The developmental profile of wild-type Thor expression shows transcripts present throughout all stages of development, with a noticeable increase in the larval stages, especially the third instar. beta-galactosidase in the P{lacW} Thor strain localizes to the embryonic nervous system and to many tissues in larvae and adults (Rodriguez, 1996). The same results were found by tissue in situ hybridizations with Thor probes, and the analysis has been extended to the reproductive system, Thor expression is found in the testes of third instar males and in the ovaries of adult females. This expression in the testes and ovarian nurse cells is part of a more complex pattern of expression in both the female and male reproductive systems. The general expression of Thor and in particular the expression in the ovaries, in which there are intricate systems of translational regulation, are consistent with the role of Thor as a 4E-BP, but the role of a 4E-BP in the immune response is not consistent with or predicted by current views in immunity (Bernal, 2000).

Effects of Mutation or Deletion

The initiation factor 4E for eukaryotic translation (eIF4E) binds the messenger RNA 5'-cap structure and is important in the regulation of protein synthesis. Mammalian eIF4E activity is inhibited when the initiation factor binds to the translational repressors, the 4E-binding proteins (4E-BPS). The Drosophila 4E-BP (d4E-BP) is a downstream target of the phosphatidylinositol-3-OH kinase [PI(3)K] signal-transduction cascade, which affects the interaction of d4E-BP with eIF4E. Ectopic expression of a highly active d4E-BP mutant in wing-imaginal discs causes a reduction of wing size, brought about by a decrease in cell size and number. A marked reduction in cell size is also observed in post-mitotic cells. Expression of d4E-BP in the eye and wing together with PI(3)K or dAkt1, the serine/threonine kinase downstream of PI(3)K, results in suppression of the growth phenotype elicited by these kinases. These results support a role for d4E-BP as an effector of cell growth (Miron, 2001).

Drosophila 4E-BP (d4E-BP) was isolated by interaction cloning from a complementary DNA expression library using ³²P-labelled deIF4EI. d4E-BP is identical to Drosophila Thor (Bernal, 2000) and homologous to 4E-BPs from other species. Phosphorylation sites in mammalian 4E-BP1 are conserved in d4E-BP, but the predicted eIF4E-binding motif in d4E-BP (YERAFMK) diverges from the canonical consensus sequence (Miron, 2001).

To examine the binding of d4E-BP to deIF4E, residues within the consensus eIF4E-binding site were mutated. Recombinant proteins were expressed in Escherichia coli, and far Western blotting was performed using ³²P-labelled deIF4EI. Mutation of Tyr 54 to Ala (Y54A) or Phe (Y54F), and Met 59 to Ala (M59A) abrogates the interaction of d4E-BP with deIF4E. Mutation of Lys 60 to Ala (K60A) decreases deIF4E binding by 87%, indicating that Lys 60 contributes to deIF4E binding. However, when either Met 59 or Lys 60 are mutated to the consensus Leu, the interaction of d4E-BP with deIF4EI is 2.5-fold higher than with the wild type, and when both Met 59 and Lys 60 are so changed, deIF4E binding increases by 3.4-fold. These results indicate that d4E-BP interacts with deIF4E, albeit more weakly than previously characterized 4E-BPs, owing to its divergent eIF4E-binding motif (Miron, 2001).

4E-BP1 is hyperphosphorylated in response to insulin in many cell types. To test whether this response operates in Drosophila, Schneider-2 (S2) cells were deprived of serum and treated with insulin. Increasing levels of a slower migrating form of d4E-BP (d4E-BP) were observed consequent to insulin treatment. To determine whether the ß-form corresponds to phosphorylated d4E-BP, extracts from insulin-stimulated S2 cells were treated with either calf intestine alkaline phosphatase (CIP) or protein phosphatase 2A (PP2A). Untreated extracts (or extracts kept on ice) contain both the faster migrating alpha- and the slower migrating ß-forms. In contrast, phosphatase-treated extracts contained only the alpha-form (Miron, 2001).

