Controlling Cell Death through Post-translational Modifications of DED Proteins

TICB 1585 No. of Pages 16

Trends in Cell Biology

Review

Controlling Cell Death through Post-translational Modifications of DED Proteins Kamil Seyrek,1,4 Nikita V. Ivanisenko,2,3,4 Max Richter,1 Laura K. Hillert,1 Corinna König,1 and Inna N. Lavrik1,2,* Apoptosis is a form of programmed cell death, deregulation of which occurs in multiple disorders, including neurodegenerative and autoimmune diseases as well as cancer. The formation of a death-inducing signaling complex (DISC) and death effector domain (DED) filaments are critical for initiation of the extrinsic apoptotic pathway. Post-translational modifications (PTMs) of DED-containing DISC components such as FADD, procaspase-8, and c-FLIP comprise an additional level of apoptosis regulation, which is necessary to overcome the threshold for apoptosis induction. In this review we discuss the influence of PTMs of FADD, procaspase-8, and c-FLIP on DED filament assembly and cell death induction, with a focus on the 3D organization of the DED filament.

Highlights Death-inducing signaling complex (DISC) and death effector domain (DED) filaments control extrinsic apoptosis. DED filaments comprise procaspase-8/10, and c-FLIP.

Post-translational modifications (PTMs) of DED filament proteins create the additional checkpoint of apoptosis. A central role in understanding apoptosis control is in deciphering the 3D architecture of PTMs in the DED filament.

Death Effector Domain (DED) Proteins in Extrinsic Apoptosis Signaling In multicellular organisms, tissue homeostasis is maintained through a fine-tuned balance between cell proliferation and cell death [1–5]. Several physiological and pathological stimuli have been reported to trigger programmed cell death [6]. Apoptosis can be induced via two pathways: the extrinsic and the intrinsic or mitochondrial pathway [2] (Figure 1). The extrinsic pathway is triggered upon binding of death ligands, including CD95 ligand (CD95L) and tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) to death receptors (DRs) such as Fas/CD95 and TRAIL receptor-1/2 (TRAILR1/2), respectively [7]. This association leads to the recruitment of proteins with death domains (DD) such as Fas-associated protein with death domain (FADD) to the DD of CD95 or TRAIL-R1/2, resulting in the formation of the death-inducing signaling complex (DISC) [8,9], which in turn directs cleavage and activation of caspases for inducing apoptosis. DISC is comprised of procaspase-8/10 and cellular FLICE-like inhibitory protein (c-FLIP). Upon induction of apoptosis, the DED of FADD interacts with DEDs of procaspase-8, procaspase-10, and c-FLIP, resulting in the formation of DED-filaments, which serve as a platform for procaspase-8 dimerization and subsequent activation [10–12] (Figure 1). DED belongs to the DD superfamily [3,13]. DED comprises six α-helixes arranged in a Greek key structural motif. FADD contains one DD and one DED, whereas both caspase-8 and c-FLIP contain two DEDs at their N terminus. DED filaments are formed through so-called type I, II, and III interactions between DEDs (Box 1) [12]. Proper architecture of DED filaments at DR complexes provide an extra layer of regulatory control of cell death. Recent findings are revealing a role for post-translational modifications (PTMs) such as phosphorylation, ubiquitylation, SUMOylation, and nitrosylation in regulating these interactions [6,14–17]. These modifications assist in maintaining proper conformations of DEDs in DED filaments, which is required for efficient caspase-8 activation, and in overcoming a threshold for apoptosis induction [18,19]. In this review, we consider the role of PTMs in controlling the mechanisms of DISC and DED filament assembly, with a focus on the 3D organization of the DED filament.

FADD,

1

Translational Inflammation Research, Medical Faculty, Otto von Guericke University, Magdeburg, Germany 2 The Federal Research Center Institute of Cytology and Genetics SB RAS, Novosibirsk, Russia 3 Novosibirsk State University, Novosibirsk, Russia 4 These authors contributed equally to this work

PTMs of FADD FADD is a pivotal signaling component of DR-mediated apoptosis. The DED of FADD initiates caspase-8 filament growth and is therefore essential for apoptosis induction [12,20]

*Correspondence: [email protected] (I.N. Lavrik).

Trends in Cell Biology, Month 2020, Vol. xx, No. xx https://doi.org/10.1016/j.tcb.2020.02.006 © 2020 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

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Figure 1. Extrinsic and Intrinsic Apoptosis. After stimulation of Death Receptor (DR), for example, CD95/Fas/APO-1 or tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-R1/2, by the corresponding death ligand (DL), the death-inducing signaling complex (DISC) forms at the membrane. The DISC consists of DR, FAS-associated death domain (FADD) (depicted in brown) and death effector domain (DED) filaments comprising procaspase-8 (depicted in blue), caspase-10, and cellular FLICE-inhibitory proteins (c-FLIP) (depicted in yellow). The death domain (DD) of DR interacts with the DD of FADD, whereas the DED of FADD interacts with the DEDs of procaspase-8 and c-FLIP. DD are shown in light brown; DED are shown in light grey. Procaspase-8 undergoes dimerization and subsequent activation at the DED filament, leading to induction of the caspase cascade and cell death. There are two types of extrinsic cell death: type I and type II cells [68]. In type I cells the cell death induction is independent from the mitochondrial or intrinsic apoptotic pathway. In the intrinsic pathway a number of stimuli, including cleavage of Bid by caspase-8, lead to changes in mitochondrial outer membrane polarization (MOMP), followed by the cytochrome C release from mitochondria, apoptosome formation, and caspase activation. Apoptosis induction is controlled by a number of inhibitory proteins, including c-FLIPs that block caspase-8 activation, antiapoptotic BCl-2 family members inhibiting cytochrome C release from mitochondria, and XIAPs blocking caspases-3, -7, and -9. Abbreviations: CD95L, CD95 ligand; c-FLIPL, long c-FLIP isoform; c-FLIPS, short c-FLIP isoform.

