Loss of PINK1 Function Promotes Mitophagy through Effects on Oxidative Stress and Mitochondrial Fission

Mitochondrial dysregulation is strongly implicated in Parkinson disease. Mutations in PTEN-induced kinase 1 (PINK1) are associated with familial parkinsonism and neuropsychiatric disorders. Although overexpressed PINK1 is neuroprotective, less is known about neuronal responses to loss of PINK1 function. We found that stable knockdown of PINK1 induced mitochondrial fragmentation and autophagy in SH-SY5Y cells, which was reversed by the reintroduction of an RNA interference (RNAi)-resistant plasmid for PINK1. Moreover, stable or transient overexpression of wild-type PINK1 increased mitochondrial interconnectivity and suppressed toxin-induced autophagy/mitophagy. Mitochondrial oxidant production played an essential role in triggering mitochondrial fragmentation and autophagy in PINK1 shRNA lines. Autophagy/mitophagy served a protective role in limiting cell death, and overexpressing Parkin further enhanced this protective mitophagic response. The dominant negative Drp1 mutant inhibited both fission and mitophagy in PINK1-deficient cells. Interestingly, RNAi knockdown of autophagy proteins Atg7 and LC3/Atg8 also decreased mitochondrial fragmentation without affecting oxidative stress, suggesting active involvement of autophagy in morphologic remodeling of mitochondria for clearance. To summarize, loss of PINK1 function elicits oxidative stress and mitochondrial turnover coordinated by the autophagic and fission/fusion machineries. Furthermore, PINK1 and Parkin may cooperate through different mechanisms to maintain mitochondrial homeostasis.Parkinson disease is an age-related neurodegenerative disease that affects ∼1% of the population worldwide. The causes of sporadic cases are unknown, although mitochondrial or oxidative toxins such as 1-methyl-4-phenylpyridinium, 6-hydroxydopamine (6-OHDA),³ and rotenone reproduce features of the disease in animal and cell culture models (1). Abnormalities in mitochondrial respiration and increased oxidative stress are observed in cells and tissues from parkinsonian patients (2, 3), which also exhibit increased mitochondrial autophagy (4). Furthermore, mutations in parkinsonian genes affect oxidative stress response pathways and mitochondrial homeostasis (5). Thus, disruption of mitochondrial homeostasis represents a major factor implicated in the pathogenesis of sporadic and inherited parkinsonian disorders (PD).The PARK6 locus involved in autosomal recessive and early-onset PD encodes for PTEN-induced kinase 1 (PINK1) (6, 7). PINK1 is a cytosolic and mitochondrially localized 581-amino acid serine/threonine kinase that possesses an N-terminal mitochondrial targeting sequence (6, 8). The primary sequence also includes a putative transmembrane domain important for orientation of the PINK1 domain (8), a conserved kinase domain homologous to calcium calmodulin kinases, and a C-terminal domain that regulates autophosphorylation activity (9, 10). Overexpression of wild-type PINK1, but not its PD-associated mutants, protects against several toxic insults in neuronal cells (6, 11, 12). Mitochondrial targeting is necessary for some (13) but not all of the neuroprotective effects of PINK1 (14), implicating involvement of cytoplasmic targets that modulate mitochondrial pathobiology (8). PINK1 catalytic activity is necessary for its neuroprotective role, because a kinase-deficient K219M substitution in the ATP binding pocket of PINK1 abrogates its ability to protect neurons (14). Although PINK1 mutations do not seem to impair mitochondrial targeting, PD-associated mutations differentially destabilize the protein, resulting in loss of neuroprotective activities (13, 15).Recent studies indicate that PINK1 and Parkin interact genetically (3, 16-18) to prevent oxidative stress (19, 20) and regulate mitochondrial morphology (21). Primary cells derived from PINK1 mutant patients exhibit mitochondrial fragmentation with disorganized cristae, recapitulated by RNA interference studies in HeLa cells (3).Mitochondria are degraded by macroautophagy, a process involving sequestration of cytoplasmic cargo into membranous autophagic vacuoles (AVs) for delivery to lysosomes (22, 23). Interestingly, mitochondrial fission accompanies autophagic neurodegeneration elicited by the PD neurotoxin 6-OHDA (24, 25). Moreover, mitochondrial fragmentation and increased autophagy are observed in neurodegenerative diseases including Alzheimer and Parkinson diseases (4, 26-28). Although inclusion of mitochondria in autophagosomes was once believed to be a random process, as observed during starvation, studies involving hypoxia, mitochondrial damage, apoptotic stimuli, or limiting amounts of aerobic substrates in facultative anaerobes support the concept of selective mitochondrial autophagy (mitophagy) (29, 30). In particular, mitochondrially localized kinases may play an important role in models involving oxidative mitochondrial injury (25, 31, 32).Autophagy is involved in the clearance of protein aggregates (33-35) and normal regulation of axonal-synaptic morphology (36). Chronic disruption of lysosomal function results in accumulation of subtly impaired mitochondria with decreased calcium buffering capacity (37), implicating an important role for autophagy in mitochondrial homeostasis (37, 38). Recently, Parkin, which complements the effects of PINK1 deficiency on mitochondrial morphology (3), was found to promote autophagy of depolarized mitochondria (39). Conversely, Beclin 1-independent autophagy/mitophagy contributes to cell death elicited by the PD toxins 1-methyl-4-phenylpyridinium and 6-OHDA (25, 28, 31, 32), causing neurite retraction in cells expressing a PD-linked mutation in leucine-rich repeat kinase 2 (40). Whereas properly regulated autophagy plays a homeostatic and neuroprotective role, excessive or incomplete autophagy creates a condition of “autophagic stress” that can contribute to neurodegeneration (28).As mitochondrial fragmentation (3) and increased mitochondrial autophagy (4) have been described in human cells or tissues of PD patients, we investigated whether or not the engineered loss of PINK1 function could recapitulate these observations in human neuronal cells (SH-SY5Y). Stable knockdown of endogenous PINK1 gave rise to mitochondrial fragmentation and increased autophagy and mitophagy, whereas stable or transient overexpression of PINK1 had the opposite effect. Autophagy/mitophagy was dependent upon increased mitochondrial oxidant production and activation of fission. The data indicate that PINK1 is important for the maintenance of mitochondrial networks, suggesting that coordinated regulation of mitochondrial dynamics and autophagy limits cell death associated with loss of PINK1 function.     

Bortezomib Overcomes Tumor Necrosis Factor-related Apoptosis-inducing Ligand Resistance in Hepatocellular Carcinoma Cells in Part through the Inhibition of the Phosphatidylinositol 3-Kinase/Akt Pathway