LY294002 and rapamycin inhibit PI(3)K and target of rapamycin (TOR) activity, respectively, and block the insulin-induced hyperphosphorylation of 4E-BP1. Similarly, exposure of serum-deprived S2 cells to either drug before treatment with insulin, results in a dose-dependent decrease in d4E-BP phosphorylation. To determine whether phosphorylation of d4E-BP prevents its binding to deIF4E, m⁷GDP-agarose precipitation was performed. The alpha form is present primarily in the bound fraction, whereas the ß-form is found exclusively in the unbound fraction. These results show that d4E-BP is a downstream target of the PI(3)K pathway, and that the binding of d4E-BP to deIF4E is modulated by its phosphorylation state (Miron, 2001).

Assembly of eIF4F is essential for translational control, and overexpression of eIF4E in mammalian cells results in malignant transformation. To investigate whether eIF4F is also linked to growth control, eIF4F assembly was perturbed in Drosophila. UAS transgenic fly lines were generated that express wild-type d4E-BP or the mutant d4E-BP that binds deIF4E most strongly, d4E-BP(LL). Expression of d4E-BP was targeted to the wing-imaginal disc using MS1096-GAL4. The size and cell number of wings from males were measured. Expression of wild-type d4E-BP has no effect on wing size or pattern. However, expression of d4E-BP(LL) from one line [d4E-BP(LL)^w] causes a marked reduction of wing size without affecting cell number. Another line, [d4E-BP(LL)^s], which expresses d4E-BP(LL) more strongly, causes a larger reduction, which is partly due to a decrease in cell number. Since direct inhibition of cellular proliferation increases, rather than decreases, cell size, it is possible that d4E-BP(LL) also affects cell size directly, and cell proliferation as a consequence. This is supported by analysis of the effects of d4E-BP(LL) expression in larval-wing discs. Although discs from the d4E-BP(wt) and d4E-BP(LL)^w lines are indistinguishable from control discs, d4E-BP(LL)^s discs are 52% smaller. d4E-BP(LL)^s males also required 1-2 days longer to eclose, which would account for the smaller decrease in adult wings (Miron, 2001).

Acridine-orange staining shows that d4E-BP(LL)^s discs contain many apoptotic cells. Co-expression of p35, the baculovirus inhibitor of apoptosis, with d4E-BP(LL)^s partially rescues the size of adult wings. To distinguish between apoptosis and decreased proliferation, cell clones expressing d4E-BP(LL), with or without p35, and co-expressing green fluorescent protein (GFP), were induced 72 h after egg deposition in developing wing discs using the flip-out technique. Clones expressing d4E-BP(LL)^w contain fewer cells than wild-type clones, but co-expression of p35 with d4E-BP(LL)^w does not affect the number of cells per clone, indicating that decreased proliferation, but not increased apoptosis, is the cause of reduction. Few clones expressing d4E-BP(LL)^s are recovered, and they usually contain 1-2 cells. Co-expression of p35 greatly increases the number of clones recovered, but only marginally increases the number of cells per clone (1-4 cells) (Miron, 2001).

Direct interference with cell proliferation using string mutants results in increased cell size. To help distinguish effects on size from effects on proliferation, cell size was evaluated by flow cytometry (FACS). Mean forward-light scatter values for GFP-positive cells that expressed d4E-BP(LL) were reduced by 6%-8%. Because cells that expressed d4E-BP(LL) are smaller and proliferate more slowly than their wild-type counterparts, it is conceivable that d4E-BP(LL) directly affects cell growth and consequently retards proliferation, which would lead to reduced viability and ultimately apoptosis. Similar results were observed in dTOR mutants, and interpreted as a primary defect in cellular growth coupled with a consequent decrease in cell proliferation. The possibility that growth and proliferation are affected independently by d4E-BP(LL) expression cannot be excluded (Miron, 2001).