(Figure 2A). The proper structural conformation of FADD is a requirement for the initiation of DED filament growth (Box 2). Hence, its modulation by PTM might have major consequences for apoptosis initiation [20]. Several putative PTM sites in FADD DED have been predicted in silico and even identified by mass spectrometry, including phosphorylation and ubiquitylation. However, their relevance for signal transduction has not been confirmed [21]. 2

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Roles for Phosphorylation One of the first reports on the CD95 DISC composition identified two proteins, termed CAP1 and CAP2, that turned out to be phosphorylated FADD forms [8,22]. Later, phosphorylation sites that regulate FADD function and cellular localization were identified at S194 (S191 of murine FADD) [20,23], S200 [24], and S203 [25] (Figure 2A). Phosphorylation of FADD is mediated by a number of kinases, including protein kinase Cζ, antimitotic kinase Aurora-A (Aur-A), casein kinase α (CKIα), casein kinase 2 (CK2), and polo-like kinase 1 (Plk1) [25]. Recently, both CKIα and Plk1 were shown to phosphorylate FADD at S194 [26], whereas CK2 mediates the phosphorylation at S200 [24]. Phosphorylation at S203 is mediated by Aur-A in response to treatment with the antimitotic drug Taxol. Phosphorylation at S203 is also a prerequisite for Plk1-mediated phosphorylation at S194 [25], while phosphorylation of FADD at S194 regulates its nuclear localization and its function in the cell cycle [16,24,26] (Figure 2B). Moreover, phosphorylation at S200 promotes FADD translocation into the nucleus [24]. In contrast, while phosphorylated FADD is present at the DISC, the phosphorylation of FADD seems not to interfere with DISC assembly [8,22]. In line with this finding is that phosphorylation sites of FADD are localized outside of the structured DD and DED regions at the C terminus of FADD (Figure 2A). According to available crystal structures of the complex formed by CD95 and FADD DDs, Protein Data Bank (PDB) ID 3EZQ [27] and PDB ID 3OQ9 [28], the C terminus of FADD is not involved in the association between DDs. Subsequently, the mapping of FADD phosphorylation sites on these structures combined in silico with the structure of FADD DED (Box 2) suggests that their location outside of the DED filament precludes any role of FADD phosphorylation in the control of the DED filament assembly (Figure 2A). Taken together, the function

Box 1. DED Filament Architecture The core components of the death-inducing signaling complex (DISC) that contain a death effector domain (DED), form so-called DED filaments. The structure of the DED filaments has been recently solved by cryogenic electron microscopy (cryo-EM) structural analysis of the procaspase-8 N terminal prodomain [12]. Procaspase-8 comprises DED1 and DED2 in its N terminal prodomain, followed by catalytic p18 and p10 domains (Figure IA). The cryo-EM analysis revealed that procaspase-8 DEDs assemble into oligomeric structures that were named DED filaments [12]. The DED filaments provide a platform for the dimerization and subsequent activation of procaspase-8. The structure of DED naturally provides the basis for formation of filaments. Each DED can be characterized by the presence of six interfaces, Ia/b, IIa/b, and IIIa/b, which have the ability to bind to each other (Figure IB). The assembly of the DED filament is mediated by three types of DED interactions: type I, II, and III (Figure IB). Type I interactions involve the binding of Ia and Ib interfaces of adjacent DEDs and are largely mediated by hydrophobic amino acids, including the highly conserved ‘FL’ motif residues (Phe/Leu) [10]. This motif is located at the Ib interface of the DEDs of caspase-8, caspase-10, and c-FLIP [10]. Type I interactions mediate the formation of the so-called linear DED chains, which, in turn, represent the substructure of the DED filament (Figure IC). Importantly, each DED filament is comprised of three linear chains (Figure IC–E). Moreover, the linear chains of procaspase-8 were first suggested to be the platform for the dimerization and subsequent activation of procaspase-8, but, as it turned out later, they represent only the substructure of the DED filament [10–12]. Type II and type III interactions are mediated via the binding of IIa/IIb and IIIa/IIIb interfaces, correspondingly [12]. Type II and III interactions are largely mediated by hydrophilic amino acid residues and provide a basis for the interactions between adjacent linear chains in the filament (Figure IB). In this way, the architecture of DED filament is based on the combination of all types of DED interactions: type I, II, and III. Remarkably, this includes so-called homotypic interactions between similar domains such as DED1/DED1 or DED2/DED2, as well as so-called heterotypic interactions between DED1 and DED2 (Figure IB). The intricate DED interaction network results in a DED filament structure with a helical symmetry and a right-handed rotation (Figure IC,D). As mentioned earlier, the DED filament structure was obtained using a structure of the N terminal DED-only part of procaspase-8 (e.g., PDB ID 5l08 [12]). In addition, the structure of the C terminal part of caspase-8 was solved (e.g., PDB ID 3H11 [54]). However, the structure of full-length procaspase-8, comprising both the prodomain and C terminal part, has not been reported so far. This has to be addressed in future studies.

