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Autophagy plays key roles in development, oncogenesis, cardiovascular, metabolic, and neurodegenerative diseases. Hence, understanding how autophagy is regulated can reveal opportunities to modify autophagy in a disease-relevant manner. Ideally, one would want to functionally define autophagy regulators whose enzymatic activity can potentially be modulated. Here, we describe the STK38 protein kinase (also termed NDR1) as a conserved regulator of autophagy. Using STK38 as bait in yeast-two-hybrid screens, we discovered STK38 as a novel binding partner of Beclin1, a key regulator of autophagy. By combining molecular, cell biological, and genetic approaches, we show that STK38 promotes autophagosome formation in human cells and in Drosophila. Upon autophagy induction, STK38-depleted cells display impaired LC3B-II conversion; reduced ATG14L, ATG12, and WIPI-1 puncta formation; and significantly decreased Vps34 activity, as judged by PI3P formation. Furthermore, we observed that STK38 supports the interaction of the exocyst component Exo84 with Beclin1 and RalB, which is required to initiate autophagosome formation. Upon studying the activation of STK38 during autophagy induction, we found that STK38 is stimulated in a MOB1- and exocyst-dependent manner. In contrast, RalB depletion triggers hyperactivation of STK38, resulting in STK38-dependent apoptosis under prolonged autophagy conditions. Together, our data establish STK38 as a conserved regulator of autophagy in human cells and flies. We also provide evidence demonstrating that STK38 and RalB assist the coordination between autophagic and apoptotic events upon autophagy induction, hence further proposing a role for STK38 in determining cellular fate in response to autophagic conditions., (Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.)

To obtain mechanistic insights into the cross talk between lipolysis and autophagy, two key metabolic responses to starvation, we screened the autophagy-inducing potential of a panel of fatty acids in human cancer cells. Both saturated and unsaturated fatty acids such as palmitate and oleate, respectively, triggered autophagy, but the underlying molecular mechanisms differed. Oleate, but not palmitate, stimulated an autophagic response that required an intact Golgi apparatus. Conversely, autophagy triggered by palmitate, but not oleate, required AMPK, PKR and JNK1 and involved the activation of the BECN1/PIK3C3 lipid kinase complex. Accordingly, the downregulation of BECN1 and PIK3C3 abolished palmitate-induced, but not oleate-induced, autophagy in human cancer cells. Moreover, Becn1(+/-) mice as well as yeast cells and nematodes lacking the ortholog of human BECN1 mounted an autophagic response to oleate, but not palmitate. Thus, unsaturated fatty acids induce a non-canonical, phylogenetically conserved, autophagic response that in mammalian cells relies on the Golgi apparatus., (© 2015 The Authors.)

We recently reported that BAG6/BAT3 (BCL2-associated athanogene 6) is essential for basal and starvation-induced autophagy in E18.5 bag6(-/-) mouse embryos and in mouse embryonic fibroblasts (MEFs) through the modulation of the EP300/p300-dependent acetylation of TRP53 and autophagy-related (ATG) proteins. We observed that BAG6 increases TRP53 acetylation during starvation and pro-autophagic TRP53-target gene expression. BAG6 also decreases the EP300 dependent-acetylation of ATG5, ATG7, and LC3-I, posttranslational modifications that inhibit autophagy. In addition, in the absence of BAG6 or when using a mutant of BAG6 exclusively located in the cytoplasm, autophagy is inhibited, ATG7 is hyperacetylated, TRP53 acetylation is abrogated, and EP300 accumulates in the cytoplasm indicating that BAG6 is involved in the regulation of the nuclear localization of EP300. We also reported that the interaction between BAG6 and EP300 occurs in the cytoplasm rather than the nucleus. Moreover, during starvation, EP300 is transported to the nucleus in a BAG6-dependent manner. We concluded that BAG6 regulates autophagy by controlling the localization of EP300 and its accessibility to nuclear (TRP53) and cytoplasmic (ATGs) substrates.