Hepatocellular carcinoma (HCC) is one of the most common and aggressive human malignancies. Recombinant tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising anti-tumor agent. However, many HCC cells show resistance to TRAIL-induced apoptosis. In this study, we showed that bortezomib, a proteasome inhibitor, overcame TRAIL resistance in HCC cells, including Huh-7, Hep3B, and Sk-Hep1. The combination of bortezomib and TRAIL restored the sensitivity of HCC cells to TRAIL-induced apoptosis. Comparing the molecular change in HCC cells treated with these agents, we found that down-regulation of phospho-Akt (P-Akt) played a key role in mediating TRAIL sensitization of bortezomib. The first evidence was that bortezomib down-regulated P-Akt in a dose- and time-dependent manner in TRAIL-treated HCC cells. Second, {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002, a PI3K inhibitor, also sensitized resistant HCC cells to TRAIL-induced apoptosis. Third, knocking down Akt1 by small interference RNA also enhanced TRAIL-induced apoptosis in Huh-7 cells. Finally, ectopic expression of mutant Akt (constitutive active) in HCC cells abolished TRAIL sensitization effect of bortezomib. Moreover, okadaic acid, a protein phosphatase 2A (PP2A) inhibitor, reversed down-regulation of P-Akt in bortezomib-treated cells, and PP2A knockdown by small interference RNA also reduced apoptosis induced by the combination of TRAIL and bortezomib, indicating that PP2A may be important in mediating the effect of bortezomib on TRAIL sensitization. Together, bortezomib overcame TRAIL resistance at clinically achievable concentrations in hepatocellular carcinoma cells, and this effect is mediated at least partly via inhibition of the PI3K/Akt pathway.Hepatocellular carcinoma (HCC)² is currently the fifth most common solid tumor worldwide and the fourth leading cause of cancer-related death. To date, surgery is still the only curative treatment but is only feasible in a small portion of patients (1). Drug treatment is the major therapy for patients with advanced stage disease. Unfortunately, the response rate to traditional chemotherapy for HCC patients is unsatisfactory (1). Novel pharmacological therapy is urgently needed for patients with advanced HCC. In this regard, the approval of sorafenib might open a new era of molecularly targeted therapy in the treatment of HCC patients.Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL), a type II transmembrane protein and a member of the TNF family, is a promising anti-tumor agent under clinical investigation (2). TRAIL functions by engaging its receptors expressed on the surface of target cells. Five receptors specific for TRAIL have been identified, including DR4/TRAIL-R1, DR5/TRAIL-R2, DcR1, DcR2, and osteoprotegerin. Among TRAIL receptors, only DR4 and DR5 contain an effective death domain that is essential to formation of death-inducing signaling complex (DISC), a critical step for TRAIL-induced apoptosis. Notably, the trimerization of the death domains recruits an adaptor molecule, Fas-associated protein with death domain (FADD), which subsequently recruits and activates caspase-8. In type I cells, activation of caspase-8 is sufficient to activate caspase-3 to induce apoptosis; however, in another type of cells (type II), the intrinsic mitochondrial pathway is essential for apoptosis characterized by cleavage of Bid and release of cytochrome c from mitochondria, which subsequently activates caspase-9 and caspase-3 (3).Although TRAIL induces apoptosis in malignant cells but sparing normal cells, some tumor cells are resistant to TRAIL-induced apoptosis. Mechanisms responsible for the resistance include receptors and intracellular resistance. Although the cell surface expression of DR4 or DR5 is absolutely required for TRAIL-induced apoptosis, tumor cells expressing these death receptors are not always sensitive to TRAIL due to intracellular mechanisms. For example, the cellular FLICE-inhibitory protein (c-FLIP), a homologue to caspase-8 but without protease activity, has been linked to TRAIL resistance in several studies (4, 5). In addition, inactivation of Bax, a proapoptotic Bcl-2 family protein, resulted in resistance to TRAIL in MMR-deficient tumors (6, 7), and reintroduction of Bax into Bax-deficient cells restored TRAIL sensitivity (8), indicating that the Bcl-2 family plays a critical role in intracellular mechanisms for resistance of TRAIL.Bortezomib, a proteasome inhibitor approved clinically for multiple myeloma and mantle cell lymphoma, has been investigated intensively for many types of cancer (9). Accumulating studies indicate that the combination of bortezomib and TRAIL overcomes the resistance to TRAIL in various types of cancer, including acute myeloid leukemia (4), lymphoma (10–13), prostate (14–17), colon (15, 18, 19), bladder (14, 16), renal cell carcinoma (20), thyroid (21), ovary (22), non-small cell lung (23, 24), sarcoma (25), and HCC (26, 27). Molecular targets responsible for the sensitizing effect of bortezomib on TRAIL-induced cell death include DR4 (14, 27), DR5 (14, 20, 22–23, 28), c-FLIP (4, 11, 21–23, 29), NF-κB (12, 24, 30), p21 (16, 21, 25), and p27 (25). In addition, Bcl-2 family also plays a role in the combinational effect of bortezomib and TRAIL, including Bcl-2 (10, 21), Bax (13, 22), Bak (27), Bcl-xL (21), Bik (18), and Bim (15).Recently, we have reported that Akt signaling is a major molecular determinant in bortezomib-induced apoptosis in HCC cells (31). In this study, we demonstrated that bortezomib overcame TRAIL resistance in HCC cells through inhibition of the PI3K/Akt pathway.     

The Neuromediator Glutamate, through Specific Substrate Interactions, Enhances Mitochondrial ATP Production and Reactive Oxygen Species Generation in Nonsynaptic Brain Mitochondria

The finding that upon neuronal activation glutamate is transported postsynaptically from synaptic clefts and increased lactate availability for neurons suggest that brain mitochondria (BM) utilize a mixture of substrates, namely pyruvate, glutamate, and the tricarboxylic acid cycle metabolites. We studied how glutamate affected oxidative phosphorylation and reactive oxygen species (ROS) production in rat BM oxidizing pyruvate + malate or succinate. Simultaneous oxidation of glutamate + pyruvate + malate increased state 3 and uncoupled respiration by 52 and 71%, respectively. The state 4 ROS generation increased 100% over BM oxidizing pyruvate + malate and 900% over that of BM oxidizing glutamate + malate. Up to 70% of ROS generation was associated with reverse electron transport. These effects of pyruvate + glutamate + malate were observed only with BM and not with liver or heart mitochondria. The effects of glutamate + pyruvate on succinate-supported respiration and ROS generation were not organ-specific and depended only on whether mitochondria were isolated with or without bovine serum albumin. With the non-bovine serum albumin brain and heart mitochondria oxidizing succinate, the addition of pyruvate and glutamate abrogated inhibition of Complex II by oxaloacetate. We conclude that (i) during neuronal activation, simultaneous oxidation of glutamate + pyruvate temporarily enhances neuronal mitochondrial ATP production, and (ii) intrinsic inhibition of Complex II by oxaloacetate is an inherent mechanism that protects against ROS generation during reverse electron transport.Recently, it has emerged that mitochondrial dysfunctions play an important role in the pathogenesis of degenerative diseases of the central nervous system (1–3). The processes underlying neuronal degeneration are complex, and some authors suggest that several genetic alterations are involved (4). However, another level of complexity may be derived from the fact that virtually all cellular activities depend upon energy metabolism in the cell (5). Alterations in energy metabolism processes within cells may also contribute to pathogenic mechanisms underlying neurodegenerative disease.A large body of evidence suggests that increased oxidative stress is an important pathogenic mechanism that promotes neurodegeneration (6). Because neurons have a long life span, and most neurodegenerative diseases have a clear association with age (7), it is important to understand mechanisms underlying reactive oxygen species (ROS)² production in neurons. Recently, Kudin et al. (8) analyzed the contribution of mitochondria to the total ROS production in brain tissue. They concluded that mitochondria are the major source of ROS and that at least 50% of ROS generated by brain mitochondria was associated with succinate-supported reverse electron transport (RET). Under conditions of normoxia, about 1% of the respiratory chain electron flow was redirected to form superoxide (8).Recently, we suggested that the organization of the respiratory chain complexes into supercomplexes that occurs in brain mitochondria (BM) (9) may represent one of the intrinsic mechanisms to prevent excessive ROS generation (10). In this paper, we put forward the hypothesis that inhibition of Complex II by oxaloacetate (OAA) represents another important intrinsic mechanism to prevent oxidative stress. We provide evidence that glutamate and pyruvate specifically exert control over the production of ROS at the level of Complex II. Below we present a brief account of published theoretical and experimental evidence that underlie our hypothesis.The neural processing of information is metabolically expensive (11). More than 80% of energy is spent postsynaptically to restore the ionic composition of neurons (11). When neurons are activated, reuptake of glutamate stimulates aerobic glycolysis in astroglial cells (12), thereby making lactate the major substrate for neuronal mitochondria (4, 13). However, rapid conversion of lactate to pyruvate in neurons requires activation of the malate-aspartate shuttle (MAS). The shuttle is the major pathway for cytosolic reducing equivalents from NADH to enter the mitochondria and be oxidized (14, 15). The key component of MAS is the mitochondrial aspartate/glutamate carrier (AGC) (16), and recent data suggest that the AGC is expressed mainly in neurons (14). Absence of the AGC from astrocytes in the brain implies a compartmentation of intermediary metabolism, with glycolysis taking place in astrocytes and lactate oxidation in neurons (13, 14, 17). Active operation of MAS requires that a certain amount of glutamate must be transported from synaptic clefts into activated neurons. In isolated BM, it has been shown that besides pyruvate, glutamate is also a good respiratory substrate (5, 18). In the presynaptic elements, the concentration of cytosolic glutamate is ∼10 mm at all times (19). Yudkoff et al. (18) have shown that synaptosomal mitochondria utilize glutamate and pyruvate as mitochondrial respiratory substrates. Glutamate is also oxidized by the astroglial mitochondria (13).Until recently, it was generally accepted that most of the glutamate is rapidly removed from the synaptic cleft by glutamate transporters EAAT1 and EAAT2 located on presynaptic termini and glial cells (20–24). However, recent data show that a significant fraction of glutamate is rapidly bound and transported by the glutamate transporter isoform, EAAT4, located juxtasynaptically in the membranes of spines and dendrites (20, 25–28). At the climbing fiber to Purkinje cell synapses in the cerebellum, about 17% (28) or more than 50% (29) of synaptically released glutamate may be removed by postsynaptic transporters. Besides the cerebellum, EAAT4 protein was found to be omnipresent throughout the fore- and midbrain regions (30). Moreover, it was shown that although most of the EAAT2 protein is astroglial, around 15% is distributed in nerve terminals and axons in hippocampal slices and that this protein may be responsible for more than half of the total uptake of glutamate from synaptic clefts (24). These data suggest that postsynaptic transport of glutamate into nerve terminals where mitochondria are located (31) may occur in all brain regions. According to calculations of Brasnjo and Otis (28), in a single synapse, EAAT4 (excitatory amino acid transporter 4) binds and transports postsynaptically about 1.3 ± 0.1 × 10⁶ glutamate molecules. In the brain, on average, 1 mm³ of tissue contains 1 × 10⁸ synapses (32, 33). Because of the high density of synaptic contacts, the neuronal cells may be exposed to mediators released from hundreds of firing synapses. Thus, in a narrow space of spines and dendrites, several million glutamate molecules postsynaptically transported from synaptic boutons may create local cytosolic concentration of glutamate in the low millimolar range. Consequently, neuronal mitochondria, particularly those located at the axonal or dendritic synaptic junctions, may, in addition to metabolizing pyruvate, temporarily metabolize glutamate and succinate formed during mitochondrial catabolism of γ-aminobutyric acid in postsynaptic cells (34).The purpose of this study was to examine how the neuromediator glutamate affects respiratory activity and ROS generation in nonsynaptic BM when combined with pyruvate and the tricarboxylic acid cycle intermediates succinate and malate. We show that with pyruvate + glutamate + malate, the rate of oxidative phosphorylation increased more than 50%, and in resting mitochondria the rate of ROS generation associated with the reverse electron transport increased severalfold. These effects were observed only with brain and spinal cord mitochondria, not with liver or heart mitochondria, suggesting that they may be restricted to neuronal cells.Taken together, the data presented support the hypothesis that in activated neurons, the neuromediator glutamate stimulates mitochondrial ATP production when energy demand is increased. However, in the absence of energy consumption, glutamate + pyruvate may increase the generation of ROS severalfold. We suggest that intrinsic inhibition of Complex II by oxaloacetate is an important natural protective mechanism against ROS associated with reverse electron transport.     