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Figure I. Death Effector Domain (DED) Filament Architecture. (A) Scheme of procaspase-8 isoforms. The prodomain comprising DED1 and DED2 as well catalytic domains p18 and p10 are shown. (B) Scheme of type I/II/III interfaces of DED (upper part) and tertiary structure of procaspase-8 DED1 and DED2 (lower part). The structure is based on Fu et al. [12]. (C) Scheme of the DED filament in 2D presentation. Three procaspase-8 DED chains are shown (chains 1, 2, and 3). DED1 is shown in dark blue, while DED2 is shown in light blue. (D) A 3D structure of procaspase-8 filament modified from [12]. (E) 3D scheme of procaspase-8 DED filament modified from [12]. The directions of type I, II, and III interactions are indicated with broken arrows. (C–E) The same color code for procaspase-8 DED1 and DED2 as in (B) is used.

of FADD phosphorylation might be attributed to its role in the cell cycle rather than to cell death (Figure 2B). Roles for Ubiquitylation The other reported PTM of FADD is ubiquitylation [29]. The C terminus HSC70-interacting protein (CHIP) induces K6-linked ubiquitylation of FADD at K149 and K153 located within the DD of FADD [30] (Figure 2A). CHIP is an E3 ubiquitin-ligase collaborating with molecular chaperones Hsp90 and Hsc70 [30]. The introduction of mutations of FADD at lysines 149 and 153 increase sensitivity to CD95L- or TRAIL-induced apoptosis. Even though mutations at K149 and K153 of FADD showed no effect on the interactions between DDs of FADD and CD95, the ubiquitylation of FADD does seem to down-modulate TRAIL-R and CD95 DISC amounts [30] (Figure 2B). In particular, according to the above-mentioned structures of the complex formed by CD95 and FADD DDs (PDB ID 3EZQ and PDB ID 3OQ9) [27,28] the K149 and K153 ubiquitylation sites are not involved in the association of DDs (Figure 2A). However, ubiquitylation at K149 and K153 might trigger a conformational change of the FADD DED, leading to abrogation of DED filament formation. Moreover, the FADD DED is required for interaction with CHIP [30], 4

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S203

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S194

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Figure 2. Phosphorylation, Ubiquitylation, and SUMOylation of FAS-Associated Death Domain (FADD) at the Death-Inducing Signaling Complex (DISC). (A) The structural model of FADD as a part of the death effector domain (DED) filament obtained in silico. The sites of post-translational modifications (PTMs) of FADD are mapped in all three 3D structures. PTM residues are denoted and shown in red (phosphorylation sites), blue (ubiquitylation sites), and purple (SUMOylation sites). FADD death domain (DD) is shown in light brown, FADD DED is presented in dark brown, DED filament is presented in white. The following Protein Data Bank (PDB) structures were used for mapping PTM sites: the full-length FADD (2GF5) and procaspase-8 DED filament (5L08). The regions of FADD for which the structural information is still missing are indicated with a gray line. The in silico model was obtained by superimposition of FADD DED on the DED filament with PyMOL software (http://sourceforge.net/projects/pymol/). Phosphorylation sites of FADD are located outside of the structured DD and DED regions at the C terminus of FADD, whereas ubiquitylation and SUMOylation sites are located at the DD of FADD at the solvent exposed side of DD. (B) Casein kinase α (CKIα) and polo-like kinase 1 (Plk1) phosphorylate FADD at S194, whereas casein kinase 2 (CK2) mediates the phosphorylation at S200 and Aur-A at S203. (Figure legend continued at the bottom of the next page.)

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suggesting that ubiquitylation of FADD at DD might interfere with interactions of FADD DEDs, which in turn suppresses DED filament formation and the recruitment of caspase-8/10 and cFLIP to the DISC. The relevance of this hypothesis has to be confirmed by further studies. Another important modulator of FADD is Makorin Ring Finger Protein 1 (MKRN1), which has E3 ligase activity. Contrary to CHIP-mediated ubiquitylation, MKRN1-mediated ubiquitylation of FADD results in the degradation of FADD [31] (Figure 2B). However, MKRN1-mediated ubiquitylation site(s) on FADD and the type of ubiquitylation remain to be clarified in future studies. In the light of these data, it is tempting to speculate that ubiquitylation of FADD could be an important checkpoint in the DR-mediated cell death pathway, though this regulation seems to not directly involve the DED region of the protein. Roles for SUMOylation The small ubiquitin-like modifier (SUMO) has been shown to bind to multiple lysine residues (K120, K125, and K149) located in the DD of the protein (Figure 2A). In particular, FADD is reported to be SUMOylated by E3 SUMO-protein ligase PIAS3. Moreover, this pathway seems to be connected to the mitochondrial translocation of FADD and its interactions with dynaminrelated protein 1 (Drp1) [32] (Figure 2B). Interestingly, K149 seems to present a point for crosstalk between ubiquitylation and SUMOylation, as this residue is also the site for ubiquitylation by CHIP. Moreover, SUMOylation of this residue might have similar effects on the DED interactions as ubiquitylation. The importance of this crosstalk for life/death decisions in the cell has to be addressed in future studies. Overall, PTMs such as phosphorylation, ubiquitylation, and SUMOylation of FADD seem to involve only the DD and the C terminal end of the protein. From the available structure it might be concluded that PTMs of FADD influence neither the direct association of FADD DD with CD95 DD, nor the interactions of FADD DED interfaces involved in DED filament formation (Figure 2A). Apparently, regulation through PTMs of FADD DD does not completely block DISC formation, but only fine-tunes it by decreasing the amount of the DISC and caspase-8 activation. This change might regulate the threshold for the induction of apoptosis via DR [18,19]. Furthermore, there might be a role for keeping the FADD DED in a ‘PTM-free’ stage. Given that FADD DED is the major initiating platform of DED filament assembly, the absence of PTMs of FADD DED might open opportunities for the regulation of its interactions within DED filaments that are essential for multiple cellular functions.