Autophagy is regulated by posttranslational modifications, including acetylation. Here we show that HLA-B-associated transcript 3 (BAT3) is essential for basal and starvation-induced autophagy in embryonic day 18.5 BAT3(-/-) mouse embryos and in mouse embryonic fibroblasts (MEFs) through the modulation of p300-dependent acetylation of p53 and ATG7. Specifically, BAT3 increases p53 acetylation and proautophagic p53 target gene expression, while limiting p300-dependent acetylation of ATG7, a mechanism known to inhibit autophagy. In the absence of BAT3 or when BAT3 is located exclusively in the cytosol, autophagy is abrogated, ATG7 is hyperacetylated, p53 acetylation is abolished, and p300 accumulates in the cytosol, indicating that BAT3 regulates the nuclear localization of p300. In addition, the interaction between BAT3 and p300 is stronger in the cytosol than in the nucleus and, during starvation, the level of p300 decreases in the cytosol but increases in the nucleus only in the presence of BAT3. We conclude that BAT3 tightly controls autophagy by modulating p300 intracellular localization, affecting the accessibility of p300 to its substrates, p53 and ATG7.

Acetyl-coenzyme A (AcCoA) is a major integrator of the nutritional status at the crossroads of fat, sugar, and protein catabolism. Here we show that nutrient starvation causes rapid depletion of AcCoA. AcCoA depletion entailed the commensurate reduction in the overall acetylation of cytoplasmic proteins, as well as the induction of autophagy, a homeostatic process of self-digestion. Multiple distinct manipulations designed to increase or reduce cytosolic AcCoA led to the suppression or induction of autophagy, respectively, both in cultured human cells and in mice. Moreover, maintenance of high AcCoA levels inhibited maladaptive autophagy in a model of cardiac pressure overload. Depletion of AcCoA reduced the activity of the acetyltransferase EP300, and EP300 was required for the suppression of autophagy by high AcCoA levels. Altogether, our results indicate that cytosolic AcCoA functions as a central metabolic regulator of autophagy, thus delineating AcCoA-centered pharmacological strategies that allow for the therapeutic manipulation of autophagy., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Macroautophagy (hereafter referred to as autophagy), a central mechanism mediating the lysosomal degradation of cytoplasmic components, can be stimulated by a wide panel of adverse stimuli, including a panoply of anticancer agents. The central autophagic organelle is the autophagosome, a double membrane-bound vacuole that sequesters the cytoplasmic material destined to disposal. The ultimate destiny of the autophagosome is to fuse with a lysosome, resulting in the degradation of the autophagic cargo. In this setting, it is important to discriminate whether a particular stimulus actually promotes autophagy or it simply blocks the fusion of autophagosomes with lysosomes. To this aim, the methods that assess autophagy should assess not only the number of autophagosomes but also the so-called autophagic flux, that is, the clearance of the autophagy cargo from the lysosomal compartment. Here, we present a compendium of methods to assess the autophagic flux in cultured malignant cells. This approach should allow for the study of the intimate link between autophagy and oncometabolism in several experimental paradigms., (© 2014 Elsevier Inc. All rights reserved.)

Amino acids, leucine in particular, are known to inhibit autophagy, at least in part by their ability to stimulate MTOR-mediated signaling. Evidence is presented showing that glutamate dehydrogenase, the central enzyme in amino acid catabolism, contributes to leucine sensing in the regulation of autophagy. The data suggest a dual mechanism by which glutamate dehydrogenase activity modulates autophagy, i.e., by activating MTORC1 and by limiting the formation of reactive oxygen species.

Breast cancer tissue contains a small population of cells that have the ability to self-renew; these cells are known as cancer stem-like cells (CSCs). We have recently shown that autophagy is essential for the tumorigenicity of these CSCs. Salinomycin (Sal), a K (+) /H (+) ionophore, has recently been shown to be at least 100 times more effective than paclitaxel in reducing the proportion of breast CSCs. However, its mechanisms of action are still unclear. We show here that Sal blocked both autophagy flux and lysosomal proteolytic activity in both CSCs and non-CSCs derived from breast cancer cells. GFP-LC3 staining combined with fluorescent dextran uptake and LysoTracker-Red staining showed that autophagosome/lysosome fusion was not altered by Sal treatment. Acridine orange staining provided evidence that lysosomes display the characteristics of acidic compartments in Sal-treated cells. However, tandem mCherry-GFP-LC3 assay indicated that the degradation of mCherry-GFP-LC3 is blocked by Sal. Furthermore, the protein degradation activity of lysosomes was inhibited, as demonstrated by the rate of long-lived protein degradation, DQ-BSA assay and measurement of cathepsin activity. Our data indicated that Sal has a relatively greater suppressant effect on autophagic flux in the ALDH (+) population in HMLER cells than in the ALDH (-) population; moreover, this differential effect on autophagic flux correlated with an increase in apoptosis in the ALDH (+) population. ATG7 depletion accelerated the proapoptotic capacity of Sal in the ALDH (+) population. Our findings provide new insights into how the autophagy-lysosomal pathway contributes to the ability of Sal to target CSCs in vitro.