Glutathione Participates in the Regulation of Mitophagy in Yeast

The antioxidant N-acetyl-l-cysteine prevented the autophagy-dependent delivery of mitochondria to the vacuoles, as examined by fluorescence microscopy of mitochondria-targeted green fluorescent protein, transmission electron microscopy, and Western blot analysis of mitochondrial proteins. The effect of N-acetyl-l-cysteine was specific to mitochondrial autophagy (mitophagy). Indeed, autophagy-dependent activation of alkaline phosphatase and the presence of hallmarks of non-selective microautophagy were not altered by N-acetyl-l-cysteine. The effect of N-acetyl-l-cysteine was not related to its scavenging properties, but rather to its fueling effect of the glutathione pool. As a matter of fact, the decrease of the glutathione pool induced by chemical or genetical manipulation did stimulate mitophagy but not general autophagy. Conversely, the addition of a cell-permeable form of glutathione inhibited mitophagy. Inhibition of glutathione synthesis had no effect in the strain Δuth1, which is deficient in selective mitochondrial degradation. These data show that mitophagy can be regulated independently of general autophagy, and that its implementation may depend on the cellular redox status.Autophagy is a major pathway for the lysosomal/vacuolar delivery of long-lived proteins and organelles, where they are degraded and recycled. Autophagy plays a crucial role in differentiation and cellular response to stress and is conserved in eukaryotic cells from yeast to mammals (1, 2). The main form of autophagy, macroautophagy, involves the non-selective sequestration of large portions of the cytoplasm into double-membrane structures termed autophagosomes, and their delivery to the vacuole/lysosome for degradation. Another process, microautophagy, involves the direct sequestration of parts of the cytoplasm by vacuole/lysosomes. The two processes coexist in yeast cells but their extent may depend on different factors including metabolic state: for example, we have observed that nitrogen-starved lactate-grown yeast cells develop microautophagy, whereas nitrogen-starved glucose-grown cells preferentially develop macroautophagy (3).Both macroautophagy and microautophagy are essentially non-selective, in the way that autophagosomes and vacuole invaginations do not appear to discriminate the sequestered material. However, selective forms of autophagy have been observed (4) that target namely peroxisomes (5, 6), chromatin (7, 8), endoplasmic reticulum (9), ribosomes (10), and mitochondria (3, 11–13). Although non-selective autophagy plays an essential role in survival by nitrogen starvation, by providing amino acids to the cell, selective autophagy is more likely to have a function in the maintenance of cellular structures, both under normal conditions as a “housecleaning” process, and under stress conditions by eliminating altered organelles and macromolecular structures (14–16). Selective autophagy targeting mitochondria, termed mitophagy, may be particularly relevant to stress conditions. The mitochondrial respiratory chain is both the main site and target of ROS⁴ production (17). Consequently, the maintenance of a pool of healthy mitochondria is a crucial challenge for the cells. The progressive accumulation of altered mitochondria (18) caused by the loss of efficiency of the maintenance process (degradation/biogenesis de novo) is often considered as a major cause of cellular aging (19–23). In mammalian cells, autophagic removal of mitochondria has been shown to be triggered following induction/blockade of apoptosis (23), suggesting that autophagy of mitochondria was required for cell survival following mitochondria injury (14). Consistent with this idea, a direct alteration of mitochondrial permeability properties has been shown to induce mitochondrial autophagy (13, 24, 25). Furthermore, inactivation of catalase induced the autophagic elimination of altered mitochondria (26). In the yeast Saccharomyces cerevisiae, the alteration of F₀F₁-ATPase biogenesis in a conditional mutant has been shown to trigger autophagy (27). Alterations of mitochondrial ion homeostasis caused by the inactivation of the K⁺/H⁺ exchanger was shown to cause both autophagy and mitophagy (28). We have reported that treatment of cells with rapamycin induced early ROS production and mitochondrial lipid oxidation that could be inhibited by the hydrophobic antioxidant resveratrol (29). Furthermore, resveratrol treatment impaired autophagic degradation of both cytosolic and mitochondrial proteins and delayed rapamycin-induced cell death, suggesting that mitochondrial oxidation events may play a crucial role in the regulation of autophagy. This existence of regulation of autophagy by ROS has received molecular support in HeLa cells (30): these authors showed that starvation stimulated ROS production, namely H₂O₂, which was essential for autophagy. Furthermore, they identified the cysteine protease hsAtg4 as a direct target for oxidation by H₂O₂. This provided a possible connection between the mitochondrial status and regulation of autophagy.Investigations of mitochondrial autophagy in nitrogen-starved lactate-grown yeast cells have established the existence of two distinct processes: the first one occurring very early, is selective for mitochondria and is dependent on the presence of the mitochondrial protein Uth1p; the second one occurring later, is not selective for mitochondria, is not dependent on Uth1p, and is a form of bulk microautophagy (3). The absence of the selective process in the Δuth1 mutant strongly delays and decreases mitochondrial protein degradation (3, 12). The putative protein phosphatase Aup1p has been also shown to be essential in inducing mitophagy (31). Additionally several Atg proteins were shown to be involved in vacuolar sequestration of mitochondrial GFP (3, 12, 32, 33). Recently, the protein Atg11p, which had been already identified as an essential protein for selective autophagy has also been reported as being essential for mitophagy (33).The question remains as to identify of the signals that trigger selective mitophagy. It is particularly intriguing that selective mitophagy is activated very early after the shift to a nitrogen-deprived medium (3). Furthermore, selective mitophagy is very active on lactate-grown cells (with fully differentiated mitochondria) but is nearly absent in glucose-grown cells (3). In the present paper, we investigated the relationships between the redox status of the cells and selective mitophagy, namely by manipulating glutathione. Our results support the view that redox imbalance is a trigger for the selective elimination of mitochondria.     