PTMs of Caspase-8 Procaspase-8 acts as the apical protease in DR-mediated apoptosis [22]. Eight isoforms of procaspase-8 have been reported [33]. Two isoforms of procaspase-8, procaspase-8a and 8b, are predominantly recruited to the DISC [33] via two DEDs (DED1 and DED2) at their N terminal (Figure 3A) (Box 1). Both isoforms are activated via dimerization at the DED filaments formed at the DISC, which provides a unique spatial platform allowing procaspase-8 homodimer formation [10] (Box 1). Procaspase-8a/b along with two DEDs comprises the large and small catalytic

Phosphorylation at S203 is a prerequisite for Plk1-mediated phosphorylation at S194. Phosphorylation at S194 and S200 leads to FADD translocation to the nucleus. Small ubiquitin-like modifier (SUMO) ligase PIAS3 SUMOylates FADD in the DD at K120, K125, and K149, leading to interaction with dynamin-related protein 1 (Drp1) that is reported to recruit FADD to the mitochondria. C terminus HSC70-interacting protein (CHIP) induces K6-linked ubiquitylation of FADD at K149 and K153. Ubiquitylation within the DD inhibits Death Receptor (DR) [e.g., CD95 or tumor necrosis factor-related apoptosisinducing ligand (TRAIL)-R1/2] DISC activity. Makorin Ring Finger Protein 1 (MKRN1)-mediated ubiquitylation of FADD results in a degradation of FADD.

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Box 2. FADD Architecture in the DED Filament FADD plays the major role in triggering DED filament growth [12]. FADD comprises both DD and DED (Figure IA). The recruitment of FADD via its DD to the DD of CD95 results in the formation of a platform that is able to nucleate caspase-8 DED filament growth [12]. However, the structure of CD95/FADD/caspase-8-DED filament is not reported yet. Moreover, though it is known that FADD DED can form filamentous structures, the structure of oligomerized FADD DEDs is also not solved. Currently, only the structure of the FADD DED monomer has been obtained [70,71]. Nevertheless, the known DED structures and the high similarity of the tertiary structure of FADD DED and caspase-8 DEDs allow in silico prediction of the structure of FADD within the DED filament. In particular, it might be suggested that FADD forms type I, II, and III interactions with caspase-8 DEDs [12] (Figure IB–D). Further evidence for FADD DED interactions within DED filament is coming from immuno-gold labeling [12]. According to these data, the nucleation point formed by the CD95/FADD complex is located at one end of the DED filament [12]. This allows the hypothesis that the procaspase-8 DED filament is elongated in one direction and that only specific interfaces of FADD DED take part in procaspase-8 binding. On the basis of mutagenesis studies of FADD DED and caspase-8 DEDs, as well as of the structure PDB ID 5l08, it might be suggested that Ib, IIa, and IIIa interfaces of FADD are involved in the binding of procaspase-8 within the DED filament (Figure IC,D) [12,34,71,72]. To consider the role of FADD within the DED filament, in silico structural models must be created. In particular, as it was done in [55], the procaspase-8 DED1 and DED2 from cryo-EM structure (PDB ID 5L08) [12] were replaced by FADD DED (PDB ID 1A1W) [70], with an approach based on the structural alignment of these structures. This type of in silico model can be used for the analysis of the FADD function in the DED filament (see Figure 2 in main text).

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Figure I. Fas-Associated Protein with Death Domain (FADD) Death Effector Domain (DED) Architecture in the DED Filament. (A) Scheme of FADD, including death domain (DD) and DED. (B) Scheme of type I/II/III interfaces of FADD DED. (C) A 2D scheme of the DED filament model with FADD in 2D presentation. Three DED chains are shown (chains 1, 2, and 3). Procaspase-8 DED1 is shown in dark blue, while procaspase-8 DED2 is shown in light blue. FADD DED is shown in brown. The presentation is modified from [55]. (D) A 3D scheme of the model of procaspase-8 filament with FADD. The direction of the type I interaction is shown with a broken arrow. The same color code for procaspase-8 and FADD DEDs as in (B) is used. This model is constructed on the basis of [55].

domains, p18 and p10, respectively. The activation and the strength of this apical enzyme activity in the DED filament is tightly regulated. One of the prominent regulators of procaspase-8 activation is c-FLIP, which interferes with the formation of procaspase-8 homodimers [34]. Additionally, PTMs of caspase-8 appear to play an important role in the modulation of caspase activation strength and provide another level of apoptosis control. Roles for SUMOylation Only a few PTMs target DED1 and DED2 of procaspase-8. SUMO-1 associates with both DED1 and DED2 of procaspase-8, leading to SUMO modification at K156 within the DED2 [35] (Figure 3A). However, according to the latest structure of caspase-8 DED filament [12], the K156 residue is exposed to the solvent and likely not involved in the interaction of DEDs within the DED filament (Figure 3A). Therefore, SUMOylation at this position most likely does not affect Trends in Cell Biology, Month 2020, Vol. xx, No. xx