Lysosome is a key subcellular organelle in the execution of the autophagic process and at present little is known whether lysosomal function is controlled in the process of autophagy. In this study, we first found that suppression of mammalian target of rapamycin (mTOR) activity by starvation or two mTOR catalytic inhibitors (PP242 and Torin1), but not by an allosteric inhibitor (rapamycin), leads to activation of lysosomal function. Second, we provided evidence that activation of lysosomal function is associated with the suppression of mTOR complex 1 (mTORC1), but not mTORC2, and the mTORC1 localization to lysosomes is not directly correlated to its regulatory role in lysosomal function. Third, we examined the involvement of transcription factor EB (TFEB) and demonstrated that TFEB activation following mTORC1 suppression is necessary but not sufficient for lysosomal activation. Finally, Atg5 or Atg7 deletion or blockage of the autophagosome-lysosome fusion process effectively diminished lysosomal activation, suggesting that lysosomal activation occurring in the course of autophagy is dependent on autophagosome-lysosome fusion. Taken together, this study demonstrates that in the course of autophagy, lysosomal function is upregulated via a dual mechanism involving mTORC1 suppression and autophagosome-lysosome fusion.

Using cultured cortical neurons, we show that the blockade of protein phosphatase 2A (PP2A), either pharmacologically by okadaic acid or by short hairpin RNA (shRNA)-mediated silencing of PP2A catalytic subunit, inhibited basal autophagy and autophagy induced in several experimental settings (including serum deprivation, endoplasmic reticulum stress, rapamycin, and proteasome inhibition) at early stages before autophagosome maturation. Conversely, PP2A upregulation by PP2A catalytic subunit overexpression stimulates neuronal autophagy. In addition, PP2A blockade resulted in the activation of the negative regulator of autophagy mammalian target of rapamycin complex 1 and 5' adenosine monophosphate (AMP)-activated protein kinase (AMPK) and led to intraneuronal accumulation of p62- and ubiquitin-positive protein inclusions, likely due to autophagy downregulation. These data are consistent with previous findings showing that specific invalidation of the autophagy process in the nervous system of mouse resulted in the accumulation of p62- and ubiquitin-positive protein inclusion bodies. Furthermore, we showed that PP2A inhibition alters the distribution of the microtubule-associated protein 1 light chain(LC) 3-I (MAP LC3-I), a key component of the autophagy molecular machinery. Whether MAP LC3-I distribution in the cell accounts for autophagy regulation remains to be determined. These data are important to human neurodegenerative diseases, especially Alzheimer's disease, because they provide links for the first time between the pathological features of Alzheimer's disease:PP2A downregulation, autophagy disruption, and protein aggregation., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Gossypol, a natural Bcl-2 homology domain 3 mimetic compound isolated from cottonseeds, is currently being evaluated in clinical trials. Here, we provide evidence that gossypol induces autophagy followed by apoptotic cell death in both the MCF-7 human breast adenocarcinoma and HeLa cell lines. We first show that knockdown of the Bcl-2 homology domain 3-only protein Beclin 1 reduces gossypol-induced autophagy in MCF-7 cells, but not in HeLa cells. Gossypol inhibits the interaction between Beclin 1 and Bcl-2 (B-cell leukemia/lymphoma 2), antagonizes the inhibition of autophagy by Bcl-2, and hence stimulates autophagy. We then show that knockdown of Vps34 reduces gossypol-induced autophagy in both cell lines, and consistent with this, the phosphatidylinositol 3-phosphate-binding protein WIPI-1 is recruited to autophagosomal membranes. Further, Atg5 knockdown also reduces gossypol-mediated autophagy. We conclude that gossypol induces autophagy in both a canonical and a noncanonical manner. Notably, we found that gossypol-mediated apoptotic cell death was potentiated by treatment with the autophagy inhibitor wortmannin or with small interfering RNA against essential autophagy genes (Vps34, Beclin 1, and Atg5). Our findings support the notion that gossypol-induced autophagy is cytoprotective and not part of the cell death process induced by this compound.

