Autophagy is a lysosomal degradation pathway important for cellular homeostasis, mammalian development, cancer and immunity. Many molecular components of autophagy have been identified, but little is known about regulatory mechanisms controlling their effector functions. Here, we show that, in contrast to other p38 MAP kinase activators, the growth arrest and DNA damage 45 beta (Gadd45b)-MAPK/ERK kinase kinase 4 (MEKK4) pathway specifically directs p38 to autophagosomes. This process results in an accumulation of autophagosomes through p38-mediated inhibition of lysosome fusion. Conversely, autophagic flux is increased in p38-deficient fibroblasts and Gadd45b-deficient cells. We further identified the underlying mechanism and demonstrate that phosphorylation of the autophagy regulator autophagy-related (Atg)5 at threonine 75 through p38 is responsible for inhibition of starvation-induced autophagy. Thus, we show for the first time that Atg5 activity is controlled by phosphorylation and, moreover, that the spatial regulation of p38 by Gadd45b/MEKK4 negatively regulates the autophagic process. Macroautophagy (hereafter referred to as autophagy) is a catabolic process, by which the cell degrades cytosolic content to supply metabolic processes with nutrients in order to maintain ATP production and macromolecular synthesis. Thus, autophagy acts as an efficient recycling mechanism in eukaryotic cells.1 Cellular stress, for example, nutrient deprivation, enhances autophagy as a survival mechanism during starvation. In addition, autophagy serves important functions in development, cancer, cell death and immunity in mammals. 1,2 Autophagy is controlled by conserved key regulators known as autophagy-related (Atg) proteins.3 At the onset of the autophagy cascade, Atg6/Beclin-1 forms a complex with the class III phosphatidylinoside kinase Vps34, which induces expansion of the precursor membrane vesicle, the phagophore, via recruitment of additional Atg proteins. During expansion, the double membrane vesicle surrounds cytosolic content, and the completed vesicle, called autophagosome, finally fuses with lysosomes to degrade the autophagosomal content.3 Maturation of autophagosomes is regulated by two ubiquitin-like conjugation systems, namely the Atg8-phosphatidylethanolamine (PE) and the Atg5-12/16L1 conjugation system. The Atg5-Atg12 conjugate interacts with Atg16L1, which tethers the complex to phagophores and autophagosomes. This complex then acts as an E3-like ubiquitin ligase for microtubule-associated protein 1 light chain 3 (LC3) lipidation. The conversion of LC3 to the PE-conjugated LC3-II form and its recruitment to the membrane serves as a wellaccepted marker for autophagy.Although Atg5-independent autophagy has been described, 4 Atg5 is crucial for autophagy under most circumstances and Atg5-deficient mouse embryonic fibroblasts (MEFs) lack LC3 conversion and autophagy. Therefore, Atg5-deficient mice die postnatal owing to their inability to cope with starvation during the neonatal period. 5 In addition, Atg5 see...