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Figure 3. Modulation of Death Receptor (DR) Signaling by Phosphorylation, Ubiquitylation, and SUMOylation of Caspase-8. (A) The sites of post-translational modifications (PTMs) are mapped on the structure of procaspase-8. Death effector domain (DED)1/DED2 of procaspase-8 are presented as embedded in the DED filament structure, whereas p18 and p10 domains are presented as located outside of the DED filament. PTM residues are denoted and shown in red (phosphorylation sites), blue (ubiquitylation sites), and purple (SUMOylation sites). The following Protein Data Bank (PDB) structures were used for mapping PTM sites: the C terminal domain of procaspase-8 (3H11, left) and procaspase-8 DED filament (5L08, right). The regions for which the structural information is still missing are indicated with a gray line. Most of the PTM sites of caspase-8 are located outside of the DED1 and DED2 and target the large and small catalytic domains. Notably, K156 residue is not involved in interactions of DEDs. (B) Src kinase phosphorylates caspase-8 at Y380, which protects cells from CD95-induced apoptosis. Serine/threonine p38-MAPK induces phosphorylation of caspase-8 at S347, whereas tyrosine kinase Lyn is responsible for phosphorylation at Y380 and Y448, which also prevents the full activation of caspase-8. Cyclin-dependent kinase 1 (CDK1) phosphorylates caspase-8 in mitotic cells at S387 in p10 subunit. The ribosomal RSK2 induces phosphorylation of caspase-8 at T263, promoting caspase-8 degradation. Polo-like kinase 3 (Plk3)-induced phosphorylation at T273 on p18 subunit promotes CD95-induced caspase-8 activity. Procaspase-8 can (Figure legend continued at the bottom of the next page.)

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DED filament assembly (Figure 3A). Accordingly, SUMOylation of procaspase-8 mediates the nuclear translocation of this protein and does not impair its apoptotic function [35] (Figure 3B). Due to the limited studies conducted on caspase-8 SUMO modification, little is known about SUMOylation-mediated alterations of procaspase-8 function. Roles for Phosphorylation Several phosphorylation events at the region of the small and large catalytic subunits are well documented to control procaspase-8 activity. Phosphorylation at Y380 by Tyrosine-protein kinase CSK, also known as Src kinase, inhibits DR-mediated apoptosis [17,36,37]. Y380 (procaspase-8a) is located within the linker loop between the p10 and p18 subunits (Figure 3A). Phosphorylation at Y380 blocks the cleavage to p43 and the subsequent release of the large subunit p18 that restricts caspase-8 activity [37] (Figure 3B). Because of its antiapoptotic control, increased caspase-8 phosphorylation at Y380 (procaspase-8a) is associated with several malignancies and, in particular, is reported to promote cell migration [36–39]. Other phosphorylation sites at the p18 domain include S347 (procaspase-8a) [40] and Y448 (procaspase-8a) [41] (Figure 3B). These phosphorylation events also impair caspase-8 activity, most likely through conformational changes in the proximity of its active center. Phosphorylation at S347 is regulated by the serine/threonine mitogen-activated protein kinase p38-MAPK, whereas tyrosine kinase Lyn is responsible for phosphorylation at Y380 and Y448 [41,42] (Figure 3B). Furthermore, phosphorylation controls cleavage at D384. In particular, in mitotic cells, procaspase-8a is phosphorylated by Cyclin-dependent kinase 1 (CDK1) at the N terminus of p10 at S387, which diminishes procaspase-8 processing at D384 and might block CD95L-induced apoptotic signaling [43] (Figure 3B). Phosphorylation of caspase-8 not only blocks the processing of caspase-8 but might trigger its proteosomal degradation. In this regard, ribosomal S6 kinase 2 (RSK2)-induced phosphorylation at T263 promotes caspase-8a ubiquitylation and subsequent degradation, resulting in inhibition of CD95L-induced cell death [44] (Figure 3B). In contrast to its antiapoptotic roles, polo-like kinase 3 (Plk3)-induced phosphorylation at T273 at the p18 subunit of caspase-8 promotes DISC-induced caspase-8 activity [45] (Figure 3B). This event probably occurs via stabilization of the conformation of the active center [17]. Roles for Ubiquitylation Ubiquitylation of procaspase-8 can have a proapoptotic function. In particular, Cullin3 (CUL3) mediates both K48- and K63-linked ubiquitylation at K461 within the p10 subunit, which stabilizes the active caspase-8 heterotetramer [46]. This stabilization is achieved via binding of p62 to CUL3-ubiquitylated caspase-8 and subsequent formation of aggregated structures, leading to enhancement of caspase-8 activity [46] (Figure 3B). Furthermore, upon endoplasmic reticulum (ER) stress, Tripartite Motif-Containing Protein 13 (TRIM13)-mediated K63-linked ubiquitylation promotes caspase-8 activity. However, conjugation sites of TRIM13-induced ubiquitylation at procaspase-8 are unknown [47]. Deletions of the Really Interesting New Gene (RING) domain of TRIM13 or p62 as well as knockdown of TRIM13 result in decreased caspase-8 activity be SUMOylated at K156 by small ubiquitin-like modifier (SUMO)-1 within the death effector domain 2 (DED2), which leads to nuclear translocation of procaspase-8. CUL3 ubiquitylates caspase-8 at K461 within the p10 subunit, leading to association with p62 that stabilizes the active caspase-8 heterotetramer. During endoplasmic reticulum stress, Tripartite Motif-Containing Protein 13 (TRIM13) modulates caspase-8 activity via ubiquitylation, which may lead cells to autophagy. Homologous to the E6-AP Carboxyl Terminus 3 (HECTD3) ubiquitylates caspase-8 at K215, which blocks subsequent activation of caspase-8 at the death-inducing signaling complex (DISC). Tumor necrosis factor receptor-associated factor 2 (TRAF2) induces caspase8 ubiquitylation at the K224, K229, and K231 triad on p18 domain, which leads to proteosomal degradation of caspase-8. The numbers of residues correspond to the caspase-8a isoform. Abbreviations: DD, death domain; DL, death ligand; FADD, FAS-associated death domain.