Galectin-8 Induces Apoptosis in Jurkat T Cells by Phosphatidic Acid-mediated ERK1/2 Activation Supported by Protein Kinase A Down-regulation

Galectins have been implicated in T cell homeostasis playing complementary pro-apoptotic roles. Here we show that galectin-8 (Gal-8) is a potent pro-apoptotic agent in Jurkat T cells inducing a complex phospholipase D/phosphatidic acid signaling pathway that has not been reported for any galectin before. Gal-8 increases phosphatidic signaling, which enhances the activity of both ERK1/2 and type 4 phosphodiesterases (PDE4), with a subsequent decrease in basal protein kinase A activity. Strikingly, rolipram inhibition of PDE4 decreases ERK1/2 activity. Thus Gal-8-induced PDE4 activation releases a negative influence of cAMP/protein kinase A on ERK1/2. The resulting strong ERK1/2 activation leads to expression of the death factor Fas ligand and caspase-mediated apoptosis. Several conditions that decrease ERK1/2 activity also decrease apoptosis, such as anti-Fas ligand blocking antibodies. In addition, experiments with freshly isolated human peripheral blood mononuclear cells, previously stimulated with anti-CD3 and anti-CD28, show that Gal-8 is pro-apoptotic on activated T cells, most likely on a subpopulation of them. Anti-Gal-8 autoantibodies from patients with systemic lupus erythematosus block the apoptotic effect of Gal-8. These results implicate Gal-8 as a novel T cell suppressive factor, which can be counterbalanced by function-blocking autoantibodies in autoimmunity.Glycan-binding proteins of the galectin family have been increasingly studied as regulators of the immune response and potential therapeutic agents for autoimmune disorders (1). To date, 15 galectins have been identified and classified according with the structural organization of their distinctive monomeric or dimeric carbohydrate recognition domain for β-galactosides (2, 3). Galectins are secreted by unconventional mechanisms and once outside the cells bind to and cross-link multiple glycoconjugates both at the cell surface and at the extracellular matrix, modulating processes as diverse as cell adhesion, migration, proliferation, differentiation, and apoptosis (4–10). Several galectins have been involved in T cell homeostasis because of their capability to kill thymocytes, activated T cells, and T cell lines (11–16). Pro-apoptotic galectins might contribute to shape the T cell repertoire in the thymus by negative selection, restrict the immune response by eliminating activated T cells at the periphery (1), and help cancer cells to escape the immune system by eliminating cancer-infiltrating T cells (17). They have also a promising therapeutic potential to eliminate abnormally activated T cells and inflammatory cells (1). Studies on the mostly explored galectins, Gal-1, -3, and -9 (14, 15, 18–20), as well as in Gal-2 (13), suggest immunosuppressive complementary roles inducing different pathways to apoptosis. Galectin-8 (Gal-8)⁴ is one of the most widely expressed galectins in human tissues (21, 22) and cancerous cells (23, 24). Depending on the cell context and mode of presentation, either as soluble stimulus or extracellular matrix, Gal-8 can promote cell adhesion, spreading, growth, and apoptosis (6, 7, 9, 10, 22, 25). Its role has been mostly studied in relation to tumor malignancy (23, 24). However, there is some evidence regarding a role for Gal-8 in T cell homeostasis and autoimmune or inflammatory disorders. For instance, the intrathymic expression and pro-apoptotic effect of Gal-8 upon CD4^highCD8^high thymocytes suggest a role for Gal-8 in shaping the T cell repertoire (16). Gal-8 could also modulate the inflammatory function of neutrophils (26), Moreover Gal-8-blocking agents have been detected in chronic autoimmune disorders (10, 27, 28). In rheumatoid arthritis, Gal-8 has an anti-inflammatory action, promoting apoptosis of synovial fluid cells, but can be counteracted by a specific rheumatoid version of CD44 (CD44vRA) (27). In systemic lupus erythematosus (SLE), a prototypic autoimmune disease, we recently described function-blocking autoantibodies against Gal-8 (10, 28). Thus it is important to define the role of Gal-8 and the influence of anti-Gal-8 autoantibodies in immune cells.In Jurkat T cells, we previously reported that Gal-8 interacts with specific integrins, such as α1β1, α3β1, and α5β1 but not α4β1, and as a matrix protein promotes cell adhesion and asymmetric spreading through activation of the extracellular signal-regulated kinases 1 and 2 (ERK1/2) (10). These early effects occur within 5–30 min. However, ERK1/2 signaling supports long term processes such as T cell survival or death, depending on the moment of the immune response. During T cell activation, ERK1/2 contributes to enhance the expression of interleukin-2 (IL-2) required for T cell clonal expansion (29). It also supports T cell survival against pro-apoptotic Fas ligand (FasL) produced by themselves and by other previously activated T cells (30, 31). Later on, ERK1/2 is required for activation-induced cell death, which controls the extension of the immune response by eliminating recently activated and restimulated T cells (32, 33). In activation-induced cell death, ERK1/2 signaling contributes to enhance the expression of FasL and its receptor Fas/CD95 (32, 33), which constitute a preponderant pro-apoptotic system in T cells (34). Here, we ask whether Gal-8 is able to modulate the intensity of ERK1/2 signaling enough to participate in long term processes involved in T cell homeostasis.The functional integration of ERK1/2 and PKA signaling (35) deserves special attention. cAMP/PKA signaling plays an immunosuppressive role in T cells (36) and is altered in SLE (37). Phosphodiesterases (PDEs) that degrade cAMP release the immunosuppressive action of cAMP/PKA during T cell activation (38, 39). PKA has been described to control the activity of ERK1/2 either positively or negatively in different cells and processes (35). A little explored integration among ERK1/2 and PKA occurs via phosphatidic acid (PA) and PDE signaling. Several stimuli activate phospholipase D (PLD) that hydrolyzes phosphatidylcholine into PA and choline. Such PLD-generated PA plays roles in signaling interacting with a variety of targeting proteins that bear PA-binding domains (40). In this way PA recruits Raf-1 to the plasma membrane (41). It is also converted by phosphatidic acid phosphohydrolase (PAP) activity into diacylglycerol (DAG), which among other functions, recruits and activates the GTPase Ras (42). Both Ras and Raf-1 are upstream elements of the ERK1/2 activation pathway (43). In addition, PA binds to and activates PDEs of the type 4 subfamily (PDE4s) leading to decreased cAMP levels and PKA down-regulation (44). The regulation and role of PA-mediated control of ERK1/2 and PKA remain relatively unknown in T cell homeostasis, because it is also unknown whether galectins stimulate the PLD/PA pathway.Here we found that Gal-8 induces apoptosis in Jurkat T cells by triggering cross-talk between PKA and ERK1/2 pathways mediated by PLD-generated PA. Our results for the first time show that a galectin increases the PA levels, down-regulates the cAMP/PKA system by enhancing rolipram-sensitive PDE activity, and induces an ERK1/2-dependent expression of the pro-apoptotic factor FasL. The enhanced PDE activity induced by Gal-8 is required for the activation of ERK1/2 that finally leads to apoptosis. Gal-8 also induces apoptosis in human peripheral blood mononuclear cells (PBMC), especially after activating T cells with anti-CD3/CD28. Therefore, Gal-8 shares with other galectins the property of killing activated T cells contributing to the T cell homeostasis. The pathway involves a particularly integrated signaling context, engaging PLD/PA, cAMP/PKA, and ERK1/2, which so far has not been reported for galectins. The pro-apoptotic function of Gal-8 also seems to be unique in its susceptibility to inhibition by anti-Gal-8 autoantibodies.     

Mechanism of Sustained Activation of Ribosomal S6 Kinase (RSK) and ERK by Kaposi Sarcoma-associated Herpesvirus ORF45: MULTIPROTEIN COMPLEXES RETAIN ACTIVE PHOSPHORYLATED ERK AND RSK AND PROTECT THEM FROM DEPHOSPHORYLATION*