A group of phosphoinositide 3-kinase (PI3K) inhibitors, such as 3-methyladenine (3-MA) and wortmannin, have been widely used as autophagy inhibitors based on their inhibitory effect on class III PI3K activity, which is known to be essential for induction of autophagy. In this study, we systematically examined and compared the effects of these two inhibitors on autophagy under both nutrient-rich and deprivation conditions. To our surprise, 3-MA is found to promote autophagy flux when treated under nutrient-rich conditions with a prolonged period of treatment, whereas it is still capable of suppressing starvation-induced autophagy. We first observed that there are marked increases of the autophagic markers in cells treated with 3-MA in full medium for a prolonged period of time (up to 9 h). Second, we provide convincing evidence that the increase of autophagic markers is the result of enhanced autophagic flux, not due to suppression of maturation of autophagosomes or lysosomal function. More importantly, we found that the autophagy promotion activity of 3-MA is due to its differential temporal effects on class I and class III PI3K; 3-MA blocks class I PI3K persistently, whereas its suppressive effect on class III PI3K is transient. Because 3-MA has been widely used as an autophagy inhibitor in the literature, understanding the dual role of 3-MA in autophagy thus suggests that caution should be exercised in the application of 3-MA in autophagy study.

Sphingosine 1-phosphate (S1P) is a bioactive sphingolipid metabolite involved in cancer development through stimulation of cell survival, proliferation, migration, and angiogenesis. Irreversible degradation of S1P is catalyzed by S1P lyase (SPL). The human SGPL1 gene that encodes SPL maps to a region often mutated in cancers. To investigate the effect of SPL deficiency on cell survival and transformation, the susceptibility to anticancer drugs of fibroblasts generated from SPL-deficient mouse embryos (Sgpl1(-/-)) was compared with that of cells from heterozygous (Sgpl1(+/-)) or wild-type (Sgpl1(+/+)) embryos. First, loss of SPL caused resistance to the toxic effects of etoposide and doxorubicin. Interestingly, heterozygosity for the Sgpl1 gene resulted in partial resistance to apoptosis. Secondly, doxorubicin-induced apoptotic signaling was strongly inhibited in Sgpl1(-/-) cells (phosphatidylserine externalization, caspase activation, and cytochrome c release). This was accompanied by a strong increase in Bcl-2 and Bcl-xL protein content. Whereas correction of SPL deficiency in Sgpl1(-/-) cells led to downregulation of antiapoptotic proteins, Bcl-2 and Bcl-xL small interfering RNA-mediated knockdown in SPL-deficient cells resulted in increased sensitivity to doxorubicin, suggesting that Bcl-2 upregulation mediates SPL protective effects. Moreover, SPL deficiency led to increased cell proliferation, anchorage-independent cell growth, and formation of tumors in nude mice. Finally, transcriptomic studies showed that SPL expression is downregulated in human melanoma cell lines. Thus, by affecting S1P metabolism and the expression of Bcl-2 members, the loss of SPL enhances cell resistance to anticancer regimens and results in an increased ability of cells to acquire a transformed phenotype and become malignant.

Ceramide is a sphingolipid bioactive molecule that induces apoptosis and other forms of cell death, and triggers macroautophagy (referred to below as autophagy). Like amino acid starvation, ceramide triggers autophagy by interfering with the mTOR-signaling pathway, and by dissociating the Beclin 1:Bcl-2 complex in a c-Jun N-terminal kinase 1 (JNK1)-mediated Bcl-2 phosphorylation-dependent manner. Dissociation of the Beclin 1:Bcl-2 complex, and the subsequent stimulation of autophagy have been observed in various contexts in which the cellular level of long-chain ceramides was increased. It is notable that the conversion of short-chain ceramides (C(2)-ceramide and C(6)-ceramide) into long-chain ceramide via the activity of ceramide synthase is required to trigger autophagy. The dissociation of the Beclin 1:Bcl-2 complex has also been observed in response to tamoxifen and PDMP (an inhibitor of the enzyme that converts ceramide to glucosylceramide), drugs that increase the intracellular level of long-chain ceramides. However, and in contrast to starvation, overexpression of Bcl-2 does not blunt ceramide-induced autophagy. Whether this autophagy that is unchecked by forced dissociation of the Beclin 1:Bcl-2 complex is related to the ability of ceramide to trigger cell death remains an open question. More generally, the question of whether ceramide-induced autophagy is a dedicated cell death mechanism deserves closer scrutiny.

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 (C(2)-ceramide and C(6)-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, Thr(69), Ser(70), and Ser(87), 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 is a three-step process: (1) autophagosomes form and mature, (2) the autophagosomes fuse with lysosomes, and (3) the autophagic cargo is degraded in the lysosomes. It is this lysosomal degradation of the autophagic cargo that constitutes the autophagic flux. As in the case of metabolic pathways, the steady-state concentration of the intermediary autophagic structures alone is insufficient for investigating the flux. Assaying the degradation of long-lived proteins as described in this chapter is one of the methods that can be used to measure autophagic flux.