To exclude proliferation effects, the growth and viability of d4E-BP(LL) cells were examined in a post-mitotic tissue. Polyploid fat-body cells undergo successive rounds of DNA synthesis without mitoses. Cells that express d4E-BP(LL)^s, induced 48 h after egg deposition in the fat body, are 45%-70% smaller than neighboring wild-type cells, but their frequency is much higher than in mitotically active tissues, such as the wing-imaginal disc. Thus, viability of cells that express d4E-BP(LL) is maintained in the absence of mitogenic signals, indicating that proliferation of wild-type neighboring cells is necessary to cause the disappearance of cells expressing d4E-BP(LL). In support of this notion is the finding that when d4E-BP(LL)^s clones are induced during development of eye-imaginal discs, only the clones that are generated posterior to the morphogenetic furrow survive; the clones generated anterior to the furrow (that is, in mitotically active cells), are eliminated (Miron, 2001).

To study the possible role of d4E-BP as an effector of cell growth through the PI(3)K signaling pathway, potential interactions between d4E-BP and relevant signaling genes of this pathway were examined. Expression of wild-type d4E-BP in eye-imaginal discs, using GMR-GAL4, does not engender any discernible phenotype, whereas expression of dAkt1 results in an enlarged eye. However, co-expression of wild-type d4E-BP and dAkt1 partially suppresses the enlarged-eye phenotype, and fully suppresses the roughness induced by expression of dAkt1. Since d4E-BP by itself has no effect on eye size but is able to suppress the dAkt1 phenotype, there is a genuine epistatic relationship between d4E-BP and dAkt1 (Miron, 2001).

Other components of the PI(3)K pathway were also examined for potential epistatic interactions with d4E-BP in the wing, using dpp-GAL4 and 4E-BP(LL)^s. Ectopic expression of Dp110 and dAkt1 causes an enlargement of the region encompassed by the third and fourth longitudinal veins, the anterior crossvein and wing margin. In contrast, expression of a dominant-negative mutant form of PI(3)K (Dp110^D954A) or d4E-BP(LL)^s results in reduction of the size of this region. Co-expression of d4E-BP(LL)^s with Dp110 or dAkt1 suppresses the growth enhancement engendered by expression of these kinases, whereas co-expression of d4E-BP(LL)^s with Dp110^D954A results in further size reduction. Flies that lacked a copy of the gene encoding the adaptor protein p60 [the Drosophila homolog of mammalian PI(3)K subunit p85] are also reduced in size when d4E-BP(LL)^s is co-expressed. These results provide genetic evidence that d4E-BP is a downstream effector of the PI(3)K pathway (Miron, 2001).

Null mutants of d4E-BP are viable and although their immune response is compromised (Bernal, 2000), they do not exhibit increased growth. Furthermore, ectopic expression of Drosophila eIF4E in a wild-type or d4E-BP null background fails to produce a growth-related phenotype. Therefore, an increase in eIF4E activity alone is not sufficient to promote cell growth in Drosophila imaginal discs. This is consistent with data in primary mouse-embryo fibroblasts, in which eIF4E overexpression leads only to oncogenic transformation when co-expressed with c-myc or E1A. Attempts were made to rescue the d4E-BP(LL)-induced growth defects in imaginal discs by co-expressing deIF4E. Unexpectedly, growth is further reduced. Thus, endogenous eIF4E expression levels are optimal for cell growth and proliferation, and in the absence of activation of the PI(3)K pathway, a further increase in eIF4E expression is either without effect or deleterious (Miron, 2001).

Many studies have shown that PI(3)K and TOR-mediated signaling is important for normal cell growth and proliferation. However, one downstream target of this pathway, S6K, regulates cell size but not proliferation. Constitutive expression of dS6K in dTOR mutants only partially suppresses the dTOR phenotype, indicating that S6K-independent targets operate downstream of dTOR. Regulation of eIF4E activity, independent of S6K, contributes to the control of cell size. In Drosophila, the activity of eIF4E is modulated through 4E-BP. Phosphorylation of eIF4E is correlated with increased translation rates. Mutation of the phosphorylation site in Drosophila eIF4E causes a cell size reduction. In summary, the results presented here show that d4E-BP acts as an important downstream effector of PI(3)K in the regulation of cell proliferation and growth, independent of S6K, and further underline the importance of translation initiation in the latter process (Miron, 2001).

