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Charles Betz Daniele Stracka Cristina Prescianotto-Baschong Maud Frieden Nicolas Demaurex Michael N. Hall 《Proceedings of the National Academy of Sciences of the United States of America》2013,110(31):12526-12534
The target of rapamycin (TOR) is a highly conserved protein kinase and a central controller of growth. Mammalian TOR complex 2 (mTORC2) regulates AGC kinase family members and is implicated in various disorders, including cancer and diabetes. Here we report that mTORC2 is localized to the endoplasmic reticulum (ER) subcompartment termed mitochondria-associated ER membrane (MAM). mTORC2 localization to MAM was growth factor-stimulated, and mTORC2 at MAM interacted with the IP3 receptor (IP3R)-Grp75–voltage-dependent anion-selective channel 1 ER-mitochondrial tethering complex. mTORC2 deficiency disrupted MAM, causing mitochondrial defects including increases in mitochondrial membrane potential, ATP production, and calcium uptake. mTORC2 controlled MAM integrity and mitochondrial function via Akt mediated phosphorylation of the MAM associated proteins IP3R, Hexokinase 2, and phosphofurin acidic cluster sorting protein 2. Thus, mTORC2 is at the core of a MAM signaling hub that controls growth and metabolism.Mitochondria-associated endoplasmic reticulum (ER) membrane (MAM) is a subcompartment of the ER that forms a quasisynaptic structure with mitochondria. The main function of this membrane is to facilitate the transfer of lipids and calcium between the two organelles. MAM thereby controls mitochondrial physiology and apoptosis (1, 2). MAM also mediates ER homeostasis and lipid biosynthesis by harboring chaperones and several key lipid synthesis enzymes (3–6). In mammalian MAM, the ER and mitochondria are physically tethered to each other by the IP3 receptor (IP3R)-Grp75-VDAC1 (voltage-dependent anion-selective channel 1) trimeric complex (7) and by dimers of the mitofusin (Mfn) proteins Mfn1 and Mfn2 (8) (Fig. S1H). The σ-1 receptor also stabilizes MAM by interacting with IP3R and VDAC (9). MAM formation is regulated by multiple signaling inputs, including calcium and possibly growth factors (10–12). However, the mechanism(s) that controls MAM formation is largely unknown other than it involves recruitment of MAM components by the MAM resident proteins phosphofurin acidic cluster sorting protein 2 (PACS2) and Rab32 (13–15). Akt, an AGC family kinase that is also found at MAM (16), phosphorylates PACS2 (17), but it remains to be determined whether Akt is involved in mediating MAM integrity.Akt, often up-regulated in cancer, also phosphorylates hexokinase 2 (HK2) to promote association of HK2 with the MAM protein VDAC1 (18, 19). This association, possibly at MAM (20, 21), enables HK2, using ATP exiting mitochondria through VDAC1, to phosphorylate glucose and thereby stimulate glycolysis (22). Conversely, upon inhibition of Akt, HK2 dissociates from VDAC1, causing VDAC1 closure and increased mitochondrial membrane potential (19). This regulation of HK2 by Akt has been proposed to account for enhanced glycolysis in cancer cells, also known as the Warburg effect (23). Furthermore, Akt regulates calcium release from MAM by phosphorylating IP3R, thereby controlling apoptosis (24–26). Thus, MAM is increasingly recognized as a signaling hub controlling cell physiology (15), and is implicated in a wide spectrum of diseases, including cancer, neurodegenerative disorders, inflammation, and infection (27).The target of rapamycin (TOR) pathway is a cellular signaling cascade that, like mitochondria, is present in all eukaryotes (28, 29). TOR integrates and relays signals from both extra- and intracellular sources (e.g., growth factors, nutrients, and cellular energy levels), and thereby instructs the cell to grow. TOR is found in two structurally and functionally distinct protein complexes that in mammalian cells are termed mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2) (30). mTORC2 comprises mTOR, rictor, mammalian lethal with SEC13 protein 8 (mLST8), stress-activated protein kinase (SAPK)-interacting protein (Sin1), and protor [also known as proline-rich protein 5 (PRR5)] (31), and phosphorylates AGC kinases, such as Akt, serum/glucocorticoid-regulated kinase 1 (SGK1), and PKC, all of which are linked to cancer and diabetes (32). Growth factors activate mTORC2 by promoting mTORC2-ribosome association in a PI3K-dependent manner (33, 34). mTORC2 is antiapoptotic, presumably via its role in phosphorylating and activating Akt (34–38).Various observations indicate that mTORC2 is linked to both the ER and mitochondria. Recent findings suggest that mTORC2 is at the ER, possibly through interaction with ER-bound ribosomes (34, 39). mTORC2 phosphorylates Akt at the ER (39, 40), and mTORC2 signaling is sensitive to ER stress (41, 42). In Chlamydomonas, TOR associates with membranes from the ER (43). With regard to mitochondria, mTOR has been observed in close proximity to the outer mitochondrial membrane (44), and mTOR and mLST8 interact with the mitochondrial outer-membrane protein VDAC1 (45) and the mitochondria-associated protein Grp75 (46), respectively. mTORC2 regulates the cellular distribution of mitochondria (47), and mTORC2-activated Akt is associated with mitochondria (18, 48, 49). Pink1, a regulator of mitochondrial function, has been implicated in mTORC2 activation (50). mTORC2-addicted cancer cells exhibit enhanced dependence on mitochondria, Rab32 and HK2 (51). Finally, Barquilla et al. reported that TORC2 in trypanosomes is localized to both ER and mitochondria (52). Thus, mTORC2 has been physically and functionally linked to both the ER and mitochondria.Here we investigate the localization of mTORC2. We show that ribosome-bound mTORC2 is at MAM. Localization to MAM is growth factor-dependent. MAM-associated mTORC2 activates Akt and thereby controls MAM integrity, mitochondrial metabolism, and cell survival. Thus, our findings describe a critical role for mTORC2 in a MAM signaling hub. 相似文献
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Morassa Mohseni Justin Cidado Sarah Croessmann Karen Cravero Ashley Cimino-Mathews Hong Yuen Wong Rob Scharpf Daniel J. Zabransky Abde M. Abukhdeir Joseph P. Garay Grace M. Wang Julia A. Beaver Rory L. Cochran Brian G. Blair D. Marc Rosen Bracha Erlanger Pedram Argani Paula J. Hurley Josh Lauring Ben Ho Park 《Proceedings of the National Academy of Sciences of the United States of America》2014,111(49):17606-17611
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Shengyi Sun Guojun Shi Xuemei Han Adam B. Francisco Yewei Ji Nuno Mendon?a Xiaojing Liu Jason W. Locasale Kenneth W. Simpson Gerald E. Duhamel Sander Kersten John R. Yates III Qiaoming Long Ling Qi 《Proceedings of the National Academy of Sciences of the United States of America》2014,111(5):E582-E591
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Bin Gong Thomas Shelite Fang C. Mei Tuha Ha Yaohua Hu Guang Xu Qing Chang Maki Wakamiya Thomas G. Ksiazek Paul J. Boor Donald H. Bouyer Vsevolod L. Popov Ju Chen David H. Walker Xiaodong Cheng 《Proceedings of the National Academy of Sciences of the United States of America》2013,110(48):19615-19620
Rickettsiae are responsible for some of the most devastating human infections. A high infectivity and severe illness after inhalation make some rickettsiae bioterrorism threats. We report that deletion of the exchange protein directly activated by cAMP (Epac) gene, Epac1, in mice protects them from an ordinarily lethal dose of rickettsiae. Inhibition of Epac1 suppresses bacterial adhesion and invasion. Most importantly, pharmacological inhibition of Epac1 in vivo using an Epac-specific small-molecule inhibitor, ESI-09, completely recapitulates the Epac1 knockout phenotype. ESI-09 treatment dramatically decreases the morbidity and mortality associated with fatal spotted fever rickettsiosis. Our results demonstrate that Epac1-mediated signaling represents a mechanism for host–pathogen interactions and that Epac1 is a potential target for the prevention and treatment of fatal rickettsioses.Rickettsiae are responsible for some of the most devastating human infections (1–4). It has been forecasted that temperature increases attributable to global climate change will lead to more widespread distribution of rickettsioses (5). These tick-borne diseases are caused by obligately intracellular bacteria of the genus Rickettsia, including Rickettsia rickettsii, the causative agent of Rocky Mountain spotted fever (RMSF) in the United States and Latin America (2, 3), and Rickettsia conorii, the causative agent of Mediterranean spotted fever endemic to southern Europe, North Africa, and India (6). A high infectivity and severe illness after inhalation make some rickettsiae (including Rickettsia prowazekii, R. rickettsii, Rickettsia typhi, and R. conorii) bioterrorism threats (7). Although the majority of rickettsial infections can be controlled by appropriate broad-spectrum antibiotic therapy if diagnosed early, up to 20% of misdiagnosed or untreated (1, 3) and 5% of treated RMSF cases (8) result in a fatal outcome caused by acute disseminated vascular endothelial infection and damage (9). Fatality rates as high as 32% have been reported in hospitalized patients diagnosed with Mediterranean spotted fever (10). In addition, strains of R. prowazekii resistant to tetracycline and chloramphenicol have been developed in laboratories (11). Disseminated endothelial infection and endothelial barrier disruption with increased microvascular permeability are the central features of SFG rickettsioses (1, 2, 9). The molecular mechanisms involved in rickettsial infection remain incompletely elucidated (9, 12). A comprehensive understanding of rickettsial pathogenesis and the development of novel mechanism-based treatment are urgently needed.Living organisms use intricate signaling networks for sensing and responding to changes in the external environment. cAMP, a ubiquitous second messenger, is an important molecular switch that translates environmental signals into regulatory effects in cells (13). As such, a number of microbial pathogens have evolved a set of diverse virulence-enhancing strategies that exploit the cAMP-signaling pathways of their hosts (14). The intracellular functions of cAMP are predominantly mediated by the classic cAMP receptor, protein kinase A (PKA), and the more recently discovered exchange protein directly activated by cAMP (Epac) (15). Thus, far, two isoforms, Epac1 and Epac2, have been identified in humans (16, 17). Epac proteins function by responding to increased intracellular cAMP levels and activating the Ras superfamily small GTPases Ras-proximate 1 and 2 (Rap1 and Rap2). Accumulating evidence demonstrates that the cAMP/Epac1 signaling axis plays key regulatory roles in controlling various cellular functions in endothelial cells in vitro, including cell adhesion (18–21), exocytosis (22), tissue plasminogen activator expression (23), suppressor of cytokine signaling 3 (SOCS-3) induction (24–27), microtubule dynamics (28, 29), cell–cell junctions, and permeability and barrier functions (30–37). Considering the critical importance of endothelial cells in rickettsioses, we examined the functional roles of Epac1 in rickettsial pathogenesis in vivo, taking advantage of the recently generated Epac1 knockout mouse (38) and Epac-specific inhibitors (39, 40) generated from our laboratory. Our studies demonstrate that Epac1 plays a key role in rickettsial infection and represents a therapeutic target for fatal rickettsioses. 相似文献
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Zhiping Wang Yuangang Liu Maho Takahashi Kathryn Van Hook Kerstin M. Kampa-Schittenhelm Brett C. Sheppard Rosalie C. Sears Philip J. S. Stork Charles D. Lopez 《Proceedings of the National Academy of Sciences of the United States of America》2013,110(1):312-317
The ASPP2 (also known as 53BP2L) tumor suppressor is a proapoptotic member of a family of p53 binding proteins that functions in part by enhancing p53-dependent apoptosis via its C-terminal p53-binding domain. Mounting evidence also suggests that ASPP2 harbors important nonapoptotic p53-independent functions. Structural studies identify a small G protein Ras-association domain in the ASPP2 N terminus. Because Ras-induced senescence is a barrier to tumor formation in normal cells, we investigated whether ASPP2 could bind Ras and stimulate the protein kinase Raf/MEK/ERK signaling cascade. We now show that ASPP2 binds to Ras–GTP at the plasma membrane and stimulates Ras-induced signaling and pERK1/2 levels via promoting Ras–GTP loading, B-Raf/C-Raf dimerization, and C-Raf phosphorylation. These functions require the ASPP2 N terminus because BBP (also known as 53BP2S), an alternatively spliced ASPP2 isoform lacking the N terminus, was defective in binding Ras–GTP and stimulating Raf/MEK/ERK signaling. Decreased ASPP2 levels attenuated H-RasV12–induced senescence in normal human fibroblasts and neonatal human epidermal keratinocytes. Together, our results reveal a mechanism for ASPP2 tumor suppressor function via direct interaction with Ras–GTP to stimulate Ras-induced senescence in nontransformed human cells.ASPP2, also known as 53BP2L, is a tumor suppressor whose expression is altered in human cancers (1). Importantly, targeting