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• W2140662159 abstract "Article9 March 2010Open Access Dynamics within the CD95 death-inducing signaling complex decide life and death of cells Leo Neumann Leo Neumann Division of Theoretical Bioinformatics, German Cancer Research Center (DKFZ), Heidelberg, Germany Department for Bioinformatics and Functional Genomics, Institute for Pharmacy and Molecular Biotechnology, Bioquant, University of Heidelberg, Heidelberg, GermanyShared first authorship Search for more papers by this author Carina Pforr Carina Pforr Division of Immunogenetics, German Cancer Research Center (DKFZ), Heidelberg, GermanyShared first authorship Search for more papers by this author Joel Beaudouin Joel Beaudouin Division of Theoretical Bioinformatics, German Cancer Research Center (DKFZ), Heidelberg, Germany Department for Bioinformatics and Functional Genomics, Institute for Pharmacy and Molecular Biotechnology, Bioquant, University of Heidelberg, Heidelberg, Germany Search for more papers by this author Alexander Pappa Alexander Pappa Division of Immunogenetics, German Cancer Research Center (DKFZ), Heidelberg, Germany Medac GmbH, Theaterstrasse, Wedel/Hamburg, Germany Search for more papers by this author Nicolai Fricker Nicolai Fricker Division of Immunogenetics, German Cancer Research Center (DKFZ), Heidelberg, Germany Search for more papers by this author Peter H Krammer Peter H Krammer Division of Immunogenetics, German Cancer Research Center (DKFZ), Heidelberg, Germany Search for more papers by this author Inna N Lavrik Inna N Lavrik Division of Immunogenetics, German Cancer Research Center (DKFZ), Heidelberg, GermanyShared senior authorship Search for more papers by this author Roland Eils Corresponding Author Roland Eils Division of Theoretical Bioinformatics, German Cancer Research Center (DKFZ), Heidelberg, Germany Department for Bioinformatics and Functional Genomics, Institute for Pharmacy and Molecular Biotechnology, Bioquant, University of Heidelberg, Heidelberg, GermanyShared senior authorship Search for more papers by this author Leo Neumann Leo Neumann Division of Theoretical Bioinformatics, German Cancer Research Center (DKFZ), Heidelberg, Germany Department for Bioinformatics and Functional Genomics, Institute for Pharmacy and Molecular Biotechnology, Bioquant, University of Heidelberg, Heidelberg, GermanyShared first authorship Search for more papers by this author Carina Pforr Carina Pforr Division of Immunogenetics, German Cancer Research Center (DKFZ), Heidelberg, GermanyShared first authorship Search for more papers by this author Joel Beaudouin Joel Beaudouin Division of Theoretical Bioinformatics, German Cancer Research Center (DKFZ), Heidelberg, Germany Department for Bioinformatics and Functional Genomics, Institute for Pharmacy and Molecular Biotechnology, Bioquant, University of Heidelberg, Heidelberg, Germany Search for more papers by this author Alexander Pappa Alexander Pappa Division of Immunogenetics, German Cancer Research Center (DKFZ), Heidelberg, Germany Medac GmbH, Theaterstrasse, Wedel/Hamburg, Germany Search for more papers by this author Nicolai Fricker Nicolai Fricker Division of Immunogenetics, German Cancer Research Center (DKFZ), Heidelberg, Germany Search for more papers by this author Peter H Krammer Peter H Krammer Division of Immunogenetics, German Cancer Research Center (DKFZ), Heidelberg, Germany Search for more papers by this author Inna N Lavrik Inna N Lavrik Division of Immunogenetics, German Cancer Research Center (DKFZ), Heidelberg, GermanyShared senior authorship Search for more papers by this author Roland Eils Corresponding Author Roland Eils Division of Theoretical Bioinformatics, German Cancer Research Center (DKFZ), Heidelberg, Germany Department for Bioinformatics and Functional Genomics, Institute for Pharmacy and Molecular Biotechnology, Bioquant, University of Heidelberg, Heidelberg, GermanyShared senior authorship Search for more papers by this author Author Information Leo Neumann1,4, Carina Pforr2, Joel Beaudouin1,4, Alexander Pappa2,3, Nicolai Fricker2, Peter H Krammer2, Inna N Lavrik2 and Roland Eils 1,4 1Division of Theoretical Bioinformatics, German Cancer Research Center (DKFZ), Heidelberg, Germany 2Division of