As obligate intracellular parasites, viruses exploit diverse cellular signaling machineries, including the mitogen-activated protein-kinase pathway, during their infections. We have demonstrated previously that the open reading frame 45 (ORF45) of Kaposi sarcoma-associated herpesvirus interacts with p90 ribosomal S6 kinases (RSKs) and strongly stimulates their kinase activities (Kuang, E., Tang, Q., Maul, G. G., and Zhu, F. (2008) J. Virol. 82 ,1838 -1850). Here, we define the mechanism by which ORF45 activates RSKs. We demonstrated that binding of ORF45 to RSK increases the association of extracellular signal-regulated kinase (ERK) with RSK, such that ORF45, RSK, and ERK formed high molecular mass protein complexes. We further demonstrated that the complexes shielded active pERK and pRSK from dephosphorylation. As a result, the complex-associated RSK and ERK were activated and sustained at high levels. Finally, we provide evidence that this mechanism contributes to the sustained activation of ERK and RSK in Kaposi sarcoma-associated herpesvirus lytic replication.The extracellular signal-regulated kinase (ERK)² mitogen-activated protein kinase (MAPK) signaling pathway has been implicated in diverse cellular physiological processes including proliferation, survival, growth, differentiation, and motility (1-4) and is also exploited by a variety of viruses such as Kaposi sarcoma-associated herpesvirus (KSHV), human cytomegalovirus, human immunodeficiency virus, respiratory syncytial virus, hepatitis B virus, coxsackie, vaccinia, coronavirus, and influenza virus (5-17). The MAPK kinases relay the extracellular signaling through sequential phosphorylation to an array of cytoplasmic and nuclear substrates to elicit specific responses (1, 2, 18). Phosphorylation of MAPK is reversible. The kinetics of deactivation or duration of signaling dictates diverse biological outcomes (19, 20). For example, sustained but not transient activation of ERK signaling induces the differentiation of PC12 cells into sympathetic-like neurons and transformation of NIH3T3 cells (20-22). During viral infection, a unique biphasic ERK activation has been observed for some viruses (an early transient activation triggered by viral binding or entry and a late sustained activation correlated with viral gene expression), but the responsible viral factors and underlying mechanism for the sustained ERK activation remain largely unknown (5, 8, 13, 23).The p90 ribosomal S6 kinases (RSKs) are a family of serine/threonine kinases that lie at the terminus of the ERK pathway (1, 24-26). In mammals, four isoforms are known, RSK1 to RSK4. Each one has two catalytically functional kinase domains, the N-terminal kinase domain (NTKD) and C-terminal kinase domain (CTKD) as well as a linker region between the two. The NTKD is responsible for phosphorylation of exogenous substrates, and the CTKD and linker region regulate RSK activation (1, 24, 25). In quiescent cells ERK binds to the docking site in the C terminus of RSK (27-29). Upon mitogen stimulation, ERK is activated by its upstream MAPK/ERK kinase (MEK). The active ERK phosphorylates Thr-359/Ser-363 of RSK in the linker region (amino acid numbers refer to human RSK1) and Thr-573 in the CTKD activation loop. The activated CTKD then phosphorylates Ser-380 in the linker region, creating a docking site for 3-phosphoinositide-dependent protein kinase-1. The 3-phosphoinositide-dependent protein kinase-1 phosphorylates Ser-221 of RSK in the activation loop and activates the NTKD. The activated NTKD autophosphorylates the serine residue near the ERK docking site, causing a transient dissociation of active ERK from RSK (25, 26, 28). The stimulation of quiescent cells by a mitogen such as epidermal growth factor or a phorbol ester such as 12-O-tetradecanoylphorbol-13-acetate (TPA) usually results in a transient RSK activation that lasts less than 30 min. RSKs have been implicated in regulating cell survival, growth, and proliferation. Mutation or aberrant expression of RSK has been implicated in several human diseases including Coffin-Lowry syndrome and prostate and breast cancers (1, 24, 25, 30-32).KSHV is a human DNA tumor virus etiologically linked to Kaposi sarcoma, primary effusion lymphoma, and a subset of multicentric Castleman disease (33, 34). Infection and reactivation of KSHV activate multiple MAPK pathways (6, 12, 35). Noticeably, the ERK/RSK activation is sustained late during KSHV primary infection and reactivation from latency (5, 6, 12, 23), but the mechanism of the sustained ERK/RSK activation is unclear. Recently, we demonstrated that ORF45, an immediate early and also virion tegument protein of KSHV, interacts with RSK1 and RSK2 and strongly stimulates their kinase activities (23). We also demonstrated that the activation of RSK plays an essential role in KSHV lytic replication (23). In the present study we determined the mechanism of ORF45-induced sustained ERK/RSK activation. We found that ORF45 increases the association of RSK with ERK and protects them from dephosphorylation, causing sustained activation of both ERK and RSK.     

Ligand Reduces Galectin-1 Sensitivity to Oxidative Inactivation by Enhancing Dimer Formation

Galectin-1 (Gal-1) regulates leukocyte turnover by inducing the cell surface exposure of phosphatidylserine (PS), a ligand that targets cells for phagocytic removal, in the absence of apoptosis. Gal-1 monomer-dimer equilibrium appears to modulate Gal-1-induced PS exposure, although the mechanism underlying this regulation remains unclear. Here we show that monomer-dimer equilibrium regulates Gal-1 sensitivity to oxidation. A mutant form of Gal-1, containing C2S and V5D mutations (mGal-1), exhibits impaired dimerization and fails to induce cell surface PS exposure while retaining the ability to recognize carbohydrates and signal Ca²⁺ flux in leukocytes. mGal-1 also displayed enhanced sensitivity to oxidation, whereas ligand, which partially protected Gal-1 from oxidation, enhanced Gal-1 dimerization. Continual incubation of leukocytes with Gal-1 resulted in gradual oxidative inactivation with concomitant loss of cell surface PS, whereas rapid oxidation prevented mGal-1 from inducing PS exposure. Stabilization of Gal-1 or mGal-1 with iodoacetamide fully protected Gal-1 and mGal-1 from oxidation. Alkylation-induced stabilization allowed Gal-1 to signal sustained PS exposure in leukocytes and mGal-1 to signal both Ca²⁺ flux and PS exposure. Taken together, these results demonstrate that monomer-dimer equilibrium regulates Gal-1 sensitivity to oxidative inactivation and provides a mechanism whereby ligand partially protects Gal-1 from oxidation.Immunological homeostasis relies on efficient contraction of activated leukocytes following an inflammatory episode. Several factors, including members of the galectin and tumor necrosis factor families (1, 2), regulate leukocyte turnover by inducing apoptotic cell death. In contrast, several galectin family members, in particular galectin-1 (Gal-1),² uniquely regulate neutrophil turnover by inducing phosphatidylserine (PS) exposure, which normally sensitizes apoptotic cells to phagocytic removal (3, 4), independent of apoptosis, a process recently termed preaparesis (5).Previous studies suggested that dimerization may be required for Gal-1-induced PS exposure, as a mutant form of Gal-1 (mGal-1) containing two point mutations within the dimer interface, C2S and V5D (C2S,V5D), displays impaired Gal-1 dimerization and fails to induce PS exposure (6). However, the manner in which monomer-dimer equilibrium regulates Gal-1 signaling remains unclear. Previous studies suggest that dimerization may be required for efficient cross-linking of functional receptors or the formation of signaling lattices (7–9). Consistent with this, monomeric mutants of several other galectins fail to induce PS exposure or signal leukocytes (4, 8). Gal-1 signaling of PS exposure requires initial signaling events, such as mobilization of intracellular Ca²⁺ followed by sustained receptor engagement (10). Although mGal-1 fails to induce PS exposure (6), whether mGal-1 can induce these initial signaling events remains unknown (10).In addition to directly regulating signaling, monomer-dimer equilibrium may also regulate other aspects of Gal-1 function. Unlike many other proteins involved in the regulation of immunity, Gal-1 displays unique sensitivity to oxidative inactivation (11–15). Although engagement of ligand partially protects Gal-1 from oxidation (15), the impact of Gal-1 oxidation on signaling remains enigmatic. During oxidation, Gal-1 forms three distinct intramolecular disulfide bridges that facilitate profound conformational changes that preclude ligand binding and Gal-1 dimerization (12–14), suggesting that monomerdimer equilibrium may also regulate Gal-1 sensitivity to oxidative inactivation.Previous studies utilized dithiothreitol (DTT) in treatment conditions to protect Gal-1 from oxidative inactivation (16, 17). Indeed, failure to include DTT precluded Gal-1-induced death in T cells (3, 18), suggesting that Gal-1 undergoes rapid oxidation in vivo in the absence of reducing conditions. However, DTT itself can induce apoptosis in leukocytes (19), leaving questions regarding the impact of Gal-1 oxidation on these signaling events. In contrast, recent studies utilizing iodoacetamide-alkylated Gal-1 (iGal-1), previously shown to protect Gal-1 from oxidative inactivation (20–29), demonstrated that DTT actually primes cells to become sensitive to Gal-1-induced apoptosis regardless of Gal-1 sensitivity to oxidation (5).As the engagement of leukocyte ligands requires glycan recognition and oxidation precludes this binding (11, 15), understanding the impact of oxidation on Gal-1 signals will facilitate a greater appreciation of the factors that govern Gal-1 oxidation and therefore function. Our results demonstrate that Gal-1 monomer-dimer equilibrium provides a key regulatory point controlling both Gal-1 sensitivity to oxidation and its ability to signal PS exposure in leukocytes. These results provide novel insights into Gal-1 function and explain at a biochemical level the mechanisms regulating Gal-1 oxidative inactivation and signaling.     