of the ASPP2 allele in two different mouse models reveals that ASPP2 heterozygous mice are prone to spontaneous and γ-irradiation–induced tumors, which rigorously demonstrates the role of ASPP2 as a tumor suppressor (2, 3). ASPP2 binds p53 via the C-terminal ankyrin-repeat and SH3 domain (4–6), is damage-inducible, and can enhance damage-induced apoptosis in part through a p53-mediated pathway (1, 2, 7–10). However, it remains unclear what biologic pathways and mechanisms mediate ASPP2 tumor suppressor function (1). Indeed, accumulating evidence demonstrates that ASPP2 also mediates nonapoptotic p53-independent pathways (1, 3, 11–15).The induction of cellular senescence forms an important barrier to tumorigenesis in vivo (16–21). It is well known that oncogenic Ras signaling induces senescence in normal nontransformed cells to prevent tumor initiation and maintain complex growth arrest pathways (16, 18, 21–24). The level of oncogenic Ras activation influences its capacity to activate senescence; high levels of oncogenic H-RasV12 signaling leads to low grade tumors with senescence markers, which progress to invasive cancers upon senescence inactivation (25). Thus, tight control of Ras signaling is critical to ensure the proper biologic outcome in the correct cellular context (26–28).The ASPP2 C terminus is important for promoting p53-dependent apoptosis (7). The ASPP2 N terminus may also suppress cell growth (1, 7, 29–33). Alternative splicing can generate the ASPP2 N-terminal truncated protein BBP (also known as 53BP2S) that is less potent in suppressing cell growth (7, 34, 35). Although the ASPP2 C terminus mediates nuclear localization, full-length ASPP2 also localizes to the cytoplasm and plasma membrane to mediate extranuclear functions (7, 11, 12, 36). Structural studies of the ASPP2 N terminus reveal a β–Grasp ubiquitin-like fold as well as a potential Ras-binding (RB)/Ras-association (RA) domain (32). Moreover, ASPP2 can promote H-RasV12–induced senescence (13, 15). However, the molecular mechanism(s) of how ASPP2 directly promotes Ras signaling are complex and remain to be completely elucidated.Here, we explore the molecular mechanisms of how Ras-signaling is enhanced by ASPP2. We demonstrate that ASPP2: (i) binds Ras-GTP and stimulates Ras-induced ERK signaling via its N-terminal domain at the plasma membrane; (ii) enhances Ras-GTP loading and B-Raf/C-Raf dimerization and forms a ASPP2/Raf complex; (iii) stimulates Ras-induced C-Raf phosphorylation and activation; and (iv) potentiates H-RasV12–induced senescence in both primary human fibroblasts and neonatal human epidermal keratinocytes. These data provide mechanistic insight into ASPP2 function(s) and opens important avenues for investigation into its role as a tumor suppressor in human cancer. 相似文献
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Shusuke Yonekawa Akiko Furuno Takashi Baba Yukio Fujiki Yuta Ogasawara Akitsugu Yamamoto Mitsuo Tagaya Katsuko Tani 《Proceedings of the National Academy of Sciences of the United States of America》2011,108(31):12746-12751
Sec16 plays a key role in the formation of coat protein II vesicles, which mediate protein transport from the endoplasmic reticulum (ER) to the Golgi apparatus. Mammals have two Sec16 isoforms: Sec16A, which is a longer primary ortholog of yeast Sec16, and Sec16B, which is a shorter distant ortholog. Previous studies have shown that Sec16B, as well as Sec16A, defines ER exit sites, where coat protein II vesicles are formed in mammalian cells. Here, we reveal an unexpected role of Sec16B in the biogenesis of mammalian peroxisomes. When overexpressed, Sec16B was targeted to the entire ER, whereas Sec16A was mostly cytosolic. Concomitant with the overexpression of Sec16B, peroxisomal membrane biogenesis factors peroxin 3 (Pex3) and Pex16 were redistributed from peroxisomes to Sec16B-positive ER membranes. Knockdown of Sec16B but not Sec16A by RNAi affected the morphology of peroxisomes, inhibited the transport of Pex16 from the ER to peroxisomes, and suppressed expression of Pex3. These phenotypes were significantly reversed by the expression of RNAi-resistant Sec16B. Together, our results support the view that peroxisomes are formed, at least partly, from the ER and identify a factor responsible for this process.Most eukaryotic cells contain peroxisomes, which are single membrane-bound organelles that function in various metabolic pathways, including the