Immunogenetics, German Cancer Research Center (DKFZ), Heidelberg, Germany 3Medac GmbH, Theaterstrasse, Wedel/Hamburg, Germany 4Department for Bioinformatics and Functional Genomics, Institute for Pharmacy and Molecular Biotechnology, Bioquant, University of Heidelberg, Heidelberg, Germany *Corresponding author. B080, Division of Theoretical Bioinformatics, German Cancer Research Center (DKFZ), Im Neuenheimer Feld 580, 69120 Heidelberg, Germany. Tel.: +49 6221 5451 290; Fax: +49 6221 5451 488; E-mail: [email protected] Molecular Systems Biology (2010)6:352https://doi.org/10.1038/msb.2010.6 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions Figures & Info This study explores the dilemma in cellular signaling that triggering of CD95 (Fas/APO-1) in some situations results in cell death and in others leads to the activation of NF-κB. We established an integrated kinetic mathematical model for CD95-mediated apoptotic and NF-κB signaling. Systematic model reduction resulted in a surprisingly simple model well approximating experimentally observed dynamics. The model postulates a new link between c-FLIPL cleavage in the death-inducing signaling complex (DISC) and the NF-κB pathway. We validated experimentally that CD95 stimulation resulted in an interaction of p43-FLIP with the IKK complex followed by its activation. Furthermore, we showed that the apoptotic and NF-κB pathways diverge already at the DISC. Model and experimental analysis of DISC formation showed that a subtle balance of c-FLIPL and procaspase-8 determines life/death decisions in a nonlinear manner. We present an integrated model describing the complex dynamics of CD95-mediated apoptosis and NF-κB signaling. Synopsis The CD95 protein (APO-1/Fas; Krammer et al, 2007) is a member of the death receptor family. Signal transduction of CD95 starts with the formation of the death-inducing signaling complex (DISC) detectable within seconds after receptor stimulation (Kischkel et al, 1995). The DISC consists of CD95, the adaptor molecule FADD, procaspase-8/10 and c-FLIPL/S/R (Muzio et al, 1996; Scaffidi et al, 1999; Sprick et al, 2002; Golks et al, 2005; Krammer et al, 2007). Procaspase-8 is converted at the DISC, in a series of autoproteolytic cleavage steps, to p43/p41 and p18, which leads to the activation of effector caspase-3 and demolition of the cell. Recently, experiments have demonstrated that CD95L also activates the induction of transcription factor NF-κB (Barnhart et al, 2004; Kreuz et al, 2004; Peter et al, 2007). It was shown that DED-containing proteins at the DISC, such as procaspase-8 and c-FLIP have a complex role in NF-κB activation (Chaudhary et al, 2000; Hu et al, 2000; Kreuz et al, 2004; Dohrman et al, 2005; Su et al, 2005). These findings motivated our systems biology approach and prompted us to determine whether CD95-mediated signaling should be considered a dynamic system, resulting in life/death decisions. We observed simultaneous apoptosis and NF-κB induction on CD95 stimulation in HeLa cells stably overexpressing CD95–GFP (HeLa-CD95) using biochemical approaches and live-cell imaging. To understand the crosstalk between CD95-mediated apoptosis and NF-κB activation, we created a mathematical model of CD95 signaling. Our model assumes a trimerized ligand (L) that binds to a trimerized CD95 receptor (R) that can recruit three copies of FADD (F) leading to the DISC formation. Subsequently, DED-containing proteins, such as procaspase-8 (C8), c-FLIPL (FL) and c-FLIPS (FS) can bind to FADD. The order of protein binding gives rise to a combinatorial variety of intermediates, resulting either in the formation of the cleavage product of procaspase-8: p43/p41, or in the formation of the cleavage product of c-FLIPL: p43-FLIP. p43/p41 gives rise to signaling in the apoptotic branch of the model, whereas the cleavage product p43-FLIP triggers the activation of NF-κB. The model postulates that p43-FLIP interacts with the IKK complex leading to the phosphorylation of IκB (NF-κB·IκB·P), which entails its degradation and the translocation of p65 to the nucleus (NF-κB*). As a validation of the model topology, we confirmed experimentally that p43-FLIP interacts with the IKK complex and subsequently leads to its activation. The complete model could be