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How will we be able to determine whether each of these many uninvestigated phosphorylations identified in animal cells has a cellular function? Unfortunately, there is no quick way to assess the function of a phosphorylation. Each case requires a separate time-consuming investigation. A stan- dard approach is to examine the effect of replacing the wild- type protein with a nonphosphorylatable version (serine/ threonine to alanine or tyrosine to phenylalanine) on the cellular function of the protein. With the advent of RNA interference (RNAi) technology, this replacement can now be achieved in tissue culture through the combination of RNAi knockdown and transfection with a plasmid encoding the nonphosphorylatable mutant with RNAi-resistant silent mutations. In addition to the specific function of the protein in question, one generic property of a protein that can be examined is its lifetime. There are many examples of phosphorylation altering the half-life of a protein [6]. Proving a negative such as non-functional phosphoryl- ation is hard to do. For many of the phosphorylated proteins in the database there is, as yet, no known function to assay. Moreover, even if a function of a protein is known and phosphorylation has no effect on that function, the possibility remains that the protein has additional, as yet undiscovered, functions that are altered by phosphoryl- ation. Finally, proteins are often phosphorylated on multiple sites [7] and one must consider the possibility that several phosphorylations function redundantly.

Research Focus

Hence, the function of any one site might not be revealed unless the several sites are mutated together as well as separately.

Acknowledgements

We are indebted to Scott Gerber and Lee Witters at Dartmouth Medical School and Forest White at Massachusetts Institute of Technology for thoughtful suggestions about this topic.

References

1 Olsen, J.V. et al. (2006) Global, in vivo , and site-specific phosphorylation dynamics in signaling networks. Cell 127, 635–648

2 Manning, G. et al. (2002) The protein kinase complement of the human genome. Science 298, 1912–1934

3 Cho, C.H. et al. (2007) Oxygen uptake rates and liver-specific functions of hepatocyte and 3T3 fibroblasts co-cultures. Biotechnol. Bioeng. 97,

188–199

4 Sohlenius-Sternbeck, A-K. (2006) Determination of hepatocellularity number for human, dog, rabbit, rat, and mouse livers from protein concentration measurements. Toxicol. In Vitro 20, 1582–1586

5 Gnad, F. et al. (2007) Phosida (phosphorylation site database):

management, structural and evolutionary investigation, and prediction of phosphosites. Genome Biol. 8, R250

6 Hunter, T. (2007) The age of crosstalk: phosphorylation, ubiquitination, and beyond. Mol. Cell 28, 730–738

7 Salazar, C. and Hofer, T. (2007) Versatile regulation of multisite protein phosphorylation by the order of phosphate processing and protein– protein interactions. FEBS J. 274, 1046–1061

0968-0004/$ – see front matter 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.tibs.2008.05.004 Available online 4 July 2008

Necrotic cell death and ‘necrostatins’: now we can control cellular explosion

Peter Vandenabeele 1,2 , Wim Declercq 1,2 and Tom Vanden Berghe 1,2

1 Molecular Signaling and Cell Death Unit, Department for Molecular Biomedical Research, VIB, 9052 Ghent, Belgium 2 Department of Molecular Biology, Ghent University, 9052 Ghent, Belgium

The receptor-interacting protein 1 (RIP1) kinase activity is necessary for death-receptor-induced necrotic cell death. Recently, it has been demonstrated that ‘necros- tatins’ efficiently block tumor necrosis factor-induced necrotic cell death through the inhibition of RIP1 kinase activity. This discovery supports the concept that re- ceptor-induced necrosis, just like apoptosis, is a con- trolled cellular process. In addition, necrostatins are becoming important tools for evaluating the contri- bution of necrotic cell death in experimental disease models.

Apoptosis and necrosis: two sides of the same coin There are three major morphological types of cell death that have been described. Type-I, or apoptotic, cell death is mediated by caspases (a family of cysteine-dependent aspartate-specific proteases) and characterized by cellular shrinkage, membrane blebbing, chromatin condensation

Corresponding author: Vandenabeele, P. ( Peter.Vandenabeele@dmbr.UGent.be).