Intersectin Regulates Dendritic Spine Development and Somatodendritic Endocytosis but Not Synaptic Vesicle Recycling in Hippocampal Neurons

Intersectin-short (intersectin-s) is a multimodule scaffolding protein functioning in constitutive and regulated forms of endocytosis in non-neuronal cells and in synaptic vesicle (SV) recycling at the neuromuscular junction of Drosophila and Caenorhabditis elegans. In vertebrates, alternative splicing generates a second isoform, intersectin-long (intersectin-l), that contains additional modular domains providing a guanine nucleotide exchange factor activity for Cdc42. In mammals, intersectin-s is expressed in multiple tissues and cells, including glia, but excluded from neurons, whereas intersectin-l is a neuron-specific isoform. Thus, intersectin-I may regulate multiple forms of endocytosis in mammalian neurons, including SV endocytosis. We now report, however, that intersectin-l is localized to somatodendritic regions of cultured hippocampal neurons, with some juxtanuclear accumulation, but is excluded from synaptophysin-labeled axon terminals. Consistently, intersectin-l knockdown (KD) does not affect SV recycling. Instead intersectin-l co-localizes with clathrin heavy chain and adaptor protein 2 in the somatodendritic region of neurons, and its KD reduces the rate of transferrin endocytosis. The protein also co-localizes with F-actin at dendritic spines, and intersectin-l KD disrupts spine maturation during development. Our data indicate that intersectin-l is indeed an important regulator of constitutive endocytosis and neuronal development but that it is not a prominent player in the regulated endocytosis of SVs.Clathrin-mediated endocytosis (CME)⁴ is a major mechanism by which cells take up nutrients, control the surface levels of multiple proteins, including ion channels and transporters, and regulate the coupling of signaling receptors to downstream signaling cascades (1-5). In neurons, CME takes on additional specialized roles; it is an important process regulating synaptic vesicle (SV) availability through endocytosis and recycling of SV membranes (6, 7), it shapes synaptic plasticity (8-10), and it is crucial in maintaining synaptic membranes and membrane structure (11).Numerous endocytic accessory proteins participate in CME, interacting with each other and with core components of the endocytic machinery such as clathrin heavy chain (CHC) and adaptor protein-2 (AP-2) through specific modules and peptide motifs (12). One such module is the Eps15 homology domain that binds to proteins bearing NPF motifs (13, 14). Another is the Src homology 3 (SH3) domain, which binds to proline-rich domains in protein partners (15). Intersectin is a multimodule scaffolding protein that interacts with a wide range of proteins, including several involved in CME (16). Intersectin has two N-terminal Eps15 homology domains that are responsible for binding to epsin, SCAMP1, and numb (17-19), a central coil-coiled domain that interacts with Eps15 and SNAP-23 and -25 (17, 20, 21), and five SH3 domains in its C-terminal region that interact with multiple proline-rich domain proteins, including synaptojanin, dynamin, N-WASP, CdGAP, and mSOS (16, 22-25). The rich binding capability of intersectin has linked it to various functions from CME (17, 26, 27) and signaling (22, 28, 29) to mitogenesis (30, 31) and regulation of the actin cytoskeleton (23).Intersectin functions in SV recycling at the neuromuscular junction of Drosophila and C. elegans where it acts as a scaffold, regulating the synaptic levels of endocytic accessory proteins (21, 32-34). In vertebrates, the intersectin gene is subject to alternative splicing, and a longer isoform (intersectin-l) is generated that is expressed exclusively in neurons (26, 28, 35, 36). This isoform has all the binding modules of its short (intersectin-s) counterpart but also has additional domains: a DH and a PH domain that provide guanine nucleotide exchange factor (GEF) activity specific for Cdc42 (23, 37) and a C2 domain at the C terminus. Through its GEF activity and binding to actin regulatory proteins, including N-WASP, intersectin-l has been implicated in actin regulation and the development of dendritic spines (19, 23, 24). In addition, because the rest of the binding modules are shared between intersectin-s and -l, it is generally thought that the two intersectin isoforms have the same endocytic functions. In particular, given the well defined role for the invertebrate orthologs of intersectin-s in SV endocytosis, it is thought that intersectin-l performs this role in mammalian neurons, which lack intersectin-s. Defining the complement of intersectin functional activities in mammalian neurons is particularly relevant given that the protein is involved in the pathophysiology of Down syndrome (DS). Specifically, the intersectin gene is localized on chromosome 21q22.2 and is overexpressed in DS brains (38). Interestingly, alterations in endosomal pathways are a hallmark of DS neurons and neurons from the partial trisomy 16 mouse, Ts65Dn, a model for DS (39, 40). Thus, an endocytic trafficking defect may contribute to the DS disease process.Here, the functional roles of intersectin-l were studied in cultured hippocampal neurons. We find that intersectin-l is localized to the somatodendritic regions of neurons, where it co-localizes with CHC and AP-2 and regulates the uptake of transferrin. Intersectin-l also co-localizes with actin at dendritic spines and disrupting intersectin-l function alters dendritic spine development. In contrast, intersectin-l is absent from presynaptic terminals and has little or no role in SV recycling.     

Nuclear Import Is Required for the Pro-apoptotic Function of the Golgi Protein p115

During apoptosis the Golgi apparatus undergoes irreversible fragmentation. In part, this results from caspase-mediated cleavage of several high molecular weight coiled-coil proteins, termed golgins. These include GM130, golgin 160, and the Golgi vesicle tethering protein p115, whose caspase cleavage generates a C-terminal fragment (CTF) of 205 residues. Here we demonstrate that early during apoptosis, following the rapid cleavage of p115, endogenous CTF translocated to the cell nucleus and its nuclear import was required to enhance the apoptotic response. Expression of a series of deletion constructs identified a putative α-helical region of 26 amino acids, whose expression alone was sufficient to induce apoptosis; deletion of these 26 residues from the CTF diminished its proapoptotic activity. This region contains several potential SUMOylation sites and co-expression of SUMO together with the SUMO ligase, UBC9, resulted in SUMOylation of the p115 CTF. Significantly, when cells were treated with drugs that induce apoptosis, SUMOylation enhanced the efficiency of p115 cleavage and the kinetics of apoptosis. A construct in which a nuclear export signal was fused to the N terminus of p115 CTF accumulated in the cytoplasm and surprisingly, its expression did not induce apoptosis. In contrast, treatment of cells expressing this chimera with the antibiotic leptomycin induced its translocation into the nucleus and resulted in the concomitant induction of apoptosis. These results demonstrate that nuclear import of the p115 CTF is required for it to stimulate the apoptotic response and suggest that its mode of action is confined to the nucleus.In mammalian cells the Golgi apparatus is a highly polarized organelle comprising a series of stacked cisternae, which form a lace-like network in the perinuclear region of the cell. It receives de novo synthesized secretory and membrane proteins, as well as lipids from the endoplasmic reticulum (ER)²; these cargo molecules are then modified, sorted, and transported to lysosomes, endosomes, secretory granules, and the plasma membrane. Although it is well established that the Golgi apparatus undergoes reversible disassembly during mitosis (1, 2), indeed this appears to be a prerequisite for mitosis (3), studies from several laboratories including our own, have also established a link between the Golgi apparatus and apoptosis (programmed cell death). During apoptosis, the Golgi apparatus undergoes extensive and irreversible fragmentation (4), the ER vesiculates (5) and secretion is inhibited (6).Golgi disassembly during apoptosis results, in part, from caspase-mediated cleavage of several golgins (7). Proteolysis of golgin 160 by caspase-2, as well as GRASP65, GM130, p115, syntaxin5, and giantin by caspases-3 and -7 contributes significantly to Golgi fragmentation (6, 8–13). Consistent with this idea, overexpression of caspase-resistant forms of golgin 160, GRASP65, or p115 has been shown to delay the kinetics of Golgi fragmentation during apoptosis (8–10). In addition, immunoreactive caspase-2, an upstream caspase, localizes to the Golgi apparatus (9) and caspase-2-mediated cleavage of golgin 160 also appears to be an early event during apoptosis. Depending on the apoptotic stimulus, expression of a golgin 160 triple mutant resistant to caspase cleavage delays the onset of apoptosis (12). Recently, our laboratory demonstrated that Golgi fragmentation is an early apoptotic event that occurs close to or soon after release of cytochrome c from mitochondria, an early indicator of apoptosis (13). Together these observations demonstrate that specific Golgi proteins may function early during apoptosis, although their role in this process and the detailed molecular mechanism by which Golgi fragmentation occurs is not well understood.A key molecule in mediating Golgi fragmentation during apoptosis is the vesicle tethering protein p115 (10), a 962-residue peripheral membrane protein. p115 is an elongated homodimer consisting of two globular “head” domains, an extended “tail” region reminiscent of the myosin-II structure (14), and 4 sequential coil-coil domains distal to the globular head region, the first of which, CC1, has been implicated in soluble NSF attachment protein receptors (SNARE) binding (15). Earlier in vitro studies on mitotic Golgi reassembly demonstrated that p115 interacts with GM130 and giantin and implicated it in Golgi cisternal stacking (16). Consistent with this idea, microinjection of anti-p115 antibodies caused Golgi fragmentation (17). Based on data demonstrating p115 binding to GM130, giantin, GOS28, and syntaxin-5, Shorter et al. (15) suggested that p115 promotes formation of a GOS28-syntaxin-5 (v-/t-SNARE) complex and hypothesized that it coordinates the sequential tethering and docking of COPI vesicles to Golgi membranes. Interestingly, p115 has also been shown to be a Rab-1 effector that binds Rab-1-GTP directly and cross-linking experiments showed that it interacts with Syntaxin5, sly1, membrin, and rbet1 on microsomal membranes and COPII vesicles suggesting that p115-SNARE interactions may facilitate membrane “docking” (18).More recent in vivo studies showed that inhibition of GM130 or giantin binding to p115 had little effect on Golgi morphology or reassembly following mitosis, suggesting its role in maintaining Golgi structure might be independent of GM130 binding (19, 20). Thus post-mitotic Golgi reassembly could be rescued by p115 lacking the C-terminal GM130 binding motif (residues 935–962) but not by a mutant lacking the SNARE interacting CC1 domain (20). In addition, other studies have implicated GM130 and GRASP65 in Golgi ribbon formation and suggested that this may occur independently of interactions with p115 (21). Most significantly, knockdown of p115 using siRNA demonstrated that it is essential for maintaining Golgi structure, compartmentalization, and cargo traffic to the plasma membrane (20, 22).Earlier work from our laboratory demonstrated that p115 is cleaved in vitro by caspase-8, an initiator caspase, as well as by the executioner caspase-3 (10, 13). In response to apoptosis inducing drugs, p115 is cleaved in vivo at Asp⁷⁵⁷ to generate a 205-residue C-terminal fragment and an N-terminal polypeptide of 757 amino acids. Most significantly, expression of the p115 C-terminal fragment in otherwise healthy cells results in its translocation to the nucleus and the induction of apoptosis suggesting that this polypeptide plays a role in potentiating the apoptotic response. To further dissect p115 function during cell death, we have now determined the minimal domain in its C terminus that mediates apoptosis efficiently and analyzed the requirement of nuclear translocation in triggering the apoptotic response.     