fitted well to a data set derived from quantitative western blots of a number of key proteins of the apoptotic and NF-κB pathways. However, we tested whether all the 92 reactions were required to reproduce the observed dynamics, as a small model would yield more reliable parameter estimates, which in turn would increase its usefulness as a predictive tool. To determine the most important interactions, we simplified the complete model in a step-wise manner obtaining a model of considerably lower complexity (Figure 5A, simplification steps are listed in Figure 5B). The final reduced model still approximated well the experimental data set (Figure 5D), whereas the number of reactions decreased from 92 to 23 (Figure 5C). To better understand the interplay of DISC proteins in the determination of cell fate, we analyzed the activity of caspase-3 and NF-κB as a function of procaspase-8 and c-FLIPL levels (Figure 8A). We observed in our simulations that the decision over apoptosis and NF-κB is controlled by both proteins. Different scenarios occur that show combination or absence of either caspase-3 or NF-κB activity. The phase diagram shown in Figure 8A predicts that either increasing or decreasing the amount of c-FLIPL leads to a different signaling mode. We sought to validate this prediction by downregulating or overexpressing procaspase-8 and c-FLIPL, respectively, in HeLa-CD95 cells and measuring CD95-mediated signaling. In agreement with the phase diagram (Figure 8A), we observed that c-FLIPL overexpression resulted in a strong reduction of apoptosis (Figure 8D). Furthermore, we could further confirm by western blot analysis that the stable knockdown of c-FLIPL and procaspase-8 led to a reduction of the levels of p43-FLIP and phosphorylated IκBα after receptor stimulation (Figure 8C and D). In addition, to control the specificity of c-FLIP downregulation and further confirm the requirement of cleavage of c-FLIPL to p43-FLIP, we performed a reconstitution experiment in HeLa-CD95–c-FLIP-deficient cells (Figure 8E). Cells reconstituted with WT c-FLIPL were able to generate p43-FLIP and increased IκBα phosphorylation on CD95 stimulation. In contrast, cells reconstituted with the noncleavable mutant of c-FLIPL (D376E) did not show processing to p43-FLIP (Figure 8E; Supplementary Figure S9). Noticeably, as postulated by the model, this resulted in a strong reduction of the levels of IκBα phosphorylation on CD95 stimulation. Hence, by perturbing the ratio of procaspase-8 to c-FLIPL at the DISC, we directed the induction of apoptosis and NF-κB activation as predicted by our model. Taken together, we found that the DISC protein levels determine cell fate in a nonlinear manner, highlighting the role of signal processing within the DISC. In this study, we propose, to the best of our knowledge, the first integrated kinetic model of CD95-mediated apoptosis and NF-κB signaling. This was achieved by integrating mechanistic knowledge of DISC assembly and caspase activation with a simple scheme of NF-κB activation. We observed that c-FLIPL levels crucially determine the balance between apoptotic and NF-κB signaling by shaping the dynamics of DISC assembly. Although this finding is based on experiments performed in cell lines, we expect that the nonlinear dynamics of DISC assembly is a generic systems property of life/death decision making in CD95 signaling pathways. This is especially important for understanding the regulation of cell death in physiologically relevant cells, such as cancer cells often showing resistance against death receptor-induced apoptosis. Introduction The CD95 protein (APO-1/Fas; Krammer et al, 2007) is a member of the death receptor (DR) family, a subfamily of the tumor necrosis factor receptor (TNF-R) superfamily (Ashkenazi and Dixit, 1998; Krammer, 2000). Cross linking of CD95 with its natural ligand, CD95L, or with agonistic anti-CD95 antibodies induces apoptosis in sensitive cells (Trauth et al, 1989). The signal transduction of CD95 starts with the formation of the death-inducing signaling complex (DISC) detectable within seconds after receptor stimulation (Kischkel et al, 1995). The DISC consists of CD95, the adaptor molecule FADD, procaspase-8a/b, procaspase-10 and c-FLIPL/S/R (Muzio et al, 1996; Scaffidi