Björn Stork

Role of AMPK-mTOR-Ulk1/2 in the Regulation of Autophagy: Cross Talk, Shortcuts, and Feedbacks

ABSTRACT Living cells are adaptive self-sustaining systems. They strictly depend on the sufficient supply of oxygen, energy, and nutrients from the outside in order to sustain their internal organization. However, as autonomous entities they are able to monitor and appropriately adapt to any critical fluctuation in their environment. In the case of insufficient external nutrient supply or augmented energy demands, cells start to extensively digest their own interior. This process, known as macroautophagy, comprises the transport of cytosolic portions and entire organelles to the lysosomal compartment via specific double-membrane vesicles, called autophagosomes. Although extensively upregulated under nutrient restriction, a low level of basal autophagy is likewise crucial in order to sustain the cellular homeostasis. On the other hand, cells have to avoid excessive and enduring self-digestion. The delicate balance between external energy and nutrient supply and internal production and consumption is a demanding task. The complex protein network that senses and precisely reacts to environmental changes is thus mainly regulated by rapid and reversible posttranslational modifications such as phosphorylation. This review focuses on the serine/threonine protein kinases AMP-activated protein kinase, mammalian target of rapamycin (mTOR), and unc-51-like kinase 1/2 (Ulk1/2), three interconnected major junctions within the autophagy regulating signaling network.

AMPK-independent induction of autophagy by cytosolic Ca2+ increase.

Autophagy is a eukaryotic lysosomal bulk degradation system initiated by cytosolic cargo sequestration in autophagosomes. The Ser/Thr kinase mTOR has been shown to constitute a central role in controlling the initiation of autophagy by integrating multiple nutrient-dependent signaling pathways that crucially involves the activity of PI3K class III to generate the phosphoinositide PI(3)P. Recent reports demonstrate that the increase in cytosolic Ca(2+) can induce autophagy by inhibition of mTOR via the CaMKK-alpha/beta-mediated activation of AMPK. Here we demonstrate that Ca(2+) signaling can additionally induce autophagy independently of the Ca(2+)-mediated activation of AMPK. First, by LC3-II protein monitoring in the absence or presence of lysosomal inhibitors we confirm that the elevation of cytosolic Ca(2+) induces autophagosome generation and does not merely block autophagosome degradation. Further, we demonstrate that Ca(2+)-chelation strongly inhibits autophagy in human, mouse and chicken cells. Strikingly, we found that the PI(3)P-binding protein WIPI-1 (Atg18) responds to the increase of cytosolic Ca(2+) by localizing to autophagosomal membranes (WIPI-1 puncta) and that Ca(2+)-chelation inhibits WIPI-1 puncta formation, although PI(3)P-generation is not generally affected by these Ca(2+) flux modifications. Importantly, using AMPK-alpha1(-/-)alpha2(-/-) MEFs we show that thapsigargin application triggers autophagy in the absence of AMPK and does not involve complete mTOR inhibition, as detected by p70S6K phosphorylation. In addition, STO-609-mediated CaMKK-alpha/beta inhibition decreased the level of thapsigargin-induced autophagy only in AMPK-positive cells. We suggest that apart from reported AMPK-dependent regulation of autophagic degradation, an AMPK-independent pathway triggers Ca(2+)-mediated autophagy, involving the PI(3)P-effector protein WIPI-1 and LC3.

Vemurafenib Potently Induces Endoplasmic Reticulum Stress–Mediated Apoptosis in BRAFV600E Melanoma Cells