Role of JNK1-dependent Bcl-2 Phosphorylation in Ceramide-induced Macroautophagy

Macroautophagy is a vacuolar lysosomal catabolic pathway that is stimulated during periods of nutrient starvation to preserve cell integrity. Ceramide is a bioactive sphingolipid associated with a large range of cell processes. Here we show that short-chain ceramides (C₂-ceramide and C₆-ceramide) and stimulation of the de novo ceramide synthesis by tamoxifen induce the dissociation of the complex formed between the autophagy protein Beclin 1 and the anti-apoptotic protein Bcl-2. This dissociation is required for macroautophagy to be induced either in response to ceramide or to starvation. Three potential phosphorylation sites, Thr⁶⁹, Ser⁷⁰, and Ser⁸⁷, located in the non-structural N-terminal loop of Bcl-2, play major roles in the dissociation of Bcl-2 from Beclin 1. We further show that activation of c-Jun N-terminal protein kinase 1 by ceramide is required both to phosphorylate Bcl-2 and to stimulate macroautophagy. These findings reveal a new aspect of sphingolipid signaling in up-regulating a major cell process involved in cell adaptation to stress.Macroautophagy (referred to below as “autophagy”) is a vacuolar, lysosomal degradation pathway for cytoplasmic constituents that is conserved in eukaryotic cells (1–3). Autophagy is initiated by the formation of a multimembrane-bound autophagosome that engulfs cytoplasmic proteins and organelles. The last stage in the process results in fusion with the lysosomal compartments, where the autophagic cargo undergoes degradation. Basal autophagy is important in controlling the quality of the cytoplasm by removing damaged organelles and protein aggregates. Inhibition of basal autophagy in the brain is deleterious, and leads to neurodegeneration in mouse models (4, 5). Stimulation of autophagy during periods of nutrient starvation is a physiological response present at birth and has been shown to provide energy in various tissues of newborn pups (6). In cultured cells, starvation-induced autophagy is an autonomous cell survival mechanism, which provides nutrients to maintain a metabolic rate and level of ATP compatible with cell survival (7). In addition, starvation-induced autophagy blocks the induction of apoptosis (8). In other contexts, such as drug treatment and a hypoxic environment, autophagy has also been shown to be cytoprotective in cancer cells (9, 10). However, autophagy is also part of cell death pathways in certain situations (11). Autophagy can be a player in apoptosis-independent type-2 cell death (type-1 cell death is apoptosis), also known as autophagic cell death. This situation has been shown to occur when the apoptotic machinery is crippled in mammalian cells (12, 13). Autophagy can also be part of the apoptotic program, for instance in tumor necrosis factor-α-induced cell death when NF-κB is inhibited (14), or in human immunodeficiency virus envelope-mediated cell death in bystander naive CD4 T cells (15). Moreover autophagy has recently been shown to be required for the externalization of phosphatidylserine, the eat-me signal for phagocytic cells, at the surface of apoptotic cells (16).The complex relationship between autophagy and apoptosis reflects the intertwined regulation of these processes (17, 18). Many signaling pathways involved in the regulation of autophagy also regulate apoptosis. This intertwining has recently been shown to occur at the level of the molecular machinery of autophagy. In fact the anti-apoptotic protein Bcl-2 has been shown to inhibit starvation-induced autophagy by interacting with the autophagy protein Beclin 1 (19). Beclin 1 is one of the Atg proteins conserved from yeast to humans (it is the mammalian orthologue of yeast Atg6) and is involved in autophagosome formation (20). Beclin 1 is a platform protein that interacts with several different partners, including hVps34 (class III phosphatidylinositol 3-kinase), which is responsible for the synthesis of phosphatidylinositol 3-phosphate. The production of this lipid is important for events associated with the nucleation of the isolation membrane before it elongates and closes to form autophagosomes in response to other Atg proteins, including the Atg12 and LC3² (microtubule-associated protein light chain 3 is the mammalian orthologue of the yeast Atg8) ubiquitin-like conjugation systems (3, 21). Various partners associated with the Beclin 1 complex modulate the activity of hVps34. For instance, Bcl-2 inhibits the activity of this enzyme, whereas UVRAG, Ambra-1, and Bif-1 all up-regulate it (22, 23).In view of the intertwining between autophagy and apoptosis, it is noteworthy that Beclin 1 belongs to the BH3-only family of proteins (24–26). However, and unlike most of the proteins in this family, Beclin 1 is not able to trigger apoptosis when its expression is forced in cells (27). A BH3-mimetic drug, ABT-737, is able to dissociate the Beclin 1-Bcl-2 complex, and to trigger autophagy by mirroring the effect of starvation (25).The sphingolipids constitute a family of bioactive lipids (28–32) of which several members, such as ceramide and sphingosine 1-phosphate, are signaling molecules. These molecules constitute a “sphingolipid rheostat” that determines the fate of the cell, because in many settings ceramide is pro-apoptotic and sphingosine 1-phosphate mitigates this apoptotic effect (31, 32). However, ceramide is also engaged in a wide variety of other cell processes, such as the formation of exosomes (33), differentiation, cell proliferation, and senescence (34). Recently we showed that both ceramide and sphingosine 1-phosphate are able to stimulate autophagy (35, 36). It has also been shown that ceramide triggers autophagy in a large panel of mammalian cells (37–39). However, elucidation of the mechanism by which ceramide stimulates autophagy is still in its infancy. We have previously demonstrated that ceramide induces autophagy in breast and colon cancer cells by inhibiting the Class I phosphatidylinositol 3-phosphate/mTOR signaling pathway, which plays a central role in inhibiting autophagy (36). Inhibition of mTOR is another hallmark of starvation-induced autophagy (17). This finding led us to investigate the effect of ceramide on the Beclin 1-Bcl-2 complex. The results presented here show that ceramide is more potent than starvation in dissociating the Beclin 1-Bcl-2 complex (see Ref. 40). This dissociation is dependent on three phosphorylation sites (Thr⁶⁹, Ser⁷⁰, and Ser⁸⁷) located in a non-structural loop of Bcl-2. Ceramide induces the c-Jun N-terminal kinase 1-dependent phosphorylation of Bcl-2. Expression of a dominant negative form of JNK1 blocks Bcl-2 phosphorylation, and thus the induction of autophagy by ceramide. These findings help to explain how autophagy is regulated by a major lipid second messenger.     