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and DNA degradation. Type-II cell death is associated with the formation of autophagic vacuoles inside the dying cell. Type-III, or necrotic, cell death is characterized by cellular swelling, plasma-membrane rupture and the subsequent loss of the intracellular contents [1,2] . For a long time, necrosis has been considered to be an accidental, uncon- trolled form of cell death but evidence is accumulating that execution of necrotic cell death can be carried out by a set of controlled signal-transduction pathways and execution mechanisms [3–5] . Triggering of the Fas or tumor necrosis factor (TNF) family of death-domain (DD) receptors usually activates the canonical apoptotic pathway, which is initiated and executed by members of the caspase family. However, caspase inhibition does not always prevent cell death. Instead, it has revealed the existence of a necrotic signaling pathway leading to cell death [6] ; this pathway has been termed ‘necroptosis’ by some groups [7] . In cell culture it has been shown that TNF, Fas ligand and TRAIL (TNF-related apoptosis-inducing ligand) can induce necro- tic cell death in the presence of caspase inhibitors or in the absence of Fas-associated death domain (FADD), which is

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an adaptor molecule involved in the recruitment and acti- vation of procaspase-8 [8] . Degterev et al. [7] have reported the characterization of necrostatins as the first-in-class inhibitors of in vitro necrotic cell death. More recently, the same authors demonstrated that these necrostatins inhibit receptor-interacting protein (RIP) 1 kinase activity [9] . This discovery has important pathological implications because, owing to a lack of necrotic biochemical markers, it has, thus far, been difficult to assess the importance of necrotic cell death in disease conditions. The occurrence of cell-death-exhibiting necrotic features has been reported in an array of acute human pathologies such as myocardial infarction, cerebral ischemia or acute organ failure [5] . Importantly, necrostatins have recently been shown to prevent tissue damage in mouse models of cerebral ische- mia and myocardial infarction [7,10,11] , indicating that there is great potential in further developing this family of inhibitors for future use in the treatment of equivalent human pathologies.

RIP1 in cell death The RIP serine/threonine kinases have emerged as essen- tial sensors of cellular stress. The different members inte- grate both extracellular and intracellular stress signals, such as pathogen infections, inflammation, T-cell receptor stimulation and DNA damage. Although these stimuli activate different signal-transduction pathways, they con- verge to initiate similar responses; for example, the acti- vation of transcription factors such as nuclear factor-kB (NF- kB) and activator protein-1 [12] . The RIP kinase family members share a homologous kinase domain (KD) but have different recruitment domains that are used to target these RIP family members to their respective signaling complexes. RIP1 bears a C-terminal DD belong- ing to the structurally related DD superfamily. The inter- mediate domain (ID) of RIP1 contains a RIP homotypic interaction motif, enabling its interaction with RIP3. The RIP1-DD was shown to be important for binding to death receptors such as TNF-receptor 1, TRAIL-receptor 1 and TRAIL-receptor 2, and to DD-containing adaptor proteins such as TNF-receptor-associated death-domain (TRADD) and FADD ( Figure 1 ). Additionally, RIP1 also interacts with a plethora of adaptor proteins through its ID, which is also used to recruit other kinases such as mitogen-acti- vated protein kinase (MAPK) kinase (MEKK)-1 and MEKK3. Moreover, RIP1, together with TNF-receptor- associated factor 2 (TRAF2), is also involved in activating MAPKs such as p38 MAPK, Jun N-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK) [13,14]. The activation of ERK depends on the kinase activity of RIP1 [15] . Remarkably, RIP1 kinase activity is apparently not essential for the activation of other MAPKs or NF-k B [16] . RIP1 is a crucial kinase at the crossroads at which the cell chooses to live or die upon exposure to several stress signals such as inflammatory cytokines, pathogen infec- tions and genotoxic stress [17] . RIP1-deficient mice show massive death of lymphoid and adipose tissue, reduced NF- kB activation and perinatal death [18] . Usually, RIP1- induced survival signaling is mediated by activating the transcription factor NF- kB [18] . Seminal work from the

transcription factor NF- k B [18] . Seminal work from the Figure 1 . A simplified