et al, 1999; Sprick et al, 2002; Golks et al, 2005; Krammer et al, 2007). Procaspase-8 is converted at the DISC, through a series of autoproteolytic cleavage steps, into p43/p41 and p18, which leads to the activation of effector caspase-3 (Lavrik et al, 2003). This can occur in two different ways in type I and type II cells (Scaffidi et al, 1998). Type I cells are characterized by high levels of CD95 DISC formation and increased amounts of active caspase-8, leading to the direct activation of downstream effector caspases-3 and -7 and subsequent apoptosis. Type II cells are characterized by lower levels of CD95 DISC formation, and caspase-3 activation requires additional amplification through the release of pro-apoptotic factors from mitochondria triggered by the caspase-8-mediated cleavage of the Bcl-2 family protein Bid. Recently, experiments have demonstrated that CD95L is not only a potent apoptosis inducer but can also activate multiple nonapoptotic pathways, in particular induction of transcription factor NF-κB (Barnhart et al, 2004; Kreuz et al, 2004; Peter et al, 2007). The NF-κB family regulates the expression of genes crucial for innate and adaptive immune responses, cell growth and apoptosis. In most cells, the NF-κB dimer is sequestered in the cytosol by inhibitors of the κB protein (IκB), and its nuclear translocation can be induced by a wide variety of stimuli through activation of the IκB kinase (IKK) complex (Karin and Lin, 2002; Hayden and Ghosh, 2004). The IKK complex consists of two catalytic subunits, IKKα and IKKβ, and a regulatory subunit IKKγ/NEMO. On activation of the IKK complex, IκB is phosphorylated and degraded in an ubiquitin-dependent manner. The NF-κB dimers can then translocate into the nucleus to induce transcription of target genes. However, the exact molecular mechanism of NF-κB activation through CD95 remains unclear (Peter et al, 2007). It has been previously shown that DED-containing proteins, such as procaspase-8 and c-FLIP have a prominent role in NF-κB activation (Chaudhary et al, 2000; Hu et al, 2000; Kreuz et al, 2004; Dohrman et al, 2005; Su et al, 2005). c-FLIP N-terminal cleavage products p43-FLIP and p22-FLIP strongly induce NF-κB (Kataoka and Tschopp, 2004; Golks et al, 2006a). p43-FLIP is generated by procaspase-8 at the DISC on CD95 stimulation (Scaffidi et al, 1999; Krueger et al, 2001). It has been shown to interact with components of the TNFR-mediated NF-κB activation pathway, TNFR-associated factor 1 (TRAF1), TRAF2 and receptor-interacting protein (RIP), which together promote NF-κB activation (Kataoka and Tschopp, 2004; Dohrman et al, 2005). Interestingly, it has been shown that c-FLIPL/S/R form heterodimers with procaspase-8, resulting in the generation of the cleavage fragment p22-FLIP (Golks et al, 2006a). This protein mediates NF-κB activation by binding directly to the IKK complex. p22-FLIP differs from p43-FLIP in that it is generated in nonapoptotic cells without DR stimulation (Golks et al, 2006a). Furthermore, it was also reported that the binding of procaspase-8 in the MALT1–Bcl-10 adapter complex formed on TCR stimulation has a prominent role in NF-κB induction (Su et al, 2005). Thus, various molecules of the CD95 signaling machinery, such as procaspase-8 and c-FLIP have a complex role in CD95-mediated apoptosis and NF-κB pathways. These findings motivated our systems biology approach and prompted us to determine whether CD95-mediated signaling should be considered as a dynamic system resulting in life/death decisions. It has been shown repeatedly that decision making is often a process brought about by a reaction system rather than by the influence of a single molecule (Xiong and Ferrell, 2003; Legewie et al, 2006; Santos et al, 2007). Modeling in biology has a long tradition and can be dated back to Hodgkin and Huxley's model of a neuron (Hodgkin and Huxley, 1952). A wide gamut of modeling formalisms has been applied to biological systems ranging from qualitative approaches, such as Boolean and Bayesian networks (Sun and Zhao, 2004) to partial differential equations and stochastic simulations. A number of models typically based on ordinary differential equations (ODEs) have described apoptosis signaling. Fussenegger et al (2000) presented the first