The insulin/PI3K signaling pathway controls both tissue growth and metabolism. Melted has been identified as a new modulator of this pathway in Drosophila. Melted interacts with both Tsc1 and Foxo and can recruit these proteins to the cell membrane. Evidence is provided that in the melted mutant, Tor activity is reduced and Foxo is activated. The melted mutant condition mimics the effects of nutrient deprivation in a normal animal, producing an animal with 40% less fat than normal (Teleman, 2005).

The insulin/PI3K signaling pathway acts via a series of protein relocalization and phosphorylation events to relay information from the cell's environment -- including growth-factor levels and nutrient availability -- into the cell where it controls gene expression (via Foxo) and protein translation (via Tor). Stimulation of the insulin receptor activates PI3K, which increases the level of phosphoinositol (3, 4, 5) triphosphate (PIP3) at the plasma membrane. PIP3 binding recruits the protein kinase Akt to the membrane, where it is activated through phosphorylation by Pdk1. Insulin-regulated Akt acts through two effector pathways: winged helix-Forkhead family transcription factors and the protein kinase Tor. Tor regulates the translational machinery of the cell via two well-characterized effectors: S6K and 4E binding protein (4E-BP). S6K controls ribosome biogenesis and, thus, the biosynthetic capacity of the cell. 4E-BP binds to the translation factor eIF4E and prevents assembly of a protein complex that facilitates recruitment of the ribosome (Teleman, 2005).

In addition to mediating insulin-responsiveness, the Tor pathway also integrates information on cellular nutritional status and stress from the heterodimeric Tsc1/2 complex. Tsc2 serves as a GTPase-activating protein that inactivates Rheb and, thereby, reduces Tor activity. Tsc1/2 mutations result in Tor hyperactivation and tissue overgrowth. Mutants in mouse Tsc1 or Tsc2 have benign overgrowths called harmatomas and show increased susceptibility to tumor formation. Cellular AMP levels are sensed by AMP-activated protein kinase (AMPK) which phosphorylates and activates Tsc2 to inhibit Tor. Tor activity is also regulated by oxygen, through the hypoxia-induced transcription factor, HIF. HIF controls expression of REDD1/Scylla/Charybdis, which reduce Tsc1/2 activity (Teleman, 2005).

Mutations in the core components of the pathway -- the insulin receptor (InR), phosphoinositide-3-kinase (PI3K), Pdk1, Akt/PKB, tuberous sclerosis complex (Tsc1/Tsc2), Rheb, the target of rapamycin (Tor), and ribosomal protein S6 kinase (S6K) -- all cause tissue growth abnormalities or lethality. A considerable body of evidence indicates that the PI3K pathway controls metabolism as well as tissue growth. Flies and mice with reduced InR/IGF receptor and PI3K activity are small and have elevated fat levels. Mice mutant for Akt2 become insulin resistant and develop lipoatrophy as they age. Humans mutant for Akt2 also have abnormal metabolism and are 35% leaner than average. One output branch of the core pathway is via Foxo transcription factors. In response to insulin, Akt phosphorylates Foxo proteins: this promotes their interaction with 14-3-3 proteins and leads to cytoplasmic retention and inactivation. Foxo transcription factors have been implicated in the control of fat metabolism and lifespan in C. elegans, flies, and mice. The Tor branch of the pathway is also beginning to be implicated in fat metabolism. Tor phosphorylates and regulates S6K and 4E-BP. S6K mutant mice are resistant to diet induced obesity. 4E-BP1 mutant mice have a defect in fat metabolism. 4E-BP also controls fat metabolism in the fly. Recent studies have identified the eIF4E kinase LK6 as a modulator of growth and fat metabolism (Teleman, 2005).

A novel modulator of the insulin/PI3K pathway, Melted, has been identified. The melted gene encodes a PH domain protein that interacts with both Tsc1 and Foxo. Melted protein can recruit the Tsc1/2 complex to the cell membrane and thereby modulate its output via the Tor pathway. Melted can also recruit Foxo to the membrane in an insulin-regulated manner and thereby influence expression of Foxo targets. By reducing Tor activity and at the same time increasing Foxo activity, the melted mutant mimics the effects of nutrient deprivation in a normal animal, producing a lean phenotype (Teleman, 2005).

Melted contains two functional domains, identified as regions of high conservation between the fly and human proteins: an N-terminal protein interaction domain and a C-terminal PH domain. The PH domain targets Melted to the cell membrane. PH-GFP fusion proteins show sharp membrane localization, and fractionation assays show that in steady state, the majority of endogenous Melted protein is membrane associated. Rescue assays show that unlike the full-length protein, Melted protein missing the PH domain cannot rescue the melt^Δ1 mutant phenotypes. Thus, Melted protein requires its PH domain, and presumably its membrane localization, to have biological activity in vivo (Teleman, 2005).