Figure 1. A simplified overview of the TNF-receptor 1 (TNFR1) initiation complex. At the plasma membrane, recruitment of TRAF2 and RIP1 to TNFR1 results in rapid activation of NF-kB and MAPKs. Following receptor endocytosis, TRADD recruits FADD and procaspase-8 or -10 resulting in apoptotic cell death [25]. In caspase- inhibitory conditions, apoptosis and caspase-8-mediated cleavage (inactivation) of RIP1 are blocked, resulting in RIP1 kinase activity-mediated necrosis. Color code:

blue, DD; orange, death effector domain; black, ID; red, KD; green, caspase domain.

group of Ju¨ rg Tschopp demonstrated that RIP1 kinase activity is crucial for the induction of necrotic cell death of FADD-deficient Jurkat cells stimulated with TNF [8] . In this respect, it is not surprising that necrosis only occurs in conditions in which apoptosis is blocked because it was shown that RIP1 is cleaved by caspase-8 during apoptotic signaling [19] . Furthermore, dimerization of the RIP1 kinase domain was sufficient to induce necrosis in FADD-deficient Jurkat cells, whereas the kinase-dead mutant could not induce cell death [7] . An active KD enables RIP1 to autophosphorylate; however, no other substrates of RIP1 have been identified so far. It has also been suggested that TNF-receptor 1 induces necrosis by means of RIP1-dependent recruitment and activation of the reactive oxygen species (ROS)-generating Nox-1 NADPH oxidase complex [20] , at least, in murine fibro- blasts. However, the role of RIP1 kinase activity was not addressed in these studies. Now, using a chemical com- pound screen of potential necrotic cell-death inhibitors, Degterev et al. [7,9] have identified small compounds that target RIP1 kinase activity.

Necrostatins trip up RIP1 Necrostatin (nec)-1 was originally identified in the labora- tory of Junying Yuan, by screening for chemical inhibitors of necrotic cell death in U937 cells induced by TNF in the presence of caspase inhibitors [7] . Nec-1 inhibited death- receptor-induced necrosis in different cellular models, indi- cating that necrotic signaling is mediated by a common

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Update Trends in Biochemical Sciences Vol.33 No.8 Figure 2 . A suggested mechanistic model for RIP1

Figure 2. A suggested mechanistic model for RIP1 kinase activation and inhibition. RIP1 kinase consists of three major domains that have been linked to distinct activities. The DD is important for recruitment to death receptors via homotypic interaction. The central region of RIP1 has been implicated in NF-kB activation through polyubiquitylation of Lys377. The kinase activity of RIP1 has been found to have a role in DD receptor-induced necrosis. Nec-1 and -3 are reported to be potent and selective inhibitors of RIP1 kinase, resulting in inhibition of necrosis in cell culture. The autophosphorylation sites were identified and are all located in the KD. The Ser161!Ala mutant (an autophosphorylation site present in the catalytic pocket) showed decreased kinase activity, indicating a positive contribution to its kinase activity. Mutagenesis studies and modeling results propose a model in which RIP1 kinase exists in equilibrium between a closed [stabilized activation segment and Asp-Leu-Gly (DLG) motif-out] and open conformation (destabilized activation segment and DLG motif-in). Both nec-1 and nec-3 inhibitors seem to stabilize the closed conformation, resulting in inhibition of RIP1 kinase activity. Color code: blue, DD; black, ID; red, KD; light blue, Lys377.

mechanism. Encouraged by this observation, additional structurally different necrostatins nec-3 and nec-5, which target RIP1 kinase activity [9] , were characterized [21,22]. The necrosis-inhibiting capacity of nec-1 derivatives was closely related to its in vitro RIP1 kinase-inhibiting activity, as measured by the level of autophosphorylation. In addition, nec-1 specifically inhibits RIP1 kinase and not its family members RIP2 and RIP3. Considering the fact that nec-3 and nec-5 are structurally very different from nec-1, it was surprising that both molecules potently inhib- ited RIP1 kinase that was immunoprecipitated from Jur- kat cells. However, nec-5 did not inhibit recombinantly expressed RIP1, indicating that nec-5 probably functions