The NF-kappaB transcription factor is an important anti-apoptotic factor, which is frequently deregulated in cancer cells. We have recently demonstrated that NF-kappaB activation mediates the repression of autophagy in response to TNFa in three models of cancer cell lines. In contrast, in the absence of NF-kappaB activation, TNFa induces macroautophagy (autophagy), which requires reactive oxygen species (ROS) production and participates in the TNFalpha-induced apoptotic signaling pathway. Autophagy-dependent apoptosis was also observed following direct addition of ROS to cells. Moreover, addition of rapamycin to TNFalpha renders these cells susceptible to the cytotoxic effect of this cytokine. These findings highlight the regulation of autophagy by oxidative stress and support the idea that repression of autophagy by NF-kappaB may constitute a novel anti-apoptotic function of this transcription factor. We also bring evidence that direct stimulation of autophagy may represent a new therapeutic strategy for overcoming the NF-kappaB-dependent chemoresistance of cancer cells.

Interruption of mTOR-dependent signaling by rapamycin is known to stimulate autophagy, both in mammalian cells and in yeast. Because activation of AMPK also inhibits mTOR-dependent signaling one would expect stimulation of autophagy by AMPK activation. According to the literature, this is true for yeast but, unexpectedly, not for mammalian cells on the basis of the use of AICAR, a pharmacological activator of AMPK. In the present study, carried out with hepatocytes, HT-29 cells, and HeLa cells, we have reexamined the possible role of AMPK in the control of mammalian autophagy. Inhibition of AMPK activity by compound C or by transfection with a dominant negative form of AMPK almost completely inhibited autophagy. These results suggest that the inhibition of autophagy by AICAR is not related to its ability to activate AMPK. We conclude that in mammalian cells, as in yeast, AMPK is required for autophagy.

Activation of NF-kappaB and autophagy are two processes involved in the regulation of cell death, but the possible cross-talk between these two signaling pathways is largely unknown. Here, we show that NF-kappaB activation mediates repression of autophagy in tumor necrosis factor-alpha (TNFalpha)-treated Ewing sarcoma cells. This repression is associated with an NF-kappaB-dependent activation of the autophagy inhibitor mTOR. In contrast, in cells lacking NF-kappaB activation, TNFalpha treatment up-regulates the expression of the autophagy-promoting protein Beclin 1 and subsequently induces the accumulation of autophagic vacuoles. Both of these responses are dependent on reactive oxygen species (ROS) production and can be mimicked in NF-kappaB-competent cells by the addition of H2O2. Small interfering RNA-mediated knockdown of beclin 1 and atg7 expression, two autophagy-related genes, reduced TNFalpha- and reactive oxygen species-induced apoptosis in cells lacking NF-kappaB activation and in NF-kappaB-competent cells, respectively. These findings demonstrate that autophagy may amplify apoptosis when associated with a death signaling pathway. They are also evidence that inhibition of autophagy is a novel mechanism of the antiapoptotic function of NF-kappaB activation. We suggest that stimulation of autophagy may be a potential way bypassing the resistance of cancer cells to anti-cancer agents that activate NF-kappaB.

The sphingolipid ceramide is involved in the cellular stress response. Here we demonstrate that ceramide controls macroautophagy, a major lysosomal catabolic pathway. Exogenous C(2)-ceramide stimulates macroautophagy (proteolysis and accumulation of autophagic vacuoles) in the human colon cancer HT-29 cells by increasing the endogenous pool of long chain ceramides as demonstrated by the use of the ceramide synthase inhibitor fumonisin B(1). Ceramide reverted the interleukin 13-dependent inhibition of macroautophagy by interfering with the activation of protein kinase B. In addition, C(2)-ceramide stimulated the expression of the autophagy gene product beclin 1. Ceramide is also the mediator of the tamoxifen-dependent accumulation of autophagic vacuoles in the human breast cancer MCF-7 cells. Monodansylcadaverine staining and electron microscopy showed that this accumulation was abrogated by myriocin, an inhibitor of de novo synthesis ceramide. The tamoxifen-dependent accumulation of vacuoles was mimicked by 1-phenyl-2-decanoylamino-3-morpholino-1-propanol, an inhibitor of glucosylceramide synthase. 1-Phenyl-2-decanoylamino-3-morpholino-1-propanol, tamoxifen, and C(2)-ceramide stimulated the expression of beclin 1, whereas myriocin antagonized the tamoxifen-dependent up-regulation. Tamoxifen and C(2)-ceramide interfere with the activation of protein kinase B, whereas myriocin relieved the inhibitory effect of tamoxifen. In conclusion, the control of macroautophagy by ceramide provides a novel function for this lipid mediator in a cell process with major biological outcomes.

