Table of Contents

Staff

Introduction

CD95 (APO-1/Fas) is a member of the death receptor (DR) family, a subfamily of the TNF-R (tumor necrosis factor receptor) superfamily (Krammer et al., 2007). Stimulation of CD95 with its natural ligand, CD95L, or with agonistic anti-CD95 antibodies, such as anti-APO-1, induces apoptosis in sensitive cells. Triggering of CD95 has also been reported to induce non-apoptotic pathways, such as NF-κB, Akt, and ERK. The binding of CD95L to CD95 leads to the formation of the death-inducing signaling complex (DISC). The DISC comprises oligomerized CD95, the adaptor protein FADD, procaspase-8, procaspase-10, and cellular FLICE inhibitory proteins (c-FLIPs). The processes regulating induction of apoptotic versus non-apoptotic pathways at the DISC have not been thoroughly elucidated to date. We addressed life/death decisions at the CD95 DISC by three independent approaches:

• Systems biology studies of CD95 signaling
• Analysis of the role of known DISC proteins: CD95, procaspase-8 and c-FLIP        
• Search for new molecules regulating the CD95 pathways  

Systems biology studies of CD95-induced apoptosis

We built a number of mathematical models describing CD95 DISC-mediated activation of apoptotic and non-apoptotic signaling pathways, e.g. NF-κB, ERK1/2 and p38 MAPKs (Lavrik et al., 2007, 2009 and 2010; Neumann et al., 2010; Fricker et al., 2010; Pforr et al., 2011). The models could quantitatively define the dynamics of DED (Death Effector Domain) proteins, procaspase-8, and c-FLIP, leading to induction of apoptotic and non-apoptotic signaling pathways. The models were validated using quantitative immunoblots and single cell analysis. In conclusion, the combination of biochemical analysis with mathematical modeling provides evidence for a key role of caspase-8 in CD95-mediated signaling, while c-FLIP exerts a regulatory function depending on its concentration at the DISC.  While this finding is based on experiments performed in cell lines, we expect that the non-linear dynamics of DISC assembly is a general systems property of life/death decision-making in CD95 signaling pathways.

Activation of procaspase-8 at the DISC

Caspase-8 is the main initiator caspase in DR–induced apoptosis (Lavrik et al., 2005). Caspase-8 is activated at the DISC. Processing of procaspase-8 at the DISC has been described to follow a two-step model. In our work we further analyzed procaspase-8 processing directly at the DISC and in the cytosol upon DR stimulation. Interestingly, we found a different sequence of cleavage events for procaspase-8, which involves the generation of the C-terminal cleavage product p30. Furthermore, we could show that p30 can sensitize cells towards DR-induced apoptosis. Our data suggested an alternative mechanism of procaspase-8 activation at the DISC (Hoffmann et al., 2009).

New complex activating caspase-8

We reported that upon CD95 stimulation in several T and B cell lines a novel signaling complex is formed, which we termed complex II (Lavrik et al., 2008). Complex II is composed of the DED proteins: procaspase-8a/b, three isoforms of c-FLIP (c-FLIP_L, c-FLIP_S, c-FLIP_R) and FADD. Notably, complex II does not contain CD95. Based on our findings we suggest that CD95 signaling comprises two steps. The first step involves formation of the DISC at the cellular membrane. The second step involves formation of the cytosolic DED protein-containing complex that may play an important role in amplification of caspase activation.

Role of c-FLIP proteins in life/death of the cell

We have found a novel link between c-FLIP_L cleavage in the DISC and the NF-kB pathway. We found that CD95 stimulation resulted in c-FLIP_L cleavage to p43-FLIP. p43-FLIP interacts with the IKK complex leading to its activation. Furthermore, we showed that the apoptotic and NF-kB pathways, respectively, diverge already at the DISC. We have found that c-FLIP levels crucially determine the balance between apoptotic and NF-kB signaling by shaping the dynamics of DISC assembly (Neumann et al., 2010; Pforr et al., 2011).

The role of protein modifications of CD95 in signal transduction

Protein modifications of DR pathways play a central role in the regulation of apoptosis. It has been demonstrated that O-glycosylation of the TRAIL-receptor is essential for sensitivity and resistance towards TRAIL-mediated apoptosis. We analyzed whether and how glycosylation of CD95 influences DISC formation and procaspase-8 activation at the CD95 DISC and, thereby, the onset of apoptosis (Shatnyeva et al., 2011). We concentrated on N-glycostructure since O-glycosylation of CD95 was not found. We applied different approaches to analyze the role of CD95 N-glycosylation on signal transduction: in silico modeling of the CD95 DISC, generation of CD95 glycosylation mutants, and modulation of N-glycosylation by inhibitors of N-glycosylation. We demonstrated that N-deglycosylation of CD95 does not block DISC formation and results only in the reduction of procaspase-8 activation at the DISC. These findings are important for a better understanding of CD95 apoptosis regulation and reveal differences between apoptotic signaling pathways of the TRAIL and CD95 systems.