Drug resistance in melanomas may be overcome by therapies that trigger endoplasmic reticulum stress. Stressing Out Resistance Many melanomas harbor a form of the kinase BRAF with an amino acid substitution (V600E) that renders the protein constitutively active. The mutant BRAF drives cancer growth by activating extracellular signal–regulated kinase (ERK), which promotes cell proliferation and survival. The BRAFV600E kinase inhibitor vemurafenib is initially an effective therapy for melanoma but loses its efficacy because the tumor cells become drug-resistant. Beck et al. found that vemurafenib inhibited survival signaling mediated by ERK and induced endoplasmic reticulum (ER) stress, a form of cellular stress that can culminate in apoptosis. Combined application of vemurafenib with the ER stress inducer thapsigargin to BRAFV600E melanoma cell lines that were resistant to vemurafenib resulted in an enhanced ER stress response and apoptosis. Their findings indicate a potential strategy to overcome drug resistance in BRAF-mutated melanoma. The V600E mutation in the kinase BRAF is frequently detected in melanomas and results in constitutive activation of BRAF, which then promotes cell proliferation by the mitogen-activated protein kinase signaling pathway. Although the BRAFV600E kinase inhibitor vemurafenib has remarkable antitumor activity in patients with BRAFV600E-mutated melanoma, its effects are limited by the onset of drug resistance. We found that exposure of melanoma cell lines with the BRAFV600E mutation to vemurafenib decreased the abundance of antiapoptotic proteins and induced intrinsic mitochondrial apoptosis. Vemurafenib-treated melanoma cells showed increased cytosolic concentration of calcium, a potential trigger for endoplasmic reticulum (ER) stress, which can lead to apoptosis. Consistent with an ER stress–induced response, vemurafenib decreased the abundance of the ER chaperone protein glucose-regulated protein 78, increased the abundance of the spliced isoform of the transcription factor X-box binding protein 1 (XBP1) (which transcriptionally activates genes involved in ER stress responses), increased the phosphorylation of the translation initiation factor eIF2α (which would be expected to inhibit protein synthesis), and induced the expression of ER stress–related genes. Knockdown of the ER stress response protein activating transcription factor 4 (ATF4) significantly reduced vemurafenib-induced apoptosis. Moreover, the ER stress inducer thapsigargin prevented invasive growth of tumors formed from vemurafenib-sensitive melanoma cells in vivo. In melanoma cells with low sensitivity or resistance to vemurafenib, combination treatment with thapsigargin augmented or induced apoptosis. Thus, thapsigargin or other inducers of ER stress may be useful in combination therapies to overcome vemurafenib resistance.

Ca2+ signaling in antigen receptor‐activated B lymphocytes

Summary:  B cells respond to antigen stimulation with mobilization of the Ca2+ second messenger in two phases operated by two distinct sets of effector proteins. First, an antigen receptor‐specific Ca2+ initiation complex is assembled, activated, and targeted to the plasma membrane to trigger the transient release of Ca2+ from intracellular stores of the endoplasmic reticulum. Second, more ubiquitously expressed Ca2+ channels of the plasma membrane are opened to allow for sustained Ca2+ influx from the extracellular medium. Depending on the developmental stage of the B cell, the kinetics and profile of the two phases are adjusted at multiple levels of positive and negative regulation. A molecular basis for the Ca2+ signaling plasticity is provided by cytosolic and transmembrane adapter proteins. They act as signal organizers, which control enzyme/substrate interactions by directing the different signaling modules into specific subcellular compartments. These arrangements orchestrate a graduated activation of Ca2+‐sensitive downstream pathways, which ultimately determine appropriate cellular responses, namely elimination of autoreactive B cells or proliferation and differentiation of immunocompetent B cells into antibody‐secreting plasma cells.

Atg13 and FIP200 act independently of Ulk1 and Ulk2 in autophagy induction

Under normal growth conditions the mammalian target of rapamycin complex 1 (mTORC1) negatively regulates the central autophagy regulator complex consisting of Unc-51-like kinases 1/2 (Ulk1/2), focal adhesion kinase family-interacting protein of 200 kDa (FIP200) and Atg13. Upon starvation, mTORC1-mediated repression of this complex is released, which then leads to Ulk1/2 activation. In this scenario, Atg13 has been proposed as an adaptor mediating the interaction between Ulk1/2 and FIP200 and enhancing Ulk1/2 kinase activity. Using Atg13-deficient cells, we demonstrate that Atg13 is indispensable for autophagy induction. We further show that Atg13 function strictly depends on FIP200 binding. In contrast, the simultaneous knockout of Ulk1 and Ulk2 did not have a similar effect on autophagy induction. Accordingly, the Ulk1-dependent phosphorylation sites we identified in Atg13 are expendable for this process. This suggests that Atg13 has an additional function independent of Ulk1/2 and that Atg13 and FIP200 act in concert during autophagy induction.