mathematical model encompassing both extrinsic and intrinsic apoptotic pathways. A more detailed model with parameters estimated from quantitative western blots verified a threshold for CD95 stimulation regulated by c-FLIP (Bentele et al, 2004). Advanced models of apoptosis have been used to discriminate between different hypotheses of apoptosis inhibition by Bcl-2 family members and XIAPs (Hua et al, 2005; Rehm et al, 2006). Others have addressed aspects, such as feedback and bistability (Eissing et al, 2004; Janes et al, 2005; Bagci et al, 2006; Legewie et al, 2006). A number of mathematical models for NF-κB activation have investigated the complex interplay of various IκB subunits in NF-κB oscillatory activity (Hoffmann et al, 2002; Nelson et al, 2004; Cheong et al, 2008). Furthermore, the signaling of the TNF pathway leading to NF-κB activation was modeled (Cheong et al, 2006; Park et al, 2006). Despite much study on both apoptotic and NF-κB signaling pathways induced by CD95, there is a lack of a detailed mechanistic understanding of the divergence of these pathways. In the following text, we propose an integrated quantitative model supported by experimental data for CD95-mediated apoptosis and NF-κB activation. A direct interaction between p43-FLIP and the IKK complex is postulated by the model and validated experimentally. The model also predicts a divergence of the two pathways at the DISC and that the cellular decision depends on the balance between the levels of c-FLIPL and caspase-8. Experimental modulations of DISC protein amounts are all consistent with the model predictions. Our model gives mechanistic insights into the cellular response on CD95 activation and provides a way to predict cellular behavior. Results NF-κB and caspases show parallel activation The CD95 protein is known to mediate the activation of caspases leading to apoptosis (Lavrik et al, 2005) and mediating the activation of NF-κB (Barnhart et al, 2004). However, it is not understood whether CD95 activates apoptotic and NF-κB pathways in a mutually exclusive manner, or whether the activation of both pathways takes place in parallel. To answer this question, we investigated CD95 signaling by western blot analysis of HeLa cells stably overexpressing CD95–GFP (HeLa-CD95) and treated the cells with agonistic anti-CD95 antibodies at three concentrations: 1500, 500 and 250 ng/ml (Figure 1A–C). Procaspase-8a/b was processed to p43/p41 and subsequently to p18. Procaspase-3 was also cleaved to the active subunit p17 showing the induction of the apoptotic pathway. In parallel, we observed the phosphorylation of IκBα followed by its degradation. Stimulation of CD95 caused apoptosis at all antibody concentrations (Supplementary Figure S1). The quantified blots demonstrate that apoptotic and NF-κB pathways were activated on a similar time scale (Figure 2A–G). Interestingly, IκBα phosphorylation was at its maximum (Figure 2F) roughly three times faster than active caspases (Figure 2B, D and G) at all antibody concentrations. Using live-cell images of HeLa-CD95 cells stably overexpressing p65–mCherry, we observed simultaneous apoptosis and nuclear translocation of p65–mCherry in each cell on induction of CD95 (Figure 1D). Thus, we could exclude the possibility that a subset of cells activated NF-κB, whereas another subset activated caspases. This demonstrates a parallel rather than a mutually exclusive activation of caspases and NF-κB. The simultaneous induction of apoptosis and NF-κB activation and their different dynamics led us to investigate the divergence point of the two pathways. Figure 1.NF-κB and caspases show parallel activation. (A–C) HeLa-CD95 cells were stimulated with (A)1500 ng/ml, (B) 500 ng/ml and (C) 250 ng/ml of agonistic anti-CD95 antibodies for the indicated periods of time. The cellular lysates were analyzed by western blotting using antibodies against caspases, IκBα and p-IκBα. Nonspecific bands of anti-p-IκBα are marked (NS). Results are representative of four different experiments. (D) To follow p65 localization, Hela-CD95 cells were stably transfected with p65–mCherry. Cells were stimulated with 1500 ng/ml anti-CD95 antibody and imaged using fluorescent microscopy over the respective time. Measurements of mCherry (depicted