Tests were performed to see if bacterially expressed Melted protein binds to immobilized phosphoinositides. At higher concentration, Melted bound many phosphoinositides but at lower concentrations, showed preferential binding to PI(5)P. The PH domain of Melted expressed as a GST fusion exhibits a similar binding pattern, although with reduced selectivity. Although Melted does not show strong binding to PIP3 in vitro, a GFP fusion to the PH domain (MeltPH-GFP) was generated and tested in S2 cells for insulin-induced relocalization. MeltPH-GFP is detectable predominantly at the cell membrane when cells are serum-starved to deprive them of insulin and after insulin-stimulation without apparent difference. The MeltPH-GFP fusion is also predominantly cortical in wing imaginal disc cells, whereas a Melted-GFP fusion protein lacking the PH domain is cytoplasmic, suggesting that the majority of Melted protein is localized to the membrane under normal conditions. This was confirmed by subcellular fractionation of wing-disc cells. The majority of the endogenous Melted protein was recovered in the membrane fraction, with a lesser amount in the cytoplasm. No signal was detected in corresponding fractions prepared from melted mutant tissue, confirming the specificity of the antibody. Control proteins fractionated predominantly as cytoplasmic (Y14) or membrane-associated (PVR). These results suggest that the endogenous Melted protein is membrane localized via its PH domain and that its localization is not regulated by insulin-dependent changes in PIP3 levels. The PH domain is required for Melted function in vivo (Teleman, 2005).
To study the function of the Melted N-terminal domain, a yeast two-hybrid screen was performed with MeltDPH as bait to screen 2.7 million clones from a larval cDNA library. Six interacting genes were isolated: Tsc1 (four independent clones), 14-3-3e (three independent clones), CG16719, CG5171, CG6767, and CG8242 (one each). When expressed in S2 cells by cotransfection, Melted coimmunoprecipitated with Myc-tagged Tsc1. Interaction of Melted and Tsc1 in S2 cells was also visualized by immunofluorescence microscopy. Tsc1myc was uniformly distributed in the cytoplasm when expressed alone. Coexpression with Melted causes Tsc1-myc to shift predominantly to the cell membrane. When expressed in S2 cells, Tsc2 is uniformly distributed in the cytoplasm. Coexpression of Tsc2 with Melted does not cause a relocalization of Tsc2 to the membrane, suggesting that Melted and Tsc2 do not interact directly. However, when coexpressed with Melted and Tsc1, Tsc2 also relocates, indicating that Melted can recruit the Tsc1/2 complex to the cell membrane (Teleman, 2005).
Does this interaction affect Tor pathway activity? Previous reports have shown that increased PI3K or Tor signaling in the adipose tissue increases fat accumulation or reduces fat consumption (autophagy). The observation that melted mutants are lean, could be explained if Tor activity is reduced in the mutant. To test this more directly, the level of Tor-dependent phosphorylation of 4E-BP was assayed in control and meltΔ1 mutant fat bodies. Phosphorylation of Drosophila 4E-BP at positions 36/47 depends on Tor activity and can be visualized with an antibody specific to the corresponding phosphorylated peptide from human 4E-BP. Although the total level of 4E-BP protein is higher in meltΔ1 mutant tissue, the level of phosphorylation is not correspondingly elevated (perhaps even mildly reduced). The increase in total 4E-BP level reflects a 2- to 3-fold increase in transcript levels in the mutant fat body. 4E-BP levels were increased by a comparable amount in wild-type fat body, as a control, with ppl-Gal4 UAS-4E-BP and the level of 4E-BP phosphorylation was observed to be correspondingly elevated. This indicates that Tor activity is not limiting in wild-type fat body. Thus, the observation that 4E-BP phosphorylation does not increase, despite increased total 4E-BP, provides evidence that Tor activity is reduced in the melted mutant adipose tissue (Teleman, 2005).