The incredible ULKs.

Macroautophagy (commonly abbreviated as autophagy) is an evolutionary conserved lysosome-directed vesicular trafficking pathway in eukaryotic cells that mediates the lysosomal degradation of intracellular components. The cytoplasmic cargo is initially enclosed by a specific double membrane vesicle, termed the autophagosome. By this means, autophagy either helps to remove damaged organelles, long-lived proteins and protein aggregates, or serves as a recycling mechanism for molecular building blocks. Autophagy was once invented by unicellular organisms to compensate the fluctuating external supply of nutrients. In higher eukaryotes, it is strongly enhanced under various stress conditions, such as nutrient and growth factor deprivation or DNA damage. The serine/threonine kinase Atg1 was the first identified autophagy-related gene (ATG) product in yeast. The corresponding nematode homolog UNC-51, however, has additional neuronal functions. Vertebrate genomes finally encode five closely related kinases, of which UNC-51-like kinase 1 (Ulk1) and Ulk2 are both involved in the regulation of autophagy and further neuron-specific vesicular trafficking processes. This review will mainly focus on the vertebrate Ulk1/2-Atg13-FIP200 protein complex, its function in autophagy initiation, its evolutionary descent from the yeast Atg1-Atg13-Atg17 complex, as well as the additional non-autophagic functions of its components. Since the rapid nutrient- and stress-dependent cellular responses are mainly mediated by serine/threonine phosphorylation, it will summarize our current knowledge about the relevant upstream signaling pathways and the altering phosphorylation status within this complex during autophagy induction.

Subcellular localization of Grb2 by the adaptor protein Dok-3 restricts the intensity of Ca2+ signaling in B cells.

Spatial and temporal modulation of intracellular Ca2+ fluxes controls the cellular response of B lymphocytes to antigen stimulation. Herein, we identify the hematopoietic adaptor protein Dok‐3 (downstream of kinase‐3) as a key component of negative feedback regulation in Ca2+ signaling from the B‐cell antigen receptor. Dok‐3 localizes at the inner leaflet of the plasma membrane and is a major substrate for activated Src family kinase Lyn. Phosphorylated Dok‐3 inhibits antigen receptor‐induced Ca2+ elevation by recruiting cytosolic Grb2, which acts at this location as a negative regulator of Brutons tyrosine kinase. This leads to diminished activation of phospholipase C‐γ2 and reduced production of soluble inositol trisphosphate. Hence, the Dok‐3/Grb2 module is a membrane‐associated signaling organizer, which orchestrates the interaction efficiency of Ca2+‐mobilizing enzymes.

Triggering of a novel intrinsic apoptosis pathway by the kinase inhibitor staurosporine: activation of caspase-9 in the absence of Apaf-1