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(A) DED filament c-FLIP

C-terminal domain С259

С254

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K353 D196

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Y211 (c-FLIPS, Murine)

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Figure 4. Modulation of Death Receptor (DR) Signaling by Cellular FLICE-Inhibitory Protein (c-FLIP) Phosphorylation, Ubiquitylation, and Nitrosylation. (A) The structural model of c-FLIP in the death effector domain (DED) filament with post-translational modification (PTM) sites. DED1/DED2 of c-FLIP are presented as embedded into the DED filament structure, whereas caspase-like p20 and p10 domains are presented as located outside of the DED filament. PTM residues are denoted and shown in red (phosphorylation sites), blue (ubiquitylation sites), and green (nitrosylation sites). The following Protein Data Bank (PDB) structures were used for mapping PTM sites: the C terminal domain of c-FLIP (3H11, left), procaspase-8 DED filament (5L08, right), and ks-v-FLIP (3CL3) as a template for homology modeling of c-FLIP DED1/DED2. The homology model of c-FLIP DED1/DED2 was obtained using SWISS-MODEL webserver [69] and superimposed on the procaspase-8 DED filament with PyMOL software (http://sourceforge.net/projects/ pymol/). The regions for which the structural information is still missing are indicated with a gray line. Phosphorylation and (Figure legend continued at the bottom of the next page.)

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upon tunicamycin-induced ER stress [47]. These data indicate that TRIM13 and p62 synergistically promote the activity of caspase-8 that in turn stimulates the activation of other downstream caspases (Figure 3B). Contrary to the CUL3- or TRIM13-mediated ubiquitylation, Homologous to the E6-AP Carboxyl Terminus 3 (HECTD3)-induced K63-linked ubiquitylation of procaspase-8 at K215 blocks activity of caspase-8 at the DISC [17,48] (Figure 3B). Due to the close proximity to the procaspase-8 cleavage sites at D216 and D210, ubiquitylation at K215 appears to be critical for caspase-8 activity. In line with these data, HECTD3 is highly expressed in breast cancer cell lines and tumors at both mRNA and protein levels [48]. Another antiapoptotic ubiquitylation event is mediated by TNF receptor-associated factor 2 (TRAF2), which has been revealed to interact with caspase-8 at the DISC, leading to caspase-8 K48-linked ubiquitylation at the K224, K229, and K231 triad at the p18 domain [49]. TRAF2-mediated K48-linked ubiquitylation leads to proteosomal degradation of active caspase-8, which might down-modulate the apoptotic threshold [49]. Roles for Nitrosylation Caspase-8 was also shown to be nitrosylated, which blocks caspase-8 activity and reduces the sensitivity of hepatocytes to TNF-α/Actinomycin D (ActD)-induced apoptosis [50]. In spite of the knowledge that nitrosylation significantly inhibits caspase-8 activity, nothing is known yet about the nitrosylation site(s) on caspase-8. Further studies are needed to clarify this neglected issue. Taken together, phosphorylation and ubiquitylation of procaspase-8 seem to target only its large and small catalytic domains as well as the linker regions between them, indicating that these PTMs do not interfere with the assembly of the DED filaments (Box 1), but might modulate or inhibit caspase-8 activity after the formation of the DED filaments (Figure 3A). This, in turn, directly influences the threshold for apoptosis induction and thereby controls life/death decisions in the cell.

PTMs of c-FLIP Procaspase-8a/b activation at the DED filaments is controlled by c-FLIP proteins [34,51]. One long and two short c-FLIP isoforms, named Long (L), Short (S), and Raji (R) (i.e., c-FLIPL, c-FLIPS, and c-FLIPR ) are recruited to the DISC [52]. In addition, two c-FLIP cleavage products were described: p22-FLIP and p43-FLIP [52]. c-FLIP proteins possess two DED domains in the N terminus (DED1 and DED2, 1–73 and 92–170, correspondingly), whereas c-FLIPL also has catalytically inactive caspase-like domains (p20 and p12) in its C terminal region. All three isoforms are recruited to the DED filament via their DEDs (Figure 4A) (Box 3). c-FLIPL can act in a pro- as well as in an antiapoptotic manner on procaspase-8 activation, depending on its expression level [53]. The proapoptotic function of c-FLIPL is mediated via formation of procaspase-8/c-FLIPL heterodimers, in which the active center of procaspase-8 is stabilized by c-FLIPL, which enhances caspase-8 activity [54]. Short c-FLIP isoforms, c-FLIPS and c-FLIPR, inhibit caspase-8 activation by forming inactive heterodimers with procaspase-8 in the DED filaments [34,51,55].

ubiquitylation sites at DED2 of c-FLIP seem to not interfere with DED filament formation. (B) c-FLIP is phosphorylated at T166, S193, and S273. Phosphorylation at S193 blocks ubiquitylation of both the short c-FLIP isoform (c-FLIPS) and the long cFLIP isoform (c-FLIPL). In contrary, phosphorylation at T166 is a prerequisite for K167 ubiquitylation and proteosomal degradation of c-FLIP. Tumor necrosis factor receptor-associated factor 2 (TRAF2) induces ubiquitylation and subsequent degradation of c-FLIP. ITCH mediates ubiquitylation of c-FLIPL within the DED2 at K167, leading to the degradation of the long form of c-FLIP. c-FLIPL is nitrosylated at C254 and C259, which precludes its ubiquitylation and subsequent degradation. Nitrosylated c-FLIP impairs caspase-8 activation and transduction of apoptotic signaling. Abbreviations: DD, death domain; DL, death ligand.