in gray) indicated translocation of p65 on CD95 activation. Induced apoptosis is observed by the appearance of apoptotic bodies. Scale bar: 10 μm. Download figure Download PowerPoint Figure 2.Quantified response to anti-CD95 stimulation. Each of the panels (A–G) shows a quantification of western blots shown in Figure 1A–C. The symbols mark the average value and the lines show their linear interpolations. The s.d. was included when more than one value was obtained. Black, blue and red colors correspond to 1500, 500 and 250 ng/ml anti-CD95 antibodies, respectively, used to induce the cells at time t=0. Download figure Download PowerPoint A mechanistic model assuming a direct interaction between p43-FLIP and the IKK complex is consistent with experimental observations To understand the crosstalk between CD95-mediated apoptosis and NF-κB activation, we created a mathematical model of CD95 signaling. When we integrated known molecular interactions into a detailed model, we faced the problem that it is mechanistically not understood how signals from CD95 are transmitted to NF-κB. Taking into account earlier findings that the c-FLIPL cleavage products, p43-FLIP and p22-FLIP, are involved in NF-κB signaling (Kataoka and Tschopp, 2004; Golks et al, 2006a), and that p22-FLIP is not formed upon CD95 stimulation (Golks et al, 2006a), we hypothesized that p43-FLIP is the missing link between the DISC and the IKK complex. The kinetics of phosphorylated IκBα in Figure 2F indicate a temporal correlation between CD95 stimulation and IKK activity. The simplest scheme to explain this phenomenon would be through p43-FLIP generation at the DISC and subsequent interaction with the IKK complex. This led us to the mathematical model of CD95-mediated caspase and NF-κB activation shown in Figure 3. Figure 3.Model of CD95-mediated signaling. A graphic representation of the complete model illustrating the process of DISC formation and subsequent signaling of the apoptotic (depicted in red) and NF-κB pathway (depicted in green). Abbreviations: L, CD95 ligand; R, CD95 receptor; F, FADD; C8, procaspase-8 (p55/p53); C8*, active caspase-8; C3, procaspase-3; C3*, active caspase-3; C6, procaspase-6; C6*, active caspase-6; FL, c-FLIPL; FS, c-FLIPS; X, C8, FL or FS; p43/p41, 1st cleavage product of procaspase-8; and p43-FLIP, cleavage product of c-FLIPL. Download figure Download PowerPoint Our model assumes a trimerized ligand (L) that binds to a trimerized CD95 receptor (R) that can recruit three copies of FADD (F) leading to the DISC formation. Subsequently, DED-containing proteins, such as procaspase-8 (C8), c-FLIPL (FL) and c-FLIPS (FS) can bind to FADD. The order of protein binding gives rise to a combinatorial variety of intermediates. Assembled DISCs can be categorized into three groups: the first group contains at least two copies of procaspase-8 and is further processed by p43/p41 into active protease subunits, p18 and p10, forming an active caspase-8 heterotetramer (C8*). This apoptotic branch of the model also includes procaspase-3 and procaspase-6 (C3 and C6), their active forms (C3* and C6*), the inhibitor IAP and a feedback loop from caspase-6 to caspase-8. The second group of DISCs features at least one copy of procaspase-8 and one copy of c-FLIPL giving rise to p43-FLIP. The model postulates that p43-FLIP interacts with the IKK complex leading to phosphorylation of IκB (NF-κB·IκB·P), which entails its degradation and the translocation of p65 to the nucleus (NF-κB*). For simplicity, we assume that the entire pool of IκB is bound to NF-κB. In the third group of DISCs, we considered all remaining configurations. They do not participate in further signaling and are merged in a terminal state (L·R·F·F·F·X·X·X). The key features of the model are its partitioning into DISC formation, apoptotic and NF-κB signaling, and the confinement of all interactions between the two pathways to the DISC. In this model, the gene transcription induced by NF-κB was neglected, as we aimed to investigate early signaling events immediately after the stimulation of CD95. For model simulations, the molecular interactions depicted in Figure 3 were translated into a system of coupled ODEs (for details on reaction equations and parameters, see Supplementary Tables