The target of rapamycin (TOR) complex 1 (TORC1) signaling pathway is a critical regulator of translation and cell growth. To identify novel components of this pathway, we performed a kinome-wide RNA interference (RNAi) screen in
Drosophila melanogaster S2 cells. RNAi targeting components of the p38 stress-activated kinase cascade prevented the cell size increase elicited by depletion of the TOR negative regulator TSC2. In mammalian and
Drosophila tissue culture, as well as in
Drosophila ovaries
ex vivo, p38-activating stresses, such as H
2O
2 and anisomycin, were able to activate TORC1. This stress-induced TORC1 activation could be blocked by RNAi against mitogen-activated protein kinase kinase 3 and 6 (MKK3/6) or by the overexpression of dominant negative Rags. Interestingly, p38 was also required for the activation of TORC1 in response to amino acids and growth factors. Genetic ablation either of
p38b or
licorne, its upstream kinase, resulted in small flies consisting of small cells. Mutants with mutations in
licorne or
p38b are nutrition sensitive; low-nutrient food accentuates the small-organism phenotypes, as well as the partial lethality of the
p38b null allele. These data suggest that p38 is an important positive regulator of TORC1 in both mammalian and
Drosophila systems in response to certain stresses and growth factors.The target of rapamycin, TOR, is a highly conserved serine/threonine kinase that is a critical regulator of cell growth. It is a core component of two signaling complexes, TORC1 and TORC2 (
60,
74). TORC1 is defined by the presence of Raptor in the complex, while TORC2 contains Rictor. Rictor and Raptor are mutually exclusive. Activation of the TORC1 pathway leads to increased protein translation, increased cell size, and increased proliferation, making this pathway an important target for emerging cancer therapies. Rapamycin is an inhibitor of TORC1 that is commonly used as an immunosuppressant following kidney transplantation (
51). At least three analogs of rapamycin are currently being tested in solid and hematological tumors and have shown some promising results (
21).The TORC1 pathway responds to numerous inputs, sensing both the desirability of and the capacity for growth. Many of these pathways control TORC1 signaling through phosphorylation of the tuberous sclerosis protein TSC2. TSC2 associates with TSC1 to form a heterodimeric GTPase-activating protein complex (GAP) that inactivates the small GTPase Rheb (
24,
29,
67). While the exact molecular mechanism remains a topic of debate, activation of Rheb promotes the kinase activity of TORC1 (
24,
29,
67). Rheb is required for the activation of TORC1 in response to both amino acids and growth factors (
55,
62). In
Drosophila melanogaster, mutation of either TOR or Rheb inhibits growth, leading to reduced body size and reduced cell size in mutant clones (
42,
64). Mutation of either TSC1 or TSC2 has the predicted opposite effect, as tissue deficient for either of these proteins overgrows and contains large cells (
49,
66).TORC1 is activated via the phosphatidylinositol 3′ kinase (PI3′K) pathway by growth-promoting mitogens, such as insulin and growth factors.
Drosophila mutants with mutations of PI3′K pathway components have size phenotypes similar to those of the TOR and Rheb mutants (
71). In mammalian cells, the PI3′K-mediated activation of TORC1 occurs at least in part through the phosphorylation of TSC2 by the PI3′K target AKT (
30,
50). Interestingly, mutation of these residues in
Drosophila has no impact on TSC2 function
in vivo, suggesting that there may be other mechanisms through which PI3′K can activate
Drosophila TOR (
20). Recent work has suggested that the proline-rich AKT substrate PRAS40 may provide part of this link (
23,
59,
69,
70). In addition, signaling through RAS activates extracellular signal-regulated kinase (ERK) and ribosomal S6 kinase (RSK), which can phosphorylate TSC2 and Raptor to activate TORC1 (
13,
40,
56). There are also likely to be additional mechanisms through which growth factors activate
Drosophila TOR that have not yet been identified.TORC1 activity is also controlled by the intracellular building blocks necessary to support cellular growth. The energy-sensing AMP-activated protein kinase (AMPK) pathway relays information about the energy status of the cell to TORC1 by phosphorylating TSC2. Unlike the inactivating phosphorylation of TSC2 by Akt, phosphorylation of TSC2 by AMPK promotes the GAP activity of the TSC complex (
31). AMPK also phosphorylates Raptor, leading to decreased TORC1 activity (
28). Thus, when energy levels are low, active AMPK inhibits TORC1.Amino acids also activate the TORC1 pathway, through a mechanism that requires Rheb, as well as the type III PI3′K VPS34 and the serine/threonine kinase mitogen-activated protein kinase kinase kinase kinase 3 (MAP4K3) (
11,
22,
43). TORC1 thereby integrates information about the availability of amino acids and the amount of energy available for growth with growth factor signaling. Given its ancient function in adapting growth rates to environmental conditions, it is likely that TOR responds to a variety of stimuli, suggesting that many TOR control mechanisms remain to be uncovered. The Rag family of Ras-related small GTPases has recently been identified as a key component of the amino acid-sensing pathway, acting in parallel to Rheb (
34,
58). Rag GTPases form heterodimers; RagA or RagB interacts with RagC or RagD. RagA and RagB are active when GTP bound, while RagC and RagD are active when bound to GDP (
34,
58). Activation of the Rags by amino acids results in TOR relocalization to Rab7-containing vesicles (
58). While the function of these vesicles in TORC1 signaling remains unclear, this relocalization is associated with increased TORC1 activity.TORC1 controls cell growth and translation through the phosphorylation and activation of components of the translational machinery, such as S6 kinase (S6K) and 4EBP1, an inhibitor of eukaryotic translation initiation factor 4E (eIF4E) activity (reviewed in reference
27). S6K phosphorylates the S6 ribosomal subunit, thereby increasing translation. Mice deficient for S6K1 are small and have small pancreatic beta cells and a correspondingly low level of circulating insulin (
45). Mutation of the phosphorylation sites on S6 results in a similar phenotype, with small beta cells and fibroblasts (
57). In
Drosophila, mutation of S6K again reduces both cell and organism size (
42), as does the overexpression of 4EBP (
41).Interestingly, while mutation of the TORC1 pathway in mammalian cells reduces cell size by 10 to 15%, ablation of core TORC1 pathway components in
Drosophila cells can affect cell size by up to 40% (
73). In an attempt to identify novel components of the TORC1 pathway, we undertook an RNA interference (RNAi)-based screen of
Drosophila S2 cells. We reasoned that the extreme size phenotypes observed in
Drosophila cells upon TORC1 manipulations would facilitate the identification of modulators. In order to increase the likelihood of isolating novel regulators of TOR, we uncoupled TOR activity from many of its known nutritional controls by depleting TSC2 and screened for double-stranded RNAs (dsRNAs) that could reverse the cell size increase elicited by loss of TSC2. Depletion of multiple components of the p38 pathway was found to revert the TSC2 RNAi-induced cell size increase. Furthermore, activation of p38 is necessary and sufficient for the activation of TOR. Strikingly, mutation of components of the stress-activated p38 pathway in
Drosophila has a similar phenotype to mutations in the TOR and insulin signaling pathway: a cell-autonomous cell size decrease, reduced body size, and a sensitization to the effects of nutritional stress.
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