The product of the FUT8 gene transfers an alpha1-6 fucose on the innermost N-acetylglucosamine of the chitobiose core of N-glycans. Northern blot analysis shows four main transcripts of 3.0, 3.3, 3.9, and 4.2 kb in the embryo. The larger forms around 4-kb decrease in fetus and adult. Fourteen embryo transcripts of FUT8 were cloned. Twelve exons comprising two new 5'untranslated-exons (A and B) and two new 3'UT-ends (L1 and L2) and the complete genomic organization of the FUT8 gene (330 kb) are described. Transcripts starting with the 5'UT-exon A are always associated with exons C and D. Exon B initiates another series of transcripts associated to exon C and D or directly to exon D. A third series of transcripts starts at exon C. The data suggest an expression of FUT8 regulated by three different promoters, starting transcription in exons A, B, or C. The A or C series are better expressed than the B series. After transfection with these cDNA constructs the transcripts with 5'UT-exons A or C have higher expression of FUT8 transcripts and higher alpha6-fucosyltransferase activity, whereas the activity of the B series is about two-thirds lower for both parameters, suggesting that exon B reduces the expression of the transcripts.

AGS3 contains GoLoco or G-protein regulatory motifs in its COOH-terminal half that stabilize the GDP-bound conformation of the alpha-subunit of the trimeric Gi3 protein. The latter is part of a signaling pathway that controls the lysosomal-autophagic catabolism in human colon cancer HT-29 cells. In the present work we show that the mRNA encoding for AGS3 is expressed in human intestinal cell lines (Caco-2 and HT-29) whatever their state of differentiation. Together with the full-length form, minute amounts of the mRNA encoding a NH2-terminal truncated form of AGS3, previously characterized in cardiac tissues, were also detected. Both the endogenous form of AGS3 and a tagged expressed form have a localization compatible with a role in the Galphai3-dependent control of autophagy. Accordingly, expressing its non-Galphai3-interacting NH2-terminal domain or its Galphai3-interacting COOH-terminal domain reversed the stimulatory role of AGS3 on autophagy. On the basis of biochemical and morphometric analysis, we conclude that AGS3 is involved in an early event during the autophagic pathway probably prior to the formation of the autophagosome. These data demonstrate that AGS3 is a novel partner of the Galphai3 protein in the control of a major catabolic pathway.

Activation of ERK1/2 stimulates macroautophagy in the human colon cancer cell line HT-29 by favoring the phosphorylation of the Galpha-interacting protein (GAIP) in an amino acid-dependent manner (Ogier-Denis, E., Pattingre, S., El Benna, J., and Codogno, P. (2000) J. Biol. Chem. 275, 39090-39095). Here we show that ERK1/2 activation by aurintricarboxylic acid (ATA) treatment induces the phosphorylation of GAIP in an amino acid-dependent manner. Accordingly, ATA challenge increased the rate of macroautophagy, whereas epidermal growth factor did not significantly affect macroautophagy and GAIP phosphorylation status. In fact, ATA activated the ERK1/2 signaling pathway, whereas epidermal growth factor stimulated both the ERK1/2 pathway and the class I phosphoinositide 3-kinase pathway, known to decrease the rate of macroautophagy. Amino acids interfered with the ATA-induced macroautophagy by inhibiting the activation of the kinase Raf-1. The role of the Ras/Raf-1/ERK1/2 signaling pathway in the GAIP- and amino acid-dependent control of macroautophagy was confirmed in HT-29 cells expressing the Ras(G12V,T35S) mutant. Similar to the protein phosphatase 2A inhibitor okadaic acid, amino acids sustained the phosphorylation of Ser(259), which is involved in the negative regulation of Raf-1. In conclusion, these results add a novel target to the amino acid signaling-dependent control of macroautophagy in intestinal cells.