Oxidizable Residues Mediating Protein Stability and Cytoprotective Interaction of DJ-1 with Apoptosis Signal-regulating Kinase 1

Parkinson disease (PD)-associated genomic deletions and the destabilizing L166P point mutation lead to loss of the cytoprotective DJ-1 protein. The effects of other PD-associated point mutations are less clear. Here we demonstrate that the M26I mutation reduces DJ-1 expression, particularly in a null background (knockout mouse embryonic fibroblasts). Thus, homozygous M26I mutation causes loss of DJ-1 protein. To determine the cellular consequences, we measured suppression of apoptosis signal-regulating kinase 1 (ASK1) and cytotoxicity for [M26I]DJ-1, and systematically all other DJ-1 methionine and cysteine mutants. C106A mutation of the central redox site specifically abolished binding to ASK1 and the cytoprotective activity of DJ-1. DJ-1 was apparently recruited into the ASK1 signalosome via Cys-106-linked mixed disulfides. The designed higher order oxidation mimicking [C106DD]DJ-1 non-covalently bound to ASK1 even in the absence of hydrogen peroxide and conferred partial cytoprotection. Interestingly, mutations of peripheral redox sites (C46A and C53A) and M26I also led to constitutive ASK1 binding. Cytoprotective [wt]DJ-1 bound to the ASK1 N terminus (which is known to bind another negative regulator, thioredoxin 1), whereas [M26I]DJ-1 bound to aberrant C-terminal site(s). Consequently, the peripheral cysteine mutants retained cytoprotective activity, whereas the PD-associated mutant [M26I]DJ-1 failed to suppress ASK1 activity and nuclear export of the death domain-associated protein Daxx and did not promote cytoprotection. Thus, cytoprotective binding of DJ-1 to ASK1 depends on the central redox-sensitive Cys-106 and may be modulated by peripheral cysteine residues. We suggest that impairments in oxidative conformation changes of DJ-1 might contribute to PD neurodegeneration.Loss-of-function mutations in the DJ-1 gene (PARK7) cause autosomal-recessive hereditary Parkinson disease (PD)² (1). The most dramatic PD-associated mutation L166P impairs DJ-1 dimer formation and dramatically destabilizes the protein (2–7). Other mutations such as M26I (8) and E64D (9) have more subtle defects with unclear cellular consequences (4, 7, 10, 11). In addition to this genetic association, DJ-1 is neuropathologically linked to PD. DJ-1 is up-regulated in reactive astrocytes, and it is oxidatively modified in brains of sporadic PD patients (12–14).DJ-1 protects against oxidative stress and mitochondrial toxins in cell culture (15–17) as well as in diverse animal models (18–21). The cytoprotective effects of DJ-1 may be stimulated by oxidation and mediated by molecular chaperoning (22, 23), and/or facilitation of the pro-survival Akt and suppression of apoptosis signal-regulating kinase 1 (ASK1) pathways (6, 24, 25). The cytoprotective activity of DJ-1 against oxidative stress depends on its cysteine residues (15, 17, 26). Among the three cysteine residues of DJ-1, the most prominent one is the easiest oxidizable Cys-106 (27) that is in a constrained conformation (28), but the other cysteine residues Cys-46 and Cys-53 have been implicated with DJ-1 activity as well (22). However, the molecular basis of oxidation-mediated cytoprotective activity of DJ-1 is not clear. Moreover, the roles of PD-mutated and in vivo oxidized methionines are not known.Here we have mutagenized all oxidizable residues within DJ-1 and studied the effects on protein stability and function. The PD-associated mutation M26I within the DJ-1 dimer interface selectively reduced protein expression as well as ASK1 suppression and cytoprotective activity in oxidatively stressed cells. These cell culture results support a pathogenic effect of the clinical M26I mutation (8). Furthermore, oxidation-defective C106A mutation abolished binding to ASK1 and cytoprotective activity of DJ-1, whereas the designed higher order oxidation mimicking mutant [C106DD]DJ-1 bound to ASK1 even in the absence of H₂O₂ and conferred partial cytoprotection. The peripheral cysteine mutants [C46A]DJ-1 and [C53A]DJ-1 were also cytoprotective and were incorporated into the ASK1 signalosome even in the basal state. Thus, DJ-1 may be activated by a complex mechanism, which depends on the redox center Cys-106 and is modulated by the peripheral cysteine residues. Impairments of oxidative DJ-1 activation might contribute to the pathogenesis of PD.     

Glutathione Peroxidase-1 Regulates Mitochondrial Function to Modulate Redox-dependent Cellular Responses

Glutathione peroxidase-1 (GPx-1) is a selenocysteine-containing enzyme that plays a major role in the reductive detoxification of peroxides in cells. In permanently transfected cells with approximate 2-fold overexpression of GPx-1, we found that intracellular accumulation of oxidants in response to exogenous hydrogen peroxide was diminished, as was epidermal growth factor receptor (EGFR)-mediated Akt activation in response to hydrogen peroxide or EGF stimulation. Knockdown of GPx-1 augmented EGFR-mediated Akt activation, whereas overexpression of catalase decreased Akt activation, suggesting that EGFR signaling is regulated by redox mechanisms. To determine whether mitochondrial oxidants played a role in these processes, cells were pretreated with a mitochondrial uncoupler prior to EGF stimulation. Inhibition of mitochondrial function attenuated EGF-mediated activation of Akt in control cells but had no additional effect in GPx-1-overexpressing cells, suggesting that GPx-1 overexpression decreased EGFR signaling by decreasing mitochondrial oxidants. Consistent with this finding, GPx-1 overexpression decreased global protein disulfide bond formation, which is dependent on mitochondrially produced oxidants. GPx-1 overexpression, in permanently transfected or adenovirus-treated cells, also caused overall mitochondrial dysfunction with a decrease in mitochondrial potential and a decrease in ATP production. GPx-1 overexpression also decreased EGF- and serum-mediated [³H]thymidine incorporation, indicating that alterations in GPx-1 can attenuate cell proliferation. Taken together, these data suggest that GPx-1 can modulate redox-dependent cellular responses by regulating mitochondrial function.Accumulation of reactive oxygen species (ROS),² such as superoxide anion and hydrogen peroxide, is thought to contribute to cellular damage, apoptosis, and cell death (1–3); however, ROS production is part of normal cellular metabolism, and evidence is accumulating that hydrogen peroxide, in particular, may function as a signaling molecule necessary for cell growth and survival (4–8). Superoxide is generated as a byproduct of mitochondrial respiration and by cellular redox enzymes, such as NADPH oxidase, that are stimulated through receptor-mediated mechanisms (9). Hydrogen peroxide is formed from the dismutation of superoxide, which occurs spontaneously or can be catalyzed by superoxide dismutase (10) or, alternatively, is produced by the two-electron enzymatic reduction of molecular oxygen by various oxidases, such as xanthine oxidase (11). Recent studies also suggest that hydrogen peroxide may be directly generated by receptor-ligand interactions (12). One mechanism by which hydrogen peroxide may modulate signal transduction is through the reversible oxidation of proteins at redox-active cysteines, including, for example, thiols in tyrosine kinase phosphatases. Oxidation and inactivation of phosphatases, such as PTEN, have been shown to promote the activity of the pro-growth and -survival kinase, Akt (13).Antioxidant enzymes, such as glutathione peroxidase, catalase, and peroxiredoxins, serve to eliminate hydrogen peroxide, thereby regulating cellular responses to this endogenous oxidant. GPx-1 is a selenoprotein and one of a family of peroxidases that reductively inactivate peroxides using glutathione as a source of reducing equivalents (14, 15). GPx-1, in particular, is a major intracellular antioxidant enzyme that is found in the cytoplasm and mitochondria of all cell types. In cell culture models as well as in genetic mouse models, GPx-1 overexpression is associated with enhanced protection against oxidative stress (16–19); however, GPx-1-overexpressing mice can become obese and insulin-resistant, and have attenuated insulin-mediated activation of Akt (20). Thus, to study how GPx-1 modulates the effects of cellular oxidants on cell signaling and cell growth, we analyzed cellular responses to hydrogen peroxide and EGF in permanently transfected cells overexpressing GPx-1.