Receptor for Activated C-Kinase (RACK1) Homolog Cpc2 Facilitates the General Amino Acid Control Response through Gcn2 Kinase in Fission Yeast

General amino acid control (GAAC) is crucial for sensing and adaptation to nutrient availability. Amino acid starvation activates protein kinase Gcn2, which plays a central role in the GAAC response by phosphorylating the α-subunit of eukaryotic initiation factor 2 (eIF2α), leading to the translational switch to stimulate selective expression of stress-responsive genes. We report here that in fission yeast Schizosaccharomyces pombe, Cpc2, a homolog of mammalian receptor for activated C-kinase (RACK1), is important for the GAAC response. Deletion of S. pombe cpc2 impairs the amino acid starvation-induced phosphorylation of eIF2α and the expression of amino acid biosynthesis genes, thereby rendering cells severely sensitive to amino acid limitation. Unlike the Saccharomyces cerevisiae Cpc2 ortholog, which normally suppresses the GAAC response, our findings suggest that S. pombe Cpc2 promotes the GAAC response. We also found that S. pombe Cpc2 is required for starvation-induced Gcn2 autophosphorylation, which is essential for Gcn2 function. These results indicate that S. pombe Cpc2 facilitates the GAAC response through the regulation of Gcn2 activation and provide a novel insight for the regulatory function of RACK1 on Gcn2-mediated GAAC response.     

Diversity of amino acid signaling pathways on autophagy regulation: A novel pathway for arginine

Autophagy is the intracellular bulk degradation process to eliminate damaged cellular machinery and to recycle building blocks, and is crucial for cell survival and cell death. Amino acids modulate autophagy in response to nutrient starvation and oxidative stress. We investigated the relevance of reactive oxygen species (ROS) production on the regulation of autophagy using amino acids, both as a mixture and individually, in rat hepatoma H4-II-E cells. Nutrient starvation elevated ROS production and stimulated autophagy. Treatment with complete (CAA), regulatory (RegAA) and non-regulatory (NonRegAA) amino acid mixtures showed significant suppression of ROS production, whereas only CAA and RegAA exhibited significant suppression of autophagy, suggesting a dissociation of the two responses. The effects of individual amino acids were examined. Leucine from RegAA decreased ROS production and suppressed autophagy. However, methionine and proline from RegAA and arginine, cystine and glutamic acid from NonRegAA suppressed autophagy with an opposite increase in ROS production. Other amino acids from the NonRegAA group showed stimulating effects on ROS production without an autophagic response. Arginine’s effect on autophagy suppression was not blocked by rapamycin, indicating an mTOR-independent pathway. Inhibitor studies on arginine-regulated autophagy may indicate the involvement of NO pathway, which is independent from ROS and mTOR pathways.     

Diacylglycerol kinase inhibitor R59022-induced autophagy and apoptosis in the neuronal cell line NG108-15

Phosphatidic acid (PA) is a lipid second messenger and is believed to be involved in cell proliferation and survival. PA is mainly produced by phospholipase D (PLD) and diacylglycerol kinase (DGK). Elevated PLD activity is believed to suppress apoptosis via activation of the mammalian target of rapamycin (mTOR). On the other hand, DGK inhibition has been demonstrated to induce apoptosis, but it is unclear whether DGK can regulate mTOR. Here, we investigated whether DGK inhibition can induce apoptosis and autophagy in neuronal cells, since mTOR is a key mediator of autophagy and the simultaneous activation of apoptosis and autophagy has been detected. A DGK inhibitor, R59022 induced autophagy and apoptosis without serum in NG108-15 cells. Autophagy preceded apoptosis, and apoptosis inhibition did not affect R59022-induced autophagy. R59022-induced autophagy was inhibited by exogenous PA, and protein kinase C activation and increases in intracellular Ca²⁺ levels, which are assumed to be caused by diacylglycerol accumulation, did not appear to be involved in R59022-induced autophagy. We also investigated the effects of R59022 on mTOR signaling pathway, and found that the pathway was not inhibited by R59022. These results imply that DGK plays an important role in cell survival via mTOR-independent mechanism.     

IL-27 inhibits IFN-γ induced autophagy by concomitant induction of JAK/PI3 K/Akt/mTOR cascade and up-regulation of Mcl-1 in Mycobacterium tuberculosis H37Rv infected macrophages

Interleukin-27 (IL-27), a key immunoregulatory cytokine plays an important role in host response to mycobacterial infection as neutralization of IL-27 augments intracellular killing of mycobacteria. Autophagy has a pivotal role in host immunity and is regulated by various cytokines. Here, we report that IL-27 inhibits IFN-γ and starvation induced autophagy and as a result blocks phagosome maturation and promotes intracellular survival of Mycobacterium tuberculosis H37Rv. Addition of exogenous IL-27 induces the activation of mTOR through JAK/PI3 K pathway and inhibits IFN-γ stimulated autophagy. Furthermore, blockade of JAKs obstructs the inhibitory effect of IL-27 on IFN-γ induced autophagy. Besides this, IL-27 also up-regulates Mcl-1through PI3 K pathway. We further show that in mTOR or Mcl-1 silenced THP-1 cells, IL-27 could no longer inhibit IFN-γ mediated autophagy in M. tuberculosis H37Rv infected cells. Altogether, our study demonstrates that IL-27 by concurrent activation of JAK/PI3 K/Akt/mTOR cascade as well as up-regulation of Mcl-1 inhibits IFN-γ induced autophagy and elimination of intracellular mycobacteria in macrophages.     