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indirectly on RIP1. The use of the necrostatin inhibitors clearly distinguishes between kinase-dependent and kinase-independent processes by RIP1 kinase. Apparently, neither of the necrostatins affect RIP1-mediated NF- kB activation [9] , which is consistent with the observation that RIP1 kinase activity was found to be dispensable for these pathways [16] (interestingly, the same study also indicated that TNF-induced p38 MAPK and JNK kinase activation of NF-k B is also dispensable, so it is probable that kinase activity in these pathways is also unlikely to be a target of the necrostatins). These results indicate that RIP1 is at the bifurcation between pro-necrotic and pro-inflammatory activities. For several protein kinases, catalytic activity is depend- ent on phosphorylation within the so-called activation segment within the kinase domain. This 30–40 amino acid segment, also known as the T-loop, is located between two conserved Asp–Phe/Leu–Gly and Ala-Pro-Glu tripeptide motifs) within the KD [23] . Typically, the unphosphory- lated activation segment is locked into an ‘inactive’ con- formation. Phosphorylation on either one or several residues fixes the activation segment into an alternative ‘active’ conformation. Degterev et al. [9] showed that RIP1 becomes autophosphorylated at Ser14/15, Ser20, Ser161 and Ser166. A kinase alignment analysis indicated that the activation segment of RIP1 is very similar to that of the B-RAF kinase [a member of the RAF (rapidly growing fibrosarcomas) kinase family]. Interestingly, the RIP1 Ser161 autophosphorylation site corresponds to the Thr598 autophosphorylation site in the activation segment of B-RAF, which has been shown to contribute to the regulation of its catalytic activity [24] . The RIP1 mutant carrying a Ser161! Glu mutation probably results in a permanently active conformation that cannot be blocked by nec-1, indicating that the inhibitor directly or indirectly stabilizes the inactive conformation. The Ser161! Ala RIP1 mutant has reduced kinase activity and is also insensitive to nec-1, further implicating this residue in the binding of nec-1. Strictly, the latter can only be demon- strated by affinity-binding studies. Altogether, these data indicate that nec-1 functions by locking the activation segment in its inactive state. Additional experiments have indicated that nec-1 probably also competes with ATP binding. A similar analysis indicated that nec-3 functions in a manner that is partially activation-segment-indepen- dent, whereas nec-5 inhibits RIP1 through an, as yet unidentified, indirect mechanism ( Figure 2).

Concluding remarks and future perspectives The reports of the Yuan laboratory on the identification of necrostatins have many important implications [7,9,21,22] . First, it has been demonstrated that drug targeting of RIP1 kinase makes necrotic cell death con- trollable. This is a paradigm shift in view of the long persisting concept of necrotic cell death being an uncon- trolled type of cell death. Second, the findings place RIP1 in a central position as a master integrator and effector of cellular stress, controlling cellular survival, inflammation and necrosis. Third, the identification of necrostatins pro- vides us with valuable therapeutic tools with which to study the contribution of necrotic cell death in many

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experimental pathologies involving ischemia reperfusion damage such as organ transplantation, cardiac infarction, stroke and traumatic brain injury (if all these instances of necrotic cell death are RIP1 kinase dependent). However, several questions remain regarding the cellu- lar targets of RIP1, its subcellular localization, its regulation and its mechanism of activation. How can RIP1 decide between the different cellular outcomes?How is RIP1 kinase activity connected to the subcellular events happening during necrosis, such as enhanced mitochondrial ROS pro- duction, destabilization of lysosomes, and activation of phos- pholipase A2 [3–5]? Further research is also necessary to see whether different necrotic stimuli, such as Toll-like receptor 3 and Toll-like receptor 4 ligands, hydrogen peroxide, or hypoxia conditions also involve RIP1 kinase activity. For all these highly relevant questions, necrostatins will become indispensable tools for obtaining the answers.

Acknowledgements

We thank A. Bredan for editing the manuscript. This research was supported by the Flanders Institute for Biotechnology (VIB) and several grants. European grants: FP6 ApopTrain, MRTN-CT-035624; EC RTD Integrated Project, FP6 Epistem, LSHB-CT-2005–019067; EC RTD Integrated Project, Apo-Sys, FP7–200767. Belgian grants:

Interuniversity attraction poles, IAP 6/18. Flemish grants: Fonds Wetenschappelijke Onderzoek Vlaanderen, 3G.0218.06 and G.0133.05. Ghent University grants: BOF-GOA – 12.0505.02. T.V.B. is supported by a Fonds voor Wetenschappelijk Onderzoek postdoctoral fellowship.

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0968-0004/$ – see front matter 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.tibs.2008.05.007 Available online 16 July 2008

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