New molecules regulating CD95 pathways

We have performed three major screens: a proteomics screen together with Martina Schnölzer, DKFZ. An siRNA screen was performed together with Michael Boutros, DKFZ and a yeast-two hybrid screen together with the core facility of DKFZ. All screens provided a number of interesting candidates, which are currently under investigation. For our analysis we selected the proteins, which significantly modulate CD95-mediated apoptotic or non-apoptotic signaling. These studies should provide new insights into the mechanisms of life/death decisions at CD95.

Key references

1. Bentele, M. Lavrik, I. N. Ulrich, M. Stösser, S. Heermann, D. W. Kalthoff, H. Krammer, P. H. Eils, R. (2004). Mathematical modeling reveals threshold mechanism in CD95-induced apoptosis. J Cell Biol 166, 839-51.
2. Lavrik, I.N. Golks, A. Krammer, P. H. (2005). Caspases: pharmacological manipulation of cell death. J Clin Invest 115, 2665-72.
3. Golks, A. Brenner, D. Krammer, P. H. Lavrik, I. N. (2006). The c-FLIP-NH2 terminus (p22-FLIP) induces NF-kappaB activation. J Exp Med 203, 1295-305.
4. Krammer, P. H. Arnold, R. Lavrik, I. N. (2007). Life and death in peripheral T cells. Nat Rev Immunol 7, 532-42.
5. Lavrik, I. N. Golks, A., Riess, D., Bentele, M., Eils, R., and Krammer, P. H. (2007). Analysis of CD95 threshold signaling: triggering of CD95 (FAS/APO-1) at low concentrations primarily results in survival signaling. J Biol Chem 282, 13664-13671.
6. Lavrik, I. N., Mock, T., Golks, A., Hoffmann, J. C., Baumann, S., and Krammer, P. H. (2008). CD95 stimulation results in the formation of a novel death effector domain protein-containing complex. J Biol Chem 283, 26401-26408.
7. Hoffmann, J. C., Pappa, A., Krammer, P. H., and Lavrik, I. N. (2009). A new C-terminal cleavage product of procaspase-8, p30, defines an alternative pathway of procaspase-8 activation. Mol Cell Biol 29, 4431-4440.
8. Lavrik, I. N., Eils, R., Fricker, N., Pforr, C., and Krammer, P. H. (2009). Understanding apoptosis by systems biology approaches. Mol Biosyst 5, 1105-1111.
9. Fricker, N., Beaudouin, J., Richter, P., Eils, R., Krammer, P. H., and Lavrik, I. N. (2010). Model-based dissection of CD95 signaling dynamics reveals both a pro- and antiapoptotic role of c-FLIPL. J Cell Biol 190, 377-389.
10. Lavrik, I. N. (2010). Systems biology of apoptosis signaling networks. Curr Opin Biotechnol 21, 551-555.
11. Matthess, Y., Raab, M., Sanhaji, M., Lavrik, I. N., and Strebhardt, K. (2010). Cdk1/cyclin B1 controls Fas-mediated apoptosis by regulating caspase-8 activity. Mol Cell Biol 30, 5726-5740.
12. Neumann, L., Pforr, C., Beaudouin, J., Pappa, A., Fricker, N., Krammer, P. H., Lavrik, I. N., and Eils, R. (2010). Dynamics within the CD95 death-inducing signaling complex decide life and death of cells. Mol Syst Biol 6, 352.
13. Paulsen, M., Valentin, S., Mathew, B., Adam-Klages, S., Bertsch, U., Lavrik, I., Krammer, P. H., Kabelitz, D., and Janssen, O. (2011). Modulation of CD4(+) T-cell activation by CD95 co-stimulation. Cell Death Differ 18, 619-631.
14. Pforr, C., Neumann, L., Eils, R., Krammer, P. H., and Lavrik, I. N. (2011). Understanding life and death at CD95. Adv Exp Med Biol 691, 151-161.
15. Shatnyeva, O. M., Kubarenko, A. V., Weber, C. E., Pappa, A., Schwartz-Albiez, R., Weber, A. N.,  Krammer, P. H., and Lavrik, I. N. (2011). Modulation of the CD95-Induced Apoptosis: The Role of CD95 N-Glycosylation. PLoS One 6(5):e19927.
16. Kober, A.M.M., Legewie, S., Pforr, C., Fricker, N., Eils, R., Krammer, P.H. and Lavrik, I.N. (2011). Caspase-8 activity is an important regulator of CD95/Fas-mediated MAPK activation. Cell Death and Disease doi: 10.1038/cddis.2011.93