β-oxidation of fatty acids, biosynthesis of plasmalogens and bile acids, and hydrogen peroxide metabolism (1). To perform this variety of functions, peroxisomes are highly dynamic; their number, size, and function change in response to cellular conditions. In addition, unlike mitochondria, peroxisomes can be formed through de novo synthesis as well as through the growth and division of preexisting peroxisomes (2, 3).Peroxisomal matrix proteins are synthesized on free ribosomes in the cytosol and posttranslationally imported to peroxisomes (4). This import pathway includes the recognition of two distinct peroxisomal targeting signals (PTS1 and PTS2) by peroxin 5 (Pex5) and Pex7, respectively, followed by translocation across the membrane through the import machinery, including Pex14 and Really Interesting New Gene peroxins (5, 6). The import pathway for peroxisomal membrane proteins (PMPs), on the other hand, is believed to be independent of that used by matrix proteins. Genetic phenotype complementation analysis of yeast and mammalian mutants devoid of peroxisome membranes revealed that Pex3, Pex16, and Pex19 are essential for PMP import (references in ref. 7). Pex3 is a PMP import receptor (8), and Pex19 is a chaperone and import receptor for most PMPs (9). Pex16 appears to function as a Pex3-Pex19 receptor in mammals (7) and as a negative regulator of peroxisome fission in yeast Yarrowia lipolytica (10) but is absent in Saccharomyces cerevisiae (11).Although compelling evidence suggests that PMPs are transported directly from the cytosol to peroxisomes (7–9, 12), recent work has suggested that some PMPs, including the PMP import receptors Pex3 and Pex16, seem to be, at least partly, transported from the endoplasmic reticulum (ER) en route to peroxisomes (13). In addition, several lines of evidence suggest that the ER participates in the de novo formation of peroxisomes (13–20). A very recent study involving a yeast cell-free system revealed that ER-peroxisome carriers are formed in a Pex19-dependent manner (21).In this report, we show that Sec16B plays an important role in the transport of Pex16 from the ER to peroxisomes in mammalian cells. Sec16 was first characterized in yeast S. cerevisiae as a 240-kDa peripheral membrane protein that interacts with coat protein II (COPII) coat components and facilitates their assembly and vesicle budding (22–25). In yeast Pichia pastoris, Sec16 defines ER exit sites (ERESs) (26), special domains where COPII-coated vesicles are formed (27). There are two mammalian orthologs, Sec16A (250 kDa) and Sec16B (117 kDa) (also referred to as Sec16L and Sec16S, respectively) (28–30). Sec16A, which is localized in cup-like structures in ERESs (31), appears to be the primary Sec16 ortholog because its molecular mass is similar to that of Sec16 in yeast (22) and Drosophila (32). Sec16B, which appears to be conserved in vertebrates, is also localized in ERESs, but its function has not been fully examined in the context of membrane trafficking. Our results suggest that Sec16B may participate in the formation of new peroxisomes derived from the ER. 相似文献
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Sarvjeet Singh Diana C. Canseco Shilpa M. Manda John M. Shelton Rajendra R. Chirumamilla Sean C. Goetsch Qiu Ye Robert D. Gerard Jay W. Schneider James A. Richardson Beverly A. Rothermel Pradeep P. A. Mammen 《Proceedings of the National Academy of Sciences of the United States of America》2014,111(1):E129-E138
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C. Dustin Rubinstein Mariana F. Wolfner 《Proceedings of the National Academy of Sciences of the United States of America》2013,110(43):17420-17425
Across animal taxa, seminal proteins are important regulators of female reproductive physiology and behavior. However, little is understood about the physiological or molecular mechanisms by which seminal proteins effect these changes. To investigate this topic, we studied the increase in Drosophila melanogaster ovulation behavior induced by mating. Ovulation requires octopamine (OA) signaling from the central nervous system to coordinate an egg’s release from the ovary and its passage into the oviduct. The seminal protein ovulin increases ovulation rates after mating. We tested whether ovulin acts through OA to increase ovulation behavior. Increasing OA neuronal excitability compensated for a lack of ovulin received during mating. Moreover, we identified a