Nonsteroidal anti-inflammatory drugs, which inhibit cyclooxygenase (COX) activity, are powerful antineoplastic agents that exert their antiproliferative and proapoptotic effects on cancer cells by COX-dependent and/or COX-independent pathways. Celecoxib, a COX-2-specific inhibitor, has been shown to reduce the number of adenomatous colorectal polyps in patients with familial adenomatous polyposis. Here, we show that celecoxib induces apoptosis in the colon cancer cell line HT-29 by inhibiting the 3-phosphoinositide-dependent kinase 1 (PDK1) activity. This effect was correlated with inhibition of the phosphorylation of the PDK1 downstream substrate Akt/protein kinase B (PKB) on two regulatory sites, Thr(308) and Ser(473). However, expression of a constitutive active form of Akt/PKB (myristoylated PKB) has a low protective effect toward celecoxib-induced cell death. In contrast, overexpression of constitutive active mutant of PDK1 (PDK1(A280V)) was as potent as the pancaspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone, to impair celecoxib-induced apoptosis. By contrast, cells expressing a kinase-defective mutant of PDK1 (PDK1(K114G)) remained sensitive to celecoxib. Furthermore, in vitro measurement reveals that celecoxib was a potential inhibitor of PDK1 activity with an IC(50) = 3.5 microm. These data indicate that inhibition of PDK1 signaling is involved in the proapoptotic effect of celecoxib in HT-29 cells.

The tumor suppressor PTEN is a dual protein and phosphoinositide phosphatase that negatively controls the phosphatidylinositol (PI) 3-kinase/protein kinase B (Akt/PKB) signaling pathway. Interleukin-13 via the activation of the class I PI 3-kinase has been shown to inhibit the macroautophagic pathway in the human colon cancer HT-29 cells. Here we demonstrate that the wild-type PTEN is expressed in this cell line. Its overexpression directed by an inducible promoter counteracts the interleukin-13 down-regulation of macroautophagy. This effect was dependent upon the phosphoinositide phosphatase activity of PTEN as determined by using the mutant G129E, which has only protein phosphatase activity. The role of Akt/PKB in the signaling control of interleukin-13-dependent macroautophagy was investigated by expressing a constitutively active form of the kinase ((Myr)PKB). Under these conditions a dramatic inhibition of macroautophagy was observed. By contrast a high rate of autophagy was observed in cells expressing a dominant negative form of PKB. These data demonstrate that the signaling control of macroautophagy overlaps with the well known PI 3-kinase/PKB survival pathway and that the loss of PTEN function in cancer cells inhibits a major catabolic pathway.

Autophagy is a major catabolic process allowing the renewal of intracellular organelles by which cells maintain their homeostasis. We have previously shown that autophagy is controlled by two transduction pathways mediated by a heterotrimeric Gi3 protein and phosphatidylinositol 3-kinase activities in the human colon cancer cell line HT-29. Here, we show that 3-methyladenine, an inhibitor of autophagy, increases the sensitivity of HT-29 cells to apoptosis induced by sulindac sulfide, a nonsteroidal anti-inflammatory drug which inhibits the cyclooxygenases. Similarly, HT-29 cells overexpressing a GTPase-deficient mutant of the G(alpha i3) protein (Q204L), which have a low rate of autophagy, were more sensitive to sulindac sulfide-induced apoptosis than parental HT-29 cells. In both cell populations we did not observe differences in the expression patterns of COX-2, Bcl-2, Bcl(XL), Bax, and Akt/PKB activity. However, the rate of cytochrome c release was higher in Q204L-overexpressing cells than in HT-29 cells. These results suggest that autophagy could retard apoptosis in colon cancer cells by sequestering mitochondrial death-promoting factors such as cytochrome c., (Copyright 2001 Academic Press.)

The macroautophagic-lysosomal pathway is a bulk degradative process for cytosolic proteins and organelles including the endoplasmic reticulum (ER). We have previously shown that the human colonic carcinoma HT-29 cell population is characterized by a high rate of autophagic degradation of N-linked glycoproteins substituted with ER-type glycans. In the present work we demonstrate that glucosidase inhibitors [castanospermine (CST) and deoxynojirimycin] have a stabilizing effect on newly synthesized glucosylated N-linked glycoproteins and impaired their lysosomal delivery as shown by subcellular fractionation on Percoll gradients. The inhibition of macroautophagy was restricted to N-linked glycoproteins because macroautophagic parameters such as the rate of sequestration of cytosolic markers and the fractional volume occupied by autophagic vacuoles were not affected in CST-treated cells. The protection of glucosylated glycoproteins from autophagic sequestration was also observed in inhibitor-treated Chinese hamster ovary (CHO) cells and in Lec23 cells (a CHO mutant deficient in glucosidase I activity). The interaction of glucosylated glycoproteins with the ER chaperone binding protein (BiP) was prolonged in inhibitor-treated cells in comparison with untreated CHO cells. These results show that the removal of glucose from N-glycans of glycoproteins is a key event for their delivery to the autophagic pathway and that interaction with BiP could prevent or delay newly synthesized glucosylated N-linked glycoproteins from being sequestered by the autophagic pathway.

