RIPK1- and RIPK3-induced cell death mode is determined by target availability

Abstract

Both receptor-interacting protein kinase 1 (RIPK1) and RIPK3 can signal cell death following death receptor ligation. To study the requirements for RIPK-triggered cell death in the absence of death receptor signaling, we engineered inducible versions of RIPK1 and RIPK3 that can be activated by dimerization with the antibiotic coumermycin. In the absence of TNF or other death ligands, expression and dimerization of RIPK1 was sufficient to cause cell death by caspase-or RIPK3-dependent mechanisms. Dimerized RIPK3 induced cell death by an MLKL-dependent mechanism but, surprisingly, also induced death mediated by FADD, caspase 8 and RIPK1. Catalytically active RIPK3 kinase domains were essential for MLKL-dependent but not for caspase 8-dependent death. When RIPK1 or RIPK3 proteins were dimerized, the mode of cell death was determined by the availability of downstream molecules such as FADD, caspase 8 and MLKL. These observations imply that rather than a 'switch' operating between the two modes of cell death, the final mechanism depends on levels of the respective signaling and effector proteins. Mammalian cells can use a number of mechanisms to kill themselves. The best characterized depends on the Bcl-2 family members Bax and Bak that work via mitochondria to activate caspases. 1 Some caspases, notably caspase 8, can be activated independently of Bcl-2 family members, for example, after stimulation of members of the TNF receptor superfamily. 2 Recently, it has become apparent that some of these receptors, including TNFR1, can activate a third suicide mechanism that does not require caspases, and in which the morphology of the dying cell differs from classical apoptosis. This form of cell death, termed 'necroptosis', can often be blocked by necrostatin-1 (nec-1), an inhibitor of the kinase activity of receptor-interacting protein kinase 1 (RIPK1). 3,4 Accordingly, observations from several groups have shown that in some cell types, expression of RIPK1 can signal cell death by caspase-independent necroptosis. 5 It has previously been revealed that RIPK1 could function downstream of death receptors, but in those cases, cell death was usually blocked by coexpression of the viral inhibitor of caspases 1 and 8, CrmA, 6 and typically exhibited a classical 'apoptotic' morphology. It was revealed that RIPK1 engages FADD via homotypic binding of their death domains (DDs), and FADD in turn activates caspase 8. 6,7 RIPK3, like RIPK1, bears a kinase domain and RIP homology interaction motif (RHIM), but unlike RIPK1 does not have a DD. 8-11 RIPK3 is required for necroptosis. 12,13 Furthermore, RIPK1 appears to activate RIPK3 in this pathway, as cell death could be blocked by nec-1. 14 RIPK3 activates, by phosphorylation, MLKL, a pseudokinase essential for this death pathway. [15][16][17] Once activated, MLKL forms multimers that trigger breaches of the plasma membrane. [18][19][20] Although RIPK3 is necessary for necroptosis, it is unclear whether activation of RIPK3 is sufficient for cell death, because TNF activates signaling ...

Evolutionary divergence of the necroptosis effector MLKL

“…We tested this hypothesis by fusing E. coli DNA gyrase (Figures 1d and e), a domain that can be dimerized by the divalent antibiotic coumermycin, to the C termini of hMLKL (1-180; NTD) and hMLKL (1-125; 4HB) domains. 21 In the absence of coumermycin, the fusion proteins behaved the same as the unfused domains in the absence of apoptotic (TS) or necroptotic (TSQ) stimuli in U937 cells (Figures 1a, f and g). Similarly, a C-terminally StrepII-tagged version of hMLKL (1-125) did not induce stimulus-independent cell death (Supplementary Figures 1A and C).…”

“…For expression in mammalian cell lines, cDNAs were ligated into the doxycycline-inducible puromycin-resistant lentiviral vector, pFTRE3G PGK Puro, 5,10 or cDNAs lacking a stop codon were ligated inframe as BamHI-NheI fragments into derivative vectors encoding either a C-terminal StrepII tag for detection of expression 15 or E. coli DNA GyraseB domain for oligomerization studies. 21 For expression of recombinant proteins, cDNAs encoding human MLKL (2-154) was ligated in-frame C-terminal to a TEV protease-cleavable GST tag in the vector pGEX-2T-TEV, and chicken MLKL (2-156) was ligated in-frame C-terminal to a NusA-His 6 tag in pETNusH Htb, as described for mouse MLKL (1-169) previously. 10 Full-length human (2-471), chicken (2-486) and frog (2-498) MLKL and frog (195-498) pseudokinase domains were expressed from insect cells as described previously for mouse MLKL 5,10 using the Bac-to-Bac system (Life Technologies, Carlsbad, CA, USA), but with a TEV-cleavable N-terminal GST Liposomes containing the self-quenching dye 5(6)-carboxyfluorescein were exposed to 1 μM recombinant protein as indicated and dye release was monitored spectrophotometrically over 3.25 h. Data are plotted as the mean ± S.D.…”

Regulated RIPK3 Necroptosis is Produced in Cardiovascular Tissues and Cells in Dietary Magnesium Deficiency: Roles of Cytokines and Their Potential Importance in Inflammation and Atherogenesis

“…Employing transmission electron microscopy (TEM), in our Mg-deficient rabbit experimentation suggested to us that, both the vascular smooth muscle cells and macrophages of the lipid-laden arterial vessels exhibited necrosis and apoptosis [16,65,66, unpublished findings of Stempak, BT Altura, M Brust and BM Altura]. Further, investigation using TEM on cardiac and arterial muscle cells of Mg-deficient rats also showed clear signs of necrosis as well as apoptosis [66][67][68][69][70][71][72][73][74][75][76][77][78]; N Shah, BT Altura and BM Altura, unpublished findings]. Experimentation with high-power TEM revealed that, these Mg-deficient muscle cells exhibited what is now termed "necroptosis".…”

“…Moreover, similar experimentation in a rodent model system showed very similar characteristics in arterial and cardiac muscles, under high power TEM. With the help of progressive research and studies now it is clear that, "necroptosis" occurs in a very controlled and regulated fashion [67][68][69] and requires the involvement of two serine/threonine kinase, receptor-interacting proteins namely, RIPK1 and RIPK3 [67][68][69][70][71]. It has been shown that, release of the cytokine TNF-alpha (TNF-α) initiates the activation of RIPK1 and RIPK3 [70,71].…”

“…With the help of progressive research and studies now it is clear that, "necroptosis" occurs in a very controlled and regulated fashion [67][68][69] and requires the involvement of two serine/threonine kinase, receptor-interacting proteins namely, RIPK1 and RIPK3 [67][68][69][70][71]. It has been shown that, release of the cytokine TNF-alpha (TNF-α) initiates the activation of RIPK1 and RIPK3 [70,71]. Though, several lines of evidence have been reported that, RIPK1 and RIPK3 can be regulated by the activation and release of other cytokines (e.g., IL-1beta, Interferon-gamma) also [68][69][70][71].…”