mating-dependent relaxation of oviduct musculature, for which ovulin is a necessary and sufficient male contribution. We report further that oviduct muscle relaxation can be induced by activating OA neurons, requires normal metabolic production of OA, and reflects ovulin’s increasing of OA neuronal signaling. Finally, we showed that as a result of ovulin exposure, there is subsequent growth of OA synaptic sites at the oviduct, demonstrating that seminal proteins can contribute to synaptic plasticity. Together, these results demonstrate that ovulin increases ovulation through OA neuronal signaling and, by extension, that seminal proteins can alter reproductive physiology by modulating known female pathways regulating reproduction.Throughout internally fertilizing animals, seminal proteins play important roles in regulating female fertility by altering female physiology and, in some cases, behavior after mating (reviewed in refs. 1–3). Despite this, little is understood about the physiological mechanisms by which seminal proteins induce postmating changes and how their actions are linked with known networks regulating female reproductive physiology.In Drosophila melanogaster, the suite of seminal proteins has been identified, as have many seminal protein-dependent postmating responses, including changes in egg production and laying, remating behavior, locomotion, feeding, and in ovulation rate (reviewed in refs. 2 and 3). For example, the Drosophila seminal protein ovulin elevates ovulation rate to maximal levels during the 24 h following mating (4, 5), and the seminal protein sex peptide (SP) suppresses female mating receptivity and increases egg-laying behavior for several days after mating (6–10). However, although a receptor for SP has been identified (11), along with elements of the neural circuit in which it is required (12–14), SP’s mechanism of action has not yet been linked to regulatory networks known to control postmating behaviors. Thus, a crucial question remains: how do male-derived seminal proteins interact with regulatory networks in females to trigger postmating responses?We addressed this question by examining the stimulation of Drosophila ovulation by the seminal protein ovulin. In insects, ovulation, defined here as the release of an egg from the ovary to the uterus, is among the best understood reproductive processes in terms of its physiology and neurogenetics (15–27). In D. melanogaster, ovulation requires input from neurons in the abdominal ganglia that release the catecholaminergic neuromodulators octopamine (OA) and tyramine (17, 18, 28). Drosophila ovulation also requires an OA receptor, OA receptor in mushroom bodies (OAMB) (19, 20). Moreover, it has been proposed that OA may integrate extrinsic factors to regulate ovulation rates (17). Noradrenaline, the vertebrate structural and functional equivalent to OA (29, 30), is important for mammalian ovulation, and its dysregulation has been associated with ovulation disorders (31–38). In this paper we investigate the role of neurons that release OA and tyramine in ovulin’s action. For simplicity, we refer to these neurons as “OA neurons” to reflect the well-established role of OA in ovulation behavior (16–20, 22).We investigated how action of the seminal protein ovulin relates to the conserved canonical neuromodulatory pathway that regulates ovulation physiology (39–41). We found that ovulin increases ovulation and egg laying through OA neuronal signaling. We also found that ovulin relaxes oviduct muscle tonus, a postmating process that is also mediated by OA neuronal signaling. Finally, subsequent to these effects we detected an ovulin-dependent increase in synaptic sites between OA motor neurons and oviduct muscle, suggesting that ovulin’s stimulation of OA neurons could have increased their synaptic activity. These results suggest that ovulin affects ovulation by manipulating the gain of a neuromodulatory pathway regulating ovulation physiology. 相似文献
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Xi Jiang Hao Huang Zejuan Li Chunjiang He Yuanyuan Li Ping Chen Sandeep Gurbuxani Stephen Arnovitz Gia-Ming Hong Colles Price Haomin Ren Rejani B. Kunjamma Mary Beth Neilly Justin Salat Mark Wunderlich Robert K. Slany Yanming Zhang Richard A. Larson Michelle M. Le Beau James C. Mulloy Janet D. Rowley Jianjun Chen 《Proceedings of the National Academy of Sciences of the United States of America》2012,109(47):19397-19402