Autophagic sequestration is controlled by the Galphai3 protein in human colon cancer HT-29 cells. Immunofluorescence and subcellular fractionation studies showed that the Galphai3 protein is preferentially associated with Golgi membranes but co-localization was also observed with the endoplasmic reticulum (ER) membrane. The Galphai2 protein, which is not involved in the control of autophagic sequestration, is associated with the plasma membrane. Transfection of chimaeric Galphai proteins (Galphai3/2, Galphai2/3) containing the N- and C-terminal parts of the relevant Galphai demonstrated that the C-terminal part of the Galphai3 protein, by governing its membrane localization [de Almeida, Holtzman, Peters, Ercolani, Ausiello and Stow (1994) J. Cell Sci. 107, 507-515], is important in the control of macroautophagic sequestration. G alpha interacting protein (GAIP),which stimulates the GTPase activity of the Galphai3 protein and favours macroautophagic sequestration in HT-29 cells,was shown, by immunofluorescence studies using confocal microscopy, to be confined to the cytoplasm. The cytoplasmic distribution of GAIP only partially overlaps with that of the Galphai3 protein. However, the presence of the two proteins on Golgi and ER membranes was confirmed by subcellular fractionation. These results point to the importance of the cytoplasmic localization of the Galphai3 protein and GAIP in controlling autophagic sequestration in HT-29 cells.

The trimeric G(i3) protein-dependent lysosomal-autophagic pathway is responsible for the degradation of a pool of N-linked glycoproteins in the human colon cancer HT-29 cell line. Here we have followed the fate of N-glycans using HT-29 cells either overexpressing the wild-type G alpha(i3) protein or transfected with different mutants of the G alpha(i3) protein. The stabilization of N-glycans was dependent upon the inhibition of autophagic sequestration by either 3-methyladenine (3-MA) or pertussis toxin (PTX). However, PTX allowed the processing of high-mannose glycans whereas 3-MA did not. The destabilization of the Golgi apparatus by brefeldin A, which interrupts the intracellular trafficking of N-linked glycoproteins along the secretory pathway, did not interfere with the macroautophagic pathway. These results suggest that the lysosomal-autophagic pathway is not dependent upon the integrity of the Golgi apparatus and points to differences between the molecular properties of two membrane flow processes (macroautophagy, exocytic pathway) controlled by the trimeric G(i3) protein.

Large, free polymannose oligosaccharides generated during glycoprotein biosynthesis rapidly appear in the cytosol of HepG2 cells where they undergo processing by a cytosolic endo H-like enzyme and a mannosidase to yield the linear isomer of Man5GlcNAc (Man[alpha 1-2]Man[alpha 1-2]Man[alpha 1-3][Man alpha 1-6]Man[beta 1-4] GlcNAc). Here we have examined the fate of these partially trimmed oligosaccharides in intact HepG2 cells. Subsequent to pulse-chase incubations with D-[2-3H]mannose followed by permeabilization of cells with streptolysin O free oligosaccharides were isolated from the resulting cytosolic and membrane-bound compartments. Control pulse-chase experiments revealed that total cellular free oligosaccharides are lost from HepG2 cells with a half-life of 3-4 h. In contrast use of the vacuolar H+/ATPase inhibitor, concanamycin A, stabilized total cellular free oligosaccharides and enabled us to demonstrate a translocation of partially trimmed oligosaccharides from the cytosol into a membrane-bound compartment. This translocation process was unaffected by inhibitors of autophagy but inhibited if cells were treated with either 100 microM swainsonine, which provokes a cytosolic accumulation of large free oligosaccharides bearing 8-9 residues of mannose, or agents known to reduce cellular ATP levels which lead to the accumulation of the linear isomer of Man5GlcNAc in the cytosol. Subcellular fractionation studies on Percoll density gradients revealed that the cytosol-generated linear isomer of Man5GlcNAc is degraded in a membrane-bound compartment that cosediments with lysosomes.