Autophagy: regulation by energy sensing

Autophagy is inhibited by the mTOR signaling pathway, which is stimulated by increased amino acid levels. When cellular energy production is compromised, AMP-activated protein kinase is activated, mTOR is inhibited and autophagy is stimulated. Two recent studies have shed light on the molecular mechanism by which AMPK controls autophagic flux.     

Modulation of intracellular ROS levels by TIGAR controls autophagy

The p53‐inducible TIGAR protein functions as a fructose‐2,6‐bisphosphatase, promoting the pentose phosphate pathway and helping to lower intracellular reactive oxygen species (ROS). ROS functions in the regulation of many cellular responses, including autophagy—a response to stress conditions such as nutrient starvation and metabolic stress. In this study, we show that TIGAR can modulate ROS in response to nutrient starvation or metabolic stress, and functions to inhibit autophagy. The ability of TIGAR to limit autophagy correlates strongly with the suppression of ROS, with no clear effects on the mTOR pathway, and is p53 independent. The induction of autophagy in response to loss of TIGAR can function to moderate apoptotic response by restraining ROS levels. These results reveal a complex interplay in the regulation of ROS, autophagy and apoptosis in response to TIGAR expression, and shows that proteins similar to TIGAR that regulate glycolysis can have a profound effect on the autophagic response through ROS regulation.     

Mitochondrial respiration links TOR complex 2 signaling to calcium regulation and autophagy

The target of rapamycin (TOR) kinase is a conserved regulator of cell growth and functions within 2 different protein complexes, TORC1 and TORC2, where TORC2 positively controls macroautophagy/autophagy during amino acid starvation. Under these conditions, TORC2 signaling inhibits the activity of the calcium-regulated phosphatase calcineurin and promotes the general amino acid control (GAAC) response and autophagy. Here we demonstrate that TORC2 regulates calcineurin by controlling the respiratory activity of mitochondria. In particular, we find that mitochondrial oxidative stress affects the calcium channel regulatory protein Mid1, which we show is an essential upstream activator of calcineurin. Thus, these findings describe a novel regulation for autophagy that involves TORC2 signaling, mitochondrial respiration, and calcium homeostasis.     

Leucine limitation induces autophagy and activation of lysosome-dependent proteolysis in C2C12 myotubes through a mammalian target of rapamycin-independent signaling pathway

Loss of muscle mass usually characterizes different pathologies (sepsis, cancer, trauma) and also occurs during normal aging. One reason for muscle wasting relates to a decrease in food intake. This study addressed the role of leucine as a regulator of protein breakdown in mouse C2C12 myotubes and aimed to determine which cellular responses regulate the process. Determination of the rate of protein breakdown indicated that leucine is one key regulator of this process in myotubes because starvation for this amino acid is responsible for 30-40% of the total increase generated by total amino acid starvation. Leucine restriction rapidly accelerates the rate of protein breakdown (+11 to 15% (p < 0.001) after 1 h of starvation) in a dose-dependent manner. By using various inhibitors, evidence is provided that acceleration of protein catabolism results mainly from an induction of autophagy, activation of lysosome-dependent proteolysis, without modification of mRNA levels encoding the lysosomal cathepsins B, L, or D. Those results suggest that autophagy is an essential cellular response for increasing protein breakdown in muscle following food deprivation. Induction of autophagy precedes a decrease in global protein synthesis (-20% to -30% (p < 0.001)) that occurs after 3 h of leucine starvation. Inhibition of the mammalian target of rapamycin (mTOR) activity does not abolish the effect of leucine starvation and the level of phosphorylated ribosomal S6 protein is not affected by leucine withdrawal. These latter data provide clear evidence that the mTOR signaling pathway is not involved in the mediation of leucine effects on both protein synthesis and degradation in C2C12 myotubes.     

Lysosome positioning coordinates mTORC1 activity and autophagy

Under nutrient-rich conditions, the nutrient-sensitive kinase mTOR (mammalian target of rapamycin) is recruited to the surface of lysosomes where it becomes activated and can promote cell growth and inhibit autophagy. In contrast, mTOR is inhibited in nutrient-poor conditions, leading to the induction of autophagy. The intracellular positioning of lysosomes in response to nutrient availability is now shown to orchestrate mTOR activation and regulate autophagy.     

Autophagy gets a brake

Autophagy, a highly regulated catabolic process, is controlled by the action of positive and negative regulators. While many of the positive mediators of autophagy have been identified, very little is known about negative regulators that might counterbalance the process. We recently identified deathassociated protein 1 (DAP1) as a suppressor of autophagy and as a novel direct substrate of mammalian target of rapamycin (mTOR). We found that DAP1 is functionally silent in cells growing under rich nutrient supplies through mTOR-dependent inhibitory phosphorylation on two sites, which were mapped to Ser3 and Ser51. During amino acid starvation, mTOR activity is turned off resulting in a rapid reduction in the phosphorylation of DAP1. This caused the conversion of the protein into a suppressor of autophagy, thus providing a buffering mechanism that counterbalances the autophagic flux and prevents its overactivation under conditions of nutrient deprivation. Based on these studies we propose the “gas and brake” concept in which mTOR, the main sensor that regulates autophagy in response to amino acid deprivation, also controls the activity of a specific balancing brake to prevent the overactivation of autophagy.