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Phosphorylation of c-FLIP has been shown to be crucial in the modulation of apoptosis [52]. Three c-FLIP phosphorylation sites, namely at T166, S193, and S273, have been identified so far. Interestingly, two of these sites, T166 and S193, mediate the regulatory crosstalk between phosphorylation and ubiquitylation of c-FLIP. In particular, the phosphorylation within DED2 at T166 has been shown to be a prerequisite for K48 ubiquitylation at K167 and proteosomal degradation of c-FLIP [56,57]. Hence, though T166 is located at DED2, it seems to serve as the signal for proteosomal degradation of c-FLIP rather than taking part in the modulation of DED interactions. On the contrary, phosphorylation at S193 by protein kinase C (PKC) blocks ubiquitylation of both c-FLIPS and c-FLIPL isoforms at K195 and K192 [58] (Figure 4B). This phosphorylation-dependent decline in ubiquitylation increases the stability of c-FLIPS, while not affecting the stability of c-FLIPL, indicating that the stability of c-FLIP isoforms is determined by distinct mechanisms [58,59]. Of note is that interactions of c-FLIP with DISC and DED filaments are mediated by DEDs, and S193 is residing outside the DEDs (Figure 4A). Phosphorylation at S193 does not influence the binding of c-FLIP to TRAIL DISC [59]. Moreover, S193 and K195 are located in the close proximity to D196, which gives rise to the p22-FLIP cleavage product [60]. p22FLIP de facto presents the DED-containing part of c-FLIP, which is a strong inducer of the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB) pathway [52,60]. Hence, it is a question for further studies to find out how the crosstalk between phosphorylation, ubiquitylation, and cleavage at this region of c-FLIP (193–196) regulates life/death decisions in cells (Figure 4A). In addition, TNFα stimulates AKT Serine/Threonine Kinase 1 (Akt1)-mediated c-FLIPL phosphorylation at S273, which promotes proteosomal degradation of c-FLIPL, resulting in the reduction of this protein [61–63] (Figure 4B). Finally, Mycobacterium tuberculosis-induced TNFα leads to activation of c-Abl and p38-MAPK,

Box 3. The Architecture of c-FLIP in the DED Filament One long and two short c-FLIP isoforms, named Long (L), Short (S), and Raji (R) (i.e., c-FLIPL, c-FLIPS, and c-FLIPR) have been described (Figure IA). Short isoforms of c-FLIP, c-FLIPS, and c-FLIPR, comprise DED1 and DED2 and demonstrate strong antiapoptotic effects. c-FLIPL, comprising DED1 and DED2 as well as a C terminal caspase-like domain, can play both a pro- and an antiapoptotic role (Figure IA). Several mechanisms of c-FLIP incorporation into the DISC and DED filaments were proposed. They include: a cooperative binding of c-FLIP to FADD in a procaspase-8-dependent manner [34]; the ability of c-FLIP to commingle with caspase-8 in the DED filament (Figure IB) [12,51], and the direct binding of c-FLIP to FADD [72]. Both the ‘commingling’ and caspase-8cooperative binding models imply the formation of heterodimers of c-FLIP with caspase-8. It must be noted that these two mechanisms suggest different patterns of interaction of c-FLIP within the DED filament; however, both lead to the formation of c-FLIP/procaspase-8 heterodimers. The heterodimer formation is expected to reduce the amount of procaspase-8 homodimers leading to decreased caspase-8 activity at the DISC. The model of direct binding of c-FLIP to FADD proposes that c-FLIP interacts via its Ib interface with the Ia interface of FADD DED, while procaspase-8 utilizes the other binding site for docking to FADD located at FADD Ib interface [72]. The ability of c-FLIP to interact with FADD in the absence of caspase-8 was confirmed recently [55]. However, so far, no 3D structure of c-FLIP DEDs has been reported. Hence, to consider the mechanisms of c-FLIP action, a number of the structural models of c-FLIP DEDs were constructed in silico. These models are based on the structure of viral FLIP proteins, such as MC159 or ks-v-FLIP, that are homologous to c-FLIP [12,34,73]. Further insights into the role of c-FLIP in the DISC structure were obtained recently using state-of-the-art quantitative mass spectrometry supported by structural modeling [69]. Using a Rosetta molecular modeling approach, the interacting interfaces of FADD DED were ranked according to the predicted energy of binding towards different interfaces of c-FLIP or procaspase-8 DEDs [55]. This analysis suggested that FADD DED can trigger the filament growth in the ‘forward’ direction by binding to the Ib/IIIa interfaces of procaspase-8 DED1 or it can trigger the filament growth in the opposite ‘backward’ direction by binding to the Ia/IIIb interfaces of the c-FLIP DED2 domain (Figure IC). Furthermore, c-FLIP was shown to regulate the length of the DED filament [55]. Upon high expression of c-FLIP, the DED filament is composed of similar amounts of procaspase-8, c-FLIP, and FADD DEDs, resulting in short DED filaments (Figure IB). Moreover, the in silico structural model suggests that the interaction of short DED filaments via type I/II/III interactions can lead to the formation of long ‘cooperative’ DED filaments formed by multiple DISC complexes. This hypothesis has yet to be validated in future studies.

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(A)

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Figure I. Cellular FLICE-Like Inhibitory Protein (c-FLIP) Architecture at the Death Effector Domain (DED) Filament. (A) Scheme of c-FLIP isoforms. (B) Scheme of the DED filament with c-FLIP according to the ‘commingling’ mechanism [12]. Three DED chains are shown (chains 1, 2, and 3). Procaspase-8 DED1 is shown in dark blue, while procaspase-8 DED2 is shown in light blue. Fas-associated protein with death domain (FADD) DED is shown in brown. DED1 of c-FLIP is shown in orange, while DED2 of c-FLIP is shown in a light orange color. (C) Structure of procaspase-8 filament with c-FLIP according to the models by [55]. The changes in the composition stoichiometry of the DED filament are presented depending on c-FLIP levels [72]. The same color code for procaspase-8, c-FLIP, and FADD DEDs as in (A) is used. The direction of the type I interaction is shown with a broken arrow. Abbreviations: c-FLIPL, Long c-FLIP isoform; c-FLIPR, Raji c-FLIP isoform; c-FLIPS, short c-FLIP isoform.