Cellular FLICE-inhibitory protein (c-FLIP) proteins are known as potent inhibitors of death receptor-mediated apoptosis by interfering with caspase-8 activation at the death-inducing signaling complex (DISC). Among the three human isoforms, c-FLIP long , c-FLIP short and c-FLIP R , the latter isoform is poorly characterized. We report here the characterization of murine c-FLIP R and show that it is the only short c-FLIP isoform expressed in mice. By generating several mutants, we demonstrate that both death effector domains (DEDs) are required for DISC binding and the antiapoptotic function of c-FLIP R . Surprisingly, the C-terminal tail is important for both protein stability and DISC recruitment. Three-dimensional modeling of c-FLIP R revealed a substantial similarity of the overall structures and potential interaction motifs with the viral FLIP MC159. We found, however, that c-FLIP R uses different structural motifs for its DISC recruitment. Whereas MC159 interferes with interaction and selfoligomerization of the DISC component FADD by its extensive hydrophilic surface, a narrow hydrophobic patch of c-FLIP R on the surface of DED2 is crucial for DISC association. Thus, despite the presence of similar tandem DEDs, viral and cellular FLIPs inhibit apoptosis by remarkably divergent mechanisms. Death receptors like CD95 (Fas/Apo-1) transduce death signals upon ligand binding and formation of a death-inducing signaling complex (DISC), which comprises the receptor, the adaptor protein Fas-associated death domain (FADD) and procaspase-8 (FLICE) or procaspase-10. 1 This assembly brings procaspase-8/10 in close proximity to the receptor and allows them to be activated by dimerization. 2-4 Activation of the initiator caspases can be counteracted by FLICEinhibitory proteins (FLIPs). 5,6 Next to viral FLIP (v-FLIP) proteins, 7 three cellular homologs (cellular FLICE-inhibitory proteins (c-FLIPs)) have been identified, namely c-FLIP long , c-FLIP short and c-FLIP R , which are generated by differential splicing. [8][9][10] The C-terminus of c-FLIP long contains a catalytically inactive caspase-like domain, whereas c-FLIP short and c-FLIP R have only a truncated C-terminus.As a characteristic feature, FLIP proteins contain tandem death effector domains (DEDs), which mediate their recruitment into the DISC. 10,11 The DED forms a bundle of six antiparallel a-helices similar to the death domain (DD) and the caspase recruitment domain (CARD), two other signaling motifs involved in homotypic protein interactions. 12 Curiously, despite their importance in apoptosis, there are currently no reported structures for cellular FLIP proteins. Only the structure of the tandem DEDs of the v-FLIP MC159 from Molluscum contagiosum has been reported showing a rigidly associated dumbbell-shaped molecule, which is tightly packed by an extensive hydrophobic interface between its two DEDs. 13,14 Similar to FADD, each DED of MC159 contains two prominent surface features important for protein interactions that distinguish them from other death mot...

The death receptor CD95 triggers apoptosis upon formation of a death-inducing signaling complex and the activation of caspase-8. Two types of CD95-mediated apoptosis have been distinguished that differ in their efficiency of death-inducing signaling complex formation and the requirement of mitochondria for caspase activation. The validity of the type I/II model, however, has been challenged, as Bcl-2 expression or the use of various CD95 agonists resulted in different apoptosis effects. By identifying a caspase-9-deficient T cell line, we now provide genetic evidence for the two-pathway model of CD95-mediated apoptosis and demonstrate that type II cells strongly depend on caspase-9. Caspase-9-deficient cells revealed strongly impaired apoptosis, caspase activation, and mitochondrial membrane depolarization upon CD95 triggering, whereas, surprisingly, activation of Bak and cytochrome c release were not inhibited. Furthermore, caspase-9-deficient cells did not switch to necrosis, and reconstitution of caspase-9 expression restored CD95 sensitivity. Finally, we also show that different death receptors have a distinct requirement for caspase-9.CD95 (APO-1/Fas) is the prototype member of the death receptor family (1). Stimulation of CD95 with its cognate ligand or agonistic antibodies results in receptor oligomerization and recruitment of signaling proteins in a death-inducing signaling complex (DISC) 3 (2, 3). Essential for apoptosis signaling through death receptors are the DISC components Fas-associated death domain and caspase-8. Recruitment of caspase-8 into the DISC leads to its dimerization-induced activation and the autoproteolytic release of its active subunits into the cytosol. The active caspase-8 activates downstream caspases, such as caspase-3, -6 and-7, that are responsible for most of the morphological manifestations of cell death (1). This death receptor-mediated pathway is also called the extrinsic pathway and is distinguished from the intrinsic mitochondrial death pathway. Apoptosis induction via mitochondria is regulated by the Bcl-2 family and involves early loss of mitochondrial membrane potential (⌬⌿ M ), release of cytochrome c, and other apoptogenic factors (4). In the cytosol, cytochrome c binds to Apaf-1 leading to caspase-9 recruitment. At this apoptosome complex, caspase-9 is activated and initiates the caspase cascade and subsequent apoptosis.An involvement of mitochondria has also been demonstrated in CD95 signaling, suggesting a functional role of this organelle in death receptor-mediated apoptosis of certain cell types (5, 6). Two types of CD95 signaling pathways have been proposed (7). CD95 type I cells show efficient DISC formation and strong activation of caspase-8 at the receptor level, which directly triggers the caspase cascade. Type II cells, in contrast, form a weak DISC and produce very little active caspase-8 at the receptor level, which is insufficient to initiate the apoptotic process. The low amounts of active caspase-8 can cleave Bid, a proapoptotic Bcl-2 protein that tra...

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