Response of mutant Thor flies to bacterial infection.

Thor has been identified as a new type of gene involved in Drosophila host immune defense. Thor is a member of the 4E-binding protein (4E-BP) family, which in mammals has been defined as comprising critical regulators in a pathway that controls initiation of translation through binding eukaryotic initiation factor 4E (eIF4E). Without an infection, Thor is expressed during all developmental stages and transcripts localize to a wide variety of tissues, including the reproductive system. In response to bacterial infection and, to a lesser extent, by wounding, Thor is up-regulated. The Thor promoter has the canonical NFkappaB and associated GATA recognition sequences that have been shown to be essential for immune induction, as well as other sequences commonly found for Drosophila immune response genes, including interferon-related regulatory sequences. In survival tests, Thor mutants show symptoms of being immune compromised, indicating that Thor may be critical in host defense. In contrast to Thor, Drosophila eIF4E is not induced by bacterial infection. These findings for Thor provide the first evidence that a 4E-BP family member has a role in immune induction in any organism. Further, no gene in the translation initiation pathway that includes 4E-BP has been previously found to be immune induced. These results suggest either a role for translational regulation in humoral immunity or a new, nontranslational function for 4E-BP type genes (Bernal, 2000).

The most critical test of immune response is for survival after infection. To determine the effect of Thor mutation on immune response, Thor mutations were identified and then both control and Thor mutant flies were subjected to different types of bacterial infection and their survival was observed for 4 days (Bernal, 2000).

The P{lacW} insertion that led to identifying Thor also resulted in the Thor¹ mutation. P{lacW} inserted adjacent to the TATA box, separating the coding region from the promoter region, and Northern analysis of flies with this insertion reveals that only a small amount of the 0.85-kb Thor transcript is present, and it does not increase after infection. The P-element insertion has thus resulted in the production of the Thor¹ mutation, a noninducible, very weakly expressing hypomorph. To control for background effects, a wild-type revertant of the Thor¹ mutation, Thor^1rv1, was used, that was produced by precise excision of the P{lacW} insertion. To confirm that any differences between Thor¹ and Thor^1rv1 flies are the result of mutation of Thor, a second Thor mutation, Thor², was used. In Thor², P{lacW} also is excised, but leaving Thor still mutant because of imprecise excision that deleted bases between P{lacW} and the first B site. Thor¹ and Thor² mutants are homozygous viable and fertile, and in noninfected and sterile wounding controls, survival is similar to wild-type Oregon R flies, Thor^1rv1 and imd. Thor^1rv1 survives like Oregon R and is referred to as the designated wild-type control. As a control for susceptibility to infection, imd mutants were used. In similar experiments, flies with the imd mutation have been shown to be highly susceptible to bacterial infection, and imd clearly operates in controlling induction of antibacterial genes (Bernal, 2000).

Survival of mutant Thor adults is similar to wild-type adults when infected by E. coli and E. cloacae B₁₂. Both bacterial types affected imd flies as expected, with on average 11% surviving 4 days after infection with E. cloacae B₁₂ and 1% surviving with E. coli infection. After infection (4 days) with S. epidermidis, however, on average only 46% of Thor¹ and 47% of Thor² flies survived. Surprisingly, 66% of imd flies survived, which is much higher than Thor mutants and also the previously reported survival for imd flies, which was approximately 8% after E. coli infection. Tests with M. roseus showed that even wild-type flies are susceptible, with 47% surviving on average. Thor mutants and imd flies are both more susceptible, with 11% for Thor¹, 16% for Thor², and 2% for imd surviving on average 4 days after infection. The antibacterial response provides a strong defense, and failure of survival by mutants indicates that a critical facet of the response has been disrupted. These results show that Thor mutants are immune compromised, and when subjected to some types of bacteria, the disruption of the immune response leads to failure of survival (Bernal, 2000).

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date revised: 25 February 2009

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