S1 and S2). To reduce the large number of free parameters, we simplified the reactions to be irreversible as previously described with the exception of a reversible enzymatic scheme used for caspase activation (Bentele et al, 2004). The model contains unknown reaction constants and concentrations that we estimated from our data by least-squares optimization. Remarkably, the model was able to reproduce the experimental data set (Supplementary Figure S2) despite the almost exclusive use of an irreversible reaction scheme. Interestingly, it shows that the activity of effector caspases is not required for reproducing the dynamics of CD95-mediated NF-κB activation. In addition, the model postulates a direct interaction between p43-FLIP and the IKK complex. p43-FLIP interacts with the IKK complex upon CD95 stimulation To test our model hypothesis that p43-FLIP interacts with the IKK complex, we transiently co-transfected p43-FLIP with FLAG-tagged IKKα, IKKβ and IKKγ into 293T cells, and then performed an immunoprecipitation (IP) using anti-FLAG (FLAG-IP) and anti-FLIP antibodies (FLIP-IP; Figure 4A). We observed IKKα in the FLIP-IP and p43-FLIP mostly in the FLAG-IKKα-IP. Thus, we established that p43-FLIP can interact with IKKα. Next, we tested whether this interaction also occurs under endogenous conditions on CD95 stimulation in Hela-CD95 cells. An IP of the IKK complex using anti-IKKγ antibodies showed a recruitment of p43-FLIP to the IKK complex at 30 min after the stimulation of CD95 (Figure 4B). To avoid co-IP of the CD95 DISC with the IKK complex, we used LZ-CD95L and not anti-CD95 antibodies for stimulation in this experiment. The RIP protein, invoked in CD95-mediated NF-κB activation (Kreuz et al, 2004), was not detectable at the IKK complex under our experimental conditions. In the IKKγ-IP, we also detected a band corresponding to the size of c-FLIPL, however, the band was also detected in the isotype control IP and, hence, is considered unspecific. In a CD95 DISC-IP (CD95-IP), we could not detect IKK-α/β, suggesting that the IKK complex is not associated to the DISC (Supplementary Figure S3). Positive controls for the CD95 DISC-IP showed that the DISC was formed efficiently, for example, procaspase-8 and its cleavage product p43/p41 were present at the DISC. Figure 4.p43-FLIP interacts with the IKK complex on CD95 stimulation. (A) The association of p43-FLIP with components of the IKK complex was determined using co-immunoprecipitations (IPs). 293T cells were co-transfected with FLAG-tagged IKKα, IKKβ or IKKγ with or without p43-FLIP. The lysates were immunoprecipitated using antibodies against the FLAG tag or against c-FLIP and analyzed by western blotting using antibodies against the FLAG tag or c-FLIP (left side). In parallel, corresponding lysates were also analyzed using western blot analysis with the same antibodies as the immunoprecipitation (right side). (B) The association of p43-FLIP with the IKK complex under endogenous conditions was determined using co-immunoprecipitations of IKKγ (IKKγ-IP). HeLa-CD95 cells were stimulated with 500 ng/ml of LZ-CD95L for the indicated time intervals and immunoprecipitated using an antibody against IKKγ. All subunits of the IKK complex were immunoprecipitated in this procedure due to IKK complex stability. The immunoprecipitated proteins (IKKγ-IP, left side) and the corresponding lysates (lysate, right side) were analyzed by western blotting using antibodies against c-FLIP, IKKβ and RIP. IKKβ served as a loading control. (C) 0.5 × 105 293T cells were co-transfected with MEKK1, p43-FLIP and c-FLIPL, and the luciferase reporter plasmid. NF-κB–luciferase activity was determined at 16 h after transfection. The results represent the mean±s.d. values of quadruplet cultures. GFP transfection was performed to control 100% transfection efficiency. (D) 1 × 105 HeLa-CD95 cells were transfected with a NF-κB-dependent luciferase reporter plasmid (1 μg per" @default.
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• W2140662159 date "2010-01-01" @default.
• W2140662159 modified "2023-10-15" @default.
• W2140662159 title "Dynamics within the CD95 death‐inducing signaling complex decide life and death of cells" @default.
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