, Pilarsky C, Grutzmann R, Saeger HD, Janssen O, Sipos B, Kloppel G, Kalthoff H. (2007) Complementary effects of HDAC inhibitor 4-PB on gap junction communication and cellular export mechanisms support restoration of chemosensitivity of PDAC cells.

Br J Cancer 96:73-81

Ammerpohl O, Trauzold A, Schniewind B, Griep U, Pilarsky C, Grutzmann R, Saeger HD, Janssen O, Sipos B, Kloppel G, Kalthoff H. (2007) Complementary effects of HDAC inhibitor 4-PB on gap junction communication and cellular export mechanisms support restoration of chemosensitivity of PDAC cells.

Br J Cancer 96:73-81

Lettau M, Beyer A, Janssen O. (2007) Novel monoclonal antibodies for the investigation of PCH family proteins.

Signal Transduction 7:320-328

Schulte M, Reiss K, Lettau M, Maretzky T, Ludwig A, Hartmann D, de Strooper B, Janssen O, Saftig P. (2007) ADAM10 regulates FasL cell surface expression and modulates FasL-induced cytotoxicity and activation-induced cell death.

Cell Death Differ 14:1040-9

Schulte M, Reiss K, Lettau M, Maretzky T, Ludwig A, Hartmann D, de Strooper B, Janssen O, Saftig P. (2007) ADAM10 regulates FasL cell surface expression and modulates FasL-induced cytotoxicity and activation-induced cell death.

Cell Death Differ 14:1040-9

Schulte M, Reiss K, Lettau M, Maretzky T, Ludwig A, Hartmann D, de Strooper B, Janssen O, Saftig P. (2007) ADAM10 regulates FasL cell surface expression and modulates FasL-induced cytotoxicity and activation-induced cell death.

Cell Death Differ 14:1040-9

Ammerpohl O, Trauzold A, Schniewind B, Griep U, Pilarsky C, Grutzmann R, Saeger HD, Janssen O, Kloppel G, Kalthoff H. (2006) Complementary effects of HDAC inhibitor4-PB on gap junction communication and cellular export mechanisms support restoration of chemosensivity of PDAC cells

Br J Cancer, in press

Ammerpohl O, Trauzold A, Schniewind B, Griep U, Pilarsky C, Grützmann R, Saeger HD, Janssen O, Sipos B, Klöppel G, Kalthoff H. (2006) Complementary effects of HDAC inhibitor 4-PB on gap junction communication and cellular export mechanisms support restoration of chemosensitivity of PDAC cells.

Br J Cancer 96:73-81

Cahuzac N, Baum W, Kirkin V, Conchonaud F, Marguet D, Janssen O, Zörnig M, Hueber AO. (2006) Fas Ligand is localized to membrane rafts where it displays increased apoptosis-inducing activity.

Blood 107:2384-2391

Lettau M, Qian J, Linkermann A, Larose L, Latreille M, Kabelitz D, Janssen O. (2006) The adapter protein Nck interacts with FasLigand: Guiding the death factor to the cytotoxic immunological synapse.

Proc Natl Acad Sci 103:5911-5916

Qian J, Chen W, Lettau M, Podda G, Zörnig M, Kabelitz D, Janssen O. (2006) Regulation of FasL expression: A SH3 domain containing protein family involved in the lysosomal association of Fasl.

Cell Signal 18:1327-1337

Baum W, Kirkin V, Mateus Fernández SB, Pick R, Lettau M, Janssen O, Zörnig M. (2005) Binding of the intracellular Fas Ligand (FasL) domain to the adaptor protein PSTPIP results in a cytoplasmic localization of FasL.

J Biol Chem 280:40012-40024

Qian J, Lettau M, Podda G, Janssen O. (2005) FasL associated factors and their potential role in the regulation of FasL expression.

Signal Transduction 5:195-201

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

J Cell Biol. 166:369-380.

Lettau M, Qian J, Kabelitz D, Janssen O. (2004) Activation-dependent FasLigand expression in T lymphocytes and Natural Killer cells.

Signal Transduction 5-6:206-211

Oberg HH, Sipos B, Kalthoff H, Janssen O, Kabelitz D. (2004) Regulation of T-cell death-associated gene 51 (TDAG51) expression in human T-cells.

Cell Death Diff., 11:674-684

Ghadimi MP, 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, 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) 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

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.

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 KU, Engels K, Harder S, Hinz T, Janssen O, Kriener S, Rummel MJ, Siebert R, Kabelitz D, Hansmann ML, Mitrou PS. (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 SM, 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.

Janssen O, Stocker A, Sanzenbacher R, Oberg HH, Siddiqi MA, Kabelitz D. (2000) Differential regulation of activation-induced cell death (AICD) in individual human T cell clones.

Int. Arch. Allergy Immunol. 121: 183-193.

Kabelitz D, Janssen O. (1999) Antigen-induced death of T-lymphocytes.

Pediatr Pathol Mol Med 18: 329-354.

Sanzenbacher R, Kabelitz D, Janssen O (1999) A role for SLP-76 in CD4-induced desensitization of the TCR/CD3 signaling complex.

J Immunol, 163, 3143-3152

Reviews

Lettau M, Schmidt H, Kabelitz D, Janssen O. (2007) Secretory lysosomes and their cargo in T and NK cells.

Immunol Lett, 108:10-19

Adam-Klages S, Adam D, Janssen O, Kabelitz D (2005) Death receptors and caspases: role in lymphocyte proliferation, cell death, and autoimmunity.

Immunol Res 33:149-166

Linkermann A, Qian J, Kabelitz D, Janssen O. (2005) Considering Fas Ligand as a target for therapy.

Expert Opin Ther Targets 9:119-134

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

Janssen O, Sanzenbacher R, Kabelitz D. (2000) Regulation of activation-induced cell death of mature T-lymphocyte populations.

Cell Tissue Res, 301, 85-99

Other publications

Janssen O, Hass R. (2005) Apoptosis - Live and let die.

Signal Transduction 5:293-296 (Editorial)

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

in: Wajant H (Ed.) Fas signalling. Landes Bioscience pp97-102