On this basis, it is proposed that Melted may function as an adaptor, facilitating association of the TSC complex and Foxo with their upstream-signaling inputs. Foxo and Tsc2 are phosphorylated in vivo by Akt/PKB, which becomes activated at the cell membrane when it binds PIP3 and is phosphorylated by Pdk1. Although the phosphorylation of Tsc2 by PKB is not strictly necessary for viability, it is possible this phosphorylation is modulatory in function. Tsc2 is also regulated via phosphorylation by AMPK. AMPK is membrane associated through its myristoylated β subunit. Therefore, it is plausible that recruiting the Tsc complex and Foxo to the cell membrane might alter their state of activation. It is considered unlikely that Melted could sequester the TSC complex and Foxo proteins at the membrane. Instead, an intriguing possibility is that by transiently binding these proteins, Melted could facilitate their phosphorylation (Teleman, 2005).

Reduced Foxo phosphorylation is expected to increase Foxo activity, and indeed the following was found: (1) upregulation of the Foxo target 4E-BP in the melted mutant and (2) that reduced Foxo levels could suppress the fat-accumulation defect in the melted mutant. These observations indicate that Foxo activity is increased in the melted mutant. Similarly, reduced Tsc2 phosphorylation is expected to lead to increased Tsc2 activity and, thus, reduced Tor activity. Indeed, it was found that Tor activity becomes limiting for 4E-BP phosphorylation in the melted mutant (Teleman, 2005).

melt^Δ1 mutant animals are lean because of lower lipid levels in adipose tissue. This phenotype is autonomous to the fat body because it can be rescued by tissue-specific expression of Melted. Evidence is presented that elevated Foxo activity in the mutant is important in this context. Adipose tissue undergoes a dramatic transcriptional change upon loss of melted function. At the 99% confidence level, 249 genes were misregulated by more than 1.5-fold in the fat body. 63% of the misregulated genes for which a function is annotated are metabolism related. Although many of these are involved in lipid or carbohydrate metabolism, it was surprising to find a significant number of genes involved in proteolysis and amino acid metabolism. Recently, Foxo has been shown to promote protein turnover during fasting (Teleman, 2005).

Members of the Tor signaling pathway have been implicated in the control of fat metabolism in flies and mice. Both S6K and 4E-BP mutant mice are either lean or resistant to diet-induced obesity. Likewise, 4E-BP mutant flies show increased sensitivity to nutrient deprivation, leading to abnormal fat loss [Teleman, A.A., Chen, Y.-W., and Cohen, S.M. (2005). 4E-BP functions as a metabolic brake used under stress conditions but not during normal growth. Genes Dev., in press]. In the fly, activation of the Tor pathway in the fat body promotes fat accumulation. Because Tor activity is reduced in the melted mutant fat body, it is believed that this reduction contributes to the observed leanness. This idea is supported by the fact that inactivating Foxo in the melted mutant leads to a 90% rescue, whereas simultaneously inactivating Foxo and restoring Tor function (via PI3K) has a more pronounced effect, elevating fat levels to above normal. When the animal is under conditions of limited nutrition, in which Foxo activity is high, the effects of Foxo-dependent transcription appear to dominate and shift the animal to a mode of net fat consumption. When the animal is under normal feeding conditions, the ability of Tor to serve as a sensor of cellular nutritional status may be important in controlling fat accumulation (Teleman, 2005).

The effect of Melted on both tissue growth and metabolism is modulatory. In the absence of Melted protein, both tissue growth and lipid metabolism function, although with modified characteristics. The magnitude of the effect caused by Melted overexpression is smaller than that caused by Tsc1/2 loss of function. This phenotype is similar to those of other components of the PI3K/Tor pathways that have recently been studied in flies. For instance, Foxo mutant flies have no detectable growth abnormalities and are impaired in their response to oxidative stress. Whether melt^Δ1 mutant flies are also sensitive to oxidative stress was tested by feeding them food with H₂O₂, and indeed they are. Scylla and Charybdis are two genes upstream of the Tsc1/2 complex that regulate S6K activity. Scylla and Charybdis double-mutant flies show very mild growth defects but are impaired in their response to hypoxia and have abnormal lipid levels. Lk6 kinase, like 4E-BP, regulates eIF4E activity. Lk6 mutant flies are 20% smaller than controls and contain elevated lipid levels. Thus, the Tor pathway appears to be closely regulated by several modulators, Melted being a new member of this group (Teleman, 2005).

Melted is highly conserved between flies and mammals, and human Melted expression rescues the mutant fly phenotype. In view of the overall similarity in the biochemistry and biological functions of the insulin/PI3K pathway in flies and mammals, the possibility that Melted might have a comparable role in mammalian metabolism merits consideration (Teleman, 2005).

GENE STRUCTURE

cDNA clone length - 3739 bp

Bases in 5' UTR - 760

Exons - 10

PROTEIN STRUCTURE

Amino Acids - 992 (isoform A)

Structural Domains

The predicted Melted protein is conserved in C. elegans (CE25943), mice, and humans (HGNC, KIAA1692). Alignment of these sequences shows a conserved region at the N terminus (76% similar in fly and human) and a conserved Pleckstrin Homology (PH) domain at the C terminus. Ubiquitous expression of Melted lacking the PH domain does not rescue the melt^Δ1 size defect, indicating that the PH domain is required for Melted function. The truncated protein is likely to fold correctly because it was used successfully as bait in a yeast two-hybrid screen to detect proteins that also interact with full-length Melted. The human homolog of Melted also rescues the size reduction of the melt^Δ1 mutant, indicating that the molecular function of Melted is conserved (Teleman, 2005).

date revised: 25 August 2005

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