which in turn phosphorylate murine c-FLIPS on Y211 and S4, correspondingly. These phosphorylation events were reported to facilitate ubiquitination and subsequent degradation of c-FLIPs by E3 ligase c-Cbl [61–63] (Figure 4B). c-FLIP is characterized by a short half-life, which serves as an important mechanism to control the sensitivity and resistance of cells towards DR stimulation. As already mentioned earlier, the ubiquitin-proteasome pathway regulates both c-FLIPL and c-FLIPS expression [56,61,63–65] (Figure 4B). Upon CD95L treatment, c-FLIP was ubiquitylated, leading to its degradation [66]. TRAF2 was suggested to be the ubiquitin ligase for CD95L-induced c-FLIP ubiquitylation via direct binding to c-FLIP [66]. However, the detailed molecular mechanisms of these ubiquitylation events, including the sites of TRAF2-mediated c-FLIP ubiquitylation, are still unknown. Additionally, c-FLIP undergoes M1 linear ubiquitylation. The catalytic subunit of linear ubiquitination chain assembly complex (LUBAC) RNF31 interacts with the DED1 of c-FLIP, which leads to M1-linked ubiquitylation of c-FLIP [67] (Figure 4B). M1-linked ubiquitin chains on c-FLIP are revealed to reside in the p20 subunit of c-FLIPL at K351 and K353 [67]. M1-linked ubiquitin Trends in Cell Biology, Month 2020, Vol. xx, No. xx

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chains compete with K48-linked ubiquitylation of c-FLIP L, thereby inhibiting proteasomemediated degradation of c-FLIPL [67]. c-FLIPL also undergoes nitrosylation at C254 and C259 [66]. Nitrosylation of c-FLIPL precludes its ubiquitylation and subsequent degradation, leading to an increase in c-FLIPL levels (Figure 4B). Accordingly, nitrosylated c-FLIPL impairs caspase-8 activation and transduction of apoptotic signaling. Importantly, the sites of nitrosylation are located at the caspase-like domain and accordingly they most likely do not interfere with the binding of c-FLIPL to the DED filament (Figure 4B). Moreover, an increase of c-FLIPL concentration would lead to an increase of caspase-8-c-FLIPL heterodimers, which per se should lead to an increase of caspase-8 activity. However, it might be that nitrosylation of the p20 subunit of c-FLIP reduces its capacity to stabilize the active center of caspase-8 in heterodimers of caspase8-c-FLIPL. Accordingly, nitrosylated c-FLIPL might act solely as a dominant negative inhibitor of caspase-8, which is observed experimentally [66]. Thus, c-FLIP nitrosylation appears to provide an important mechanism for apoptosis regulation that may have therapeutic potential. Taken together, one of the major routes of PTM control of c-FLIP function involves the regulation of the expression level via switching on proteosomal degradation. Accordingly, it seems that many PTMs target c-FLIP turnover, which is largely controlled by the crosstalk between phosphorylation and ubiquitylation. This regulation involves PTM of cFLIP DEDs as well as of its caspase-like domains. The underlying mechanisms ensure the dynamic adjustment of c-FLIP levels in the cell, which allows the response to cell death stimuli to be fine-tuned. This regulation does not have to target c-FLIP interactions with the DED platforms, as these PTMs seem to affect the overall levels of c-FLIP proteins in the cell. Hence, based on the so-far reported PTMs of c-FLIP, one can suggest that that they do not modulate the interactions within DED platforms but rather determine the level of c-FLIP in the cell.

Concluding Remarks Although the molecular nature of the DISC formation is rather well documented, we are only now beginning to grasp the role of PTMs of DISC components in the modulation of the extrinsic apoptotic signaling pathway. Here, we have discussed the role of FADD, caspase-8, and c-FLIP modifications in the modulation of DR-mediated apoptosis. Surprisingly, there are not so many PTMs directly affecting the assembly of the DR complexes. So far only K6 ubiquitylation of the DD of FADD seems to largely decrease the level of the DISCs and caspase-8 activation. Interestingly, the reported PTMs of these proteins largely do not modulate their direct interactions within DED platform. Furthermore, it might be essential for the proper assembly of the DED filaments to have this region not undergo PTM. Another important function of PTMs is to regulate the intracellular levels of the DED-containing proteins FADD, c-FLIP, and caspase-8 and thereby control apoptosis. Furthermore, PTMs can switch DED-protein-mediated signaling towards the antiapoptotic route. Hence, PTMs of core DISC components provide an important checkpoint to determine the DED-mediated signaling outputs and a threshold for apoptosis induction. Another important factor, which must be considered in future studies, is that there may be additional unknown PTMs of the core DISC components, which determine or fine-tune the extrinsic apoptotic pathway (see Outstanding Questions). Acknowledgments We acknowledge Volkswagen Foundation (VW 90315), Wilhelm Sander-Stiftung (2017.008.01), and Center of dynamic systems (CDS), funded by the EU-programme ERDF (European Regional Development Fund) and DFG (LA 2386), Russian

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Outstanding Questions Why do PTMs not appear to directly affect the assembly of the DR complexes? Why do most PTMs act via regulation of the intracellular levels of DEDcontaining proteins? How can PTMs switch DED-proteinmediated signaling towards the antiapoptotic route? How do PTMs of core DISC components determine the DED-mediated signaling outputs and threshold for apoptosis induction?

Trends in Cell Biology

Foundation for Basic Research (19-54-45015), and Russian State Budget Project (АААА-А17-117092070032-4) for supporting our work.

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