As a second means to identify possible functions of Melted, the Eukaryotic Linear Motif server) was used to look for functional motifs conserved between fly and human Melted. The only conserved motifs found in the N-terminal region of these proteins were two Forkhead-associated domain ligand domains (LIG_FHA_1). Forkhead transcription factors FoxA2, FoxA3, FoxC2, and FoxO1 are involved in glucose and fat metabolism. Insulin signaling activates Akt, which phosphorylates Foxo and leads to its retention in the cytoplasm. It was therefore asked if Melted affects the subcellular localization of a Foxo-GFP fusion protein. Foxo-GFP is predominantly nuclear in the absence of insulin stimulation in serum-starved S2 cells and increases in the cytoplasm after insulin stimulation. In serum-starved cells cotransfected to express Melted, Foxo-GFP is still primarily nuclear, but much of the nonnuclear protein appears at the membrane colocalized with Melted. Upon insulin stimulation, a robust increase in the level of Foxo-GFP was observed at the cell membrane. The interaction was confirmed by coimmunoprecipitation of Melted with Foxo in insulin-stimulated S2 cells (Teleman, 2005).
The observation that insulin stimulation induces a shift toward membrane localization of Foxo in the presence of Melted in S2 cells raised the possibility that melted regulates Foxo activity in vivo. To address this, expression of the Foxo target 4E-BP was examined in wild-type and melted mutant animals. Under fed conditions, insulin signaling is active and 4E-BP transcript levels are relatively low. In wild-type flies that were starved for 24 hr to reduce insulin levels and thereby activate Foxo, 4E-BP transcript increased ~4-fold. In starved flies lacking Melted, 4E-BP transcript increased over 25-fold. This increase in 4EBP transcription was absent in the starved melted/Foxo double mutant, confirming that it is Foxo dependent. Thus, in the absence of Melted, Foxo activity is higher than normal, suggesting that Melted limits Foxo activity in vivo (Teleman, 2005).
To determine whether the elevated Foxo activity observed in melted mutants contributes to the lean phenotype of these animals, the normalized triglyceride levels of melted mutant and melted foxo double-mutant flies were compared. Reducing Foxo activity suppresses the leanness of the melted mutant to a considerable degree, reaching near normal fat levels. The rescue was highly statistically significant. foxo mutants did not show higher-than-normal fat levels compared to wild-type. These observations suggest that Melted acts by regulating Foxo activity to control expression of genes important in fat metabolism (Teleman, 2005).
melted mutants also exhibit reduced Tor activity. To determine whether Tor activity affects fat accumulation, the effects were tested of increasing Tor activity in wild-type and melted mutant adipose tissue. Use was made of a UAS-Tor transgene that can provide Tor activity in vivo when expressed at appropriate levels. It was confirmed that expression of UAS-Tor under ppl-Gal4 control in adipose tissue leads to increased total body fat, as does increasing PI3K activity. In contrast, a comparable elevation of Tor expression in melted mutant flies has no effect on fat levels. Both this result and the significant rescue caused by removal of Foxo indicate that in the melted mutant, the Foxo branch of the pathway becomes limiting for fat accumulation. In view of this finding, it was next asked whether elevated Tor pathway activity could increase fat levels in the melted mutant if Foxo activity was simultaneously reduced. To do so, use was made of the catalytic subunit of PI3K (Dp110) to inactivate Foxo and simultaneously activate Tor. The fat body driver lsp2-Gal4 or the UAS-Dp110 transgenes have little effect on their own in the melted mutant background, but when combined, the elevated PI3K activity in the fat body increases fat levels of the melted mutant. The effect is stronger than that of removing Foxo only, increasing fat levels to above normal. Taken together, these observations suggest that the Tor branch of the pathway contributes to the control of fat levels under conditions in which Foxo activity levels are low. This is normally the case in feeding animals in which insulin levels are relatively high (Foxo activity is elevated under starvation conditions: as seen by comparing 4E-BP levels in fed versus starved wild-type and foxo mutant flies). Under conditions in which insulin levels are low or in the melted mutant, in which Foxo activity is elevated, the effects of Foxo appear to dominate (Teleman, 2005).
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