The protein kinase inhibitor staurosporine is one of the most potent and frequently used proapoptotic stimuli, although its mechanism of action is poorly understood. Here, we show that staurosporine as well as its analog 7‐hydroxystaurosporine (UCN‐01) not only trigger the classical mitochondrial apoptosis pathway but, moreover, activate an additional novel intrinsic apoptosis pathway. Unlike conventional anticancer drugs, staurosporine and UCN‐01 induced apoptosis in a variety of tumor cells overexpressing the apoptosis inhibitors Bcl‐2 and Bcl‐xL. Furthermore, activation of this novel intrinsic apoptosis pathway by staurosporine did not rely on Apaf‐1 and apoptosome formation, an essential requirement for the mitochondrial pathway. Nevertheless, as demonstrated in caspase‐9‐deficient murine embryonic fibroblasts, human lymphoma cells, and chicken DT40 cells, staurosporine‐induced apoptosis was essentially mediated by caspase‐9. Our results therefore suggest that, in addition to the classical cytochrome c/Apaf‐1‐dependent pathway of caspase‐9 activation, staurosporine can induce caspase‐9 activation and apoptosis independently of the apoptosome. Since staurosporine derivatives have proven efficacy in clinical trials, activation of this novel pathway might represent a powerful target to induce apoptosis in multidrug‐resistant tumor cells.— Manns, J., Daubrawa, M., Driessen, S., Paasch, F., Hoffmann, N., Löffler, A., Lauber, K., Dieterle, A., Alers, S., Iftner, T., Schulze‐Osthoff, K., Stork, B., Wesselborg, S. Triggering of a novel intrinsic apoptosis pathway by the kinase inhibitor staurosporine: activation of caspase‐9 in the absence of Apaf‐1. FASEB J. 25, 3250‐3261 (2011). www.fasebj.org

Interferon-γ and tumor necrosis factor-α sensitize primarily resistant human endometrial stromal cells to Fas-mediated apoptosis

The subtle interaction between the implanting embryo and the maternal endometrium plays a pivotal role during the process of implantation. Human endometrial stromal cells (ESCs) express Fas and the implanting trophoblast cells secrete Fas ligand (FASLG, FasL), suggesting a possible role for Fas-mediated signaling during early implantation. Here we show that ESCs are primarily resistant to Fas-mediated apoptosis independently of their state of hormonal differentiation. Pre-treatment of ESCs with interferon (IFN)-γ and tumor necrosis factor (TNF)-α sensitizes them to become apoptotic upon stimulation of Fas by an agonistic anti-Fas antibody. Incubation of ESCs with the early embryonic signal human chorionic gonadotropin (hCG, CGB) does not influence their reaction to Fas stimulation. The sensitizing effect of IFN-γ and TNF-α was accompanied by a significant upregulation of Fas and FLICE-inhibitory protein (FLIP, CFLAR) expression in ESCs. Additionally, we observed an activation of caspase 3, caspase 8 and caspase 9 upon apoptotic Fas triggering. In summary, we demonstrate that IFN-γ and TNF-α sensitize primarily apoptosis-resistant ESCs to Fas-mediated cell death. This might be due to an upregulation of Fas expression, and apoptosis seems to be mediated by active caspase 3, caspase 8 and caspase 9. The observed pro-apoptotic effect of IFN-γ and TNF-α on ESCs could play an important role in the modulation of early implantation.

The Akt inhibitor triciribine sensitizes prostate carcinoma cells to TRAIL-induced apoptosis

Aberrant PI3K/Akt signaling has been implicated in many human cancers, including prostate carcinomas. Currently different therapeutic strategies target the inhibition of this survival pathway. The nucleoside analog triciribine (TCN), which was initially described as a DNA synthesis inhibitor, has recently been shown to function as an inhibitor of Akt. Here, we demonstrate that TCN inhibits Akt phosphorylation at Thr308 and Ser473 and Akt activity in the human prostate cancer cell line PC‐3. In addition, TCN sensitized PC‐3 cells to TRAIL‐ and anti‐CD95‐induced apoptosis, whereas the cells remained resistant to DNA damaging chemotherapeutics. The observed sensitization essentially depended on the phosphorylation status of Akt. Thus, prostate cancer cell lines displaying constitutively active Akt, e.g. PC‐3 or LNCaP, were sensitized to death receptor‐induced apoptosis. Most importantly with respect to therapeutic application, derivatives of both TCN and TRAIL are already tested in current clinical trials. Therefore, this combinatorial treatment might open a promising therapeutic approach for the elimination of hormone‐refractory prostate cancers, which are largely resistant to conventional DNA damaging anticancer drugs or irradiation.