BIOLOGICAL OVERVIEW

pelle mutations cause a dorsalized embryo (one lacking ventral structures) whose phenotype can be corrected by injecting mutant eggs with the cytoplasm from normal eggs. The mRNA for Pelle is provided maternally; therefore mutant mothers are unable to support the growth of embryos. Normal maternal cytoplasm or mRNA when injected into embryos from mutant mothers partially restores the normal pattern. Rescuing potential remains in the cytoplasm of normal eggs until the late blastoderm stage. After this stage has passed, eggs from mutant mothers fail to respond to injected mRNA. Thus there is a critical period in development in which the pelle gene product can act to rescue defective eggs (Müller-Holtcamp, 1985).

The cloning of pelle kinase has confirmed classic injection experiments. Kinases are proteins that can catalytically attach phosphate groups, thus passing a signal from one molecule (the kinase) to its target. The message that Pelle transduces, the activation of the Toll receptor by its ligand Spätzle, is passed to Cactus causing its degradation. Freeing the Rel-related protein Dorsal from it cytoplasmic inhibitor Cactus, allows Dorsal to translocate into ventral and ventrolateral nuclei and direct gene expression.

Pelle-mediated signaling induces the spatially graded degradation of Cactus. Using a tissue culture system which reconstitutes Pelle-dependent Cactus degradation, it can be shown that a motif in Cactus, resembling the sites of signal-dependent phosphorylation in the vertebrate Cactus homolog IkappaB is essential for Pelle-induced Cactus degradation. Substitution of four serines within this motif with nonphosphorylatable alanine residues generates a mutant Cactus that still functions as a Dorsal inhibitor but is resistant to degradation (Reach, 1996).

Although both Cactus and Dorsal are modified by phosphorylation in response to signaling, it is believed that signal-dependent modification of Cactus precedes modification of Dorsal. Signal-dependent modification of Dorsal presumably occurs downstream of Cactus degradation, since loss of Cactus function induces Dorsal modification in the absence of signaling. Phosphorylation of Dorsal upon release from Cactus could reflect unmasking of a phosphorylation site or an encounter with a protein kinase upon translocation into nuclei (Gillespie, 1994 and Reach, 1996).

The Pelle kinase appears to require Tube for its activation. Tube protein associates with the membrane of the oocyte and is required to pass the activation signal from the receptor Toll to Pelle. In the process Tube also becomes phosphorylated by Pelle. Whether Pelle can phosphorylate Cactus or Dorsal is unknown. Tube is the one known target for Pelle phosphorylation (Grosshans, 1994 and Galindo, 1995).

Biochemical interactions are described among recombinant Toll, Pelle and Tube that provide insights into early events in activation of the signaling cascade. A tertiary complex exists prior to activation. The Pelle-Toll complex is required to suppress that kinase activity of Pelle. Pelle binds directly to a region within the Toll intracytoplasmic domain, providing the first evidence that these two evolutionarily conserved molecules physically interact. Pelle contains an N-terminal putative regulatory domain, consisting largely of a region with significant similarity to the consensus death domain, and a C-terminal catalytic domain. To determine whether either (or both) of these regions are required for the interaction with the intracellular domain of Toll (Toll IC), the Pelle N terminus (Pelle-R) and C terminus (Pelle-C) were produced separately by in vitro translation, and then tests were carried out to see if these molecules could bind any of several GST-Toll IC derivatives. The results indicate that the Pelle N terminus is incapable of binding any of the GST-Toll derivatives, whereas the catalytic region binds efficiently to both GST-Toll and GST-Toll ICNae (a derivative lacking the C-terminal ID but containing the entire IL-1R homology region). The Toll C-terminal inhibitory domain (ID) is neither necessary nor sufficient for this interaction (Shen, 1998).

It is thought that upon activation Pelle is autophosphorylated, and that this prevents binding to Toll as well as Tube. Autophosphorylation occurs in the N-terminal, death-domain-containing region of Pelle, a region dispensable for binding to Toll but required for enzymatic activity. Pelle phosphorylates Toll, within the region required for Pelle interaction, but this phosphorylation can be blocked by a previously characterized inhibitory domain at the Toll C terminus. These and other results allow for the proposal of a model by which multiple phosphorylation-regulated interactions between these three proteins lead to activation of the Dorsal signaling pathway (Shen, 1998).

It is proposed that the intracytoplasmic IL-1R homology domain of the Toll receptor initially interacts directly with unphosphorylated Pelle, and that the Toll ID helps down regulate kinase activity. Tube is also recruited to the complex through its interaction with Pelle and/or Toll, forming the Toll/Pelle/Tube ternary complex (Pelle inactive). Binding of Spätzle to Toll induces Toll dimerization, and the Toll cytoplasmic domain is modified through conformational changes or proteolysis of the Toll inhibitory domain, allowing activation of kinase activity (Pelle active). Active Pelle then phosphorylates multiple substrates, including itself, Toll and Tube. This causes disruption of the Toll/Pelle/Tube complex, freeing Pelle to phosphorylate unknown downstream targets, eventually resulting in Cactus phosphorylation/degradation and Dorsal phosphorylation (e. g., by protein kinase A [PKA] and possibly Pelle itself] and nuclear translocation. Phosphorylated Tube may also translocate with Dorsal and function as a transcriptional coactivator (Shen, 1998).

The direct interactions described between unphosphorylated Pelle, Toll and Tube are consistent with the existence of a ternary complex at the plasma membrane. Pelle interacts with Toll via residues in its catalytic domain, and with Tube via Pelle's N-terminal death domain: both interactions can occur simultaneously. An important question is whether the ternary complex forms independent of signaling. Previous studies have shown that the artificial recruitment of Pelle or Tube to the plasma membrane can initiate the signaling pathway independent of ligand binding. But it is not clear whether it is recruitment to the membrane per se that results in activation, or the dimerization of the Torso fusion proteins employed in these previous studies. A possible mechanism for Pelle activation is simply dimerization, induced naturally, it is suggested, by conformational changes in the ternary complex that occur following ligand binding. How might such changes be induced? There is considerable indirect evidence suggesting that Toll molecules interact: an attractive model posits that ligand binding induces dimerization or even aggregation. It is suggested that this leads to activation of signaling, i.e., of Pelle activity, by either (or both) of two related mechanisms: (1) oligomerization of Toll receptors increases the local ternary complex concentration and hence Pelle concentration, thereby favoring Pelle dimerization and activation by simple mass action; (2) ligand-induced Toll self-association causes a conformational change in the intracytoplasmic domain such that the ID is displaced, thereby facilitating Pelle activation, again perhaps by dimerization. A speculative possibility is that the ID is actually cleaved upon activation. The product of the strong dominant gain-of-function allele Toll 10b, which contains a single C to Y change in its extracellular domain, has been found in a partially proteolyzed form, such that full-length Toll 10b is associated with a truncated form lacking most or all of its extracellular domain as well as likely sequences from the very C terminus, i.e., the ID. Perhaps relevant to this, a putative PEST degradation sequence is situated between the IL-1R homology region and the ID. It is intriguing that the structure of this truncated product is similar to mammalian IL-1RAcp, which functions in IRAK activation during IL-1 signaling. In any event, it is proposed that IL-1R homology domain interactions activate Pelle via the direct, phosphorylation-sensitive protein-protein interactions described in this paper (Shen, 1998).

Pelle modulates dFoxO-mediated cell death in Drosophila

Interleukin-1 receptor-associated kinases (IRAKs) are crucial mediators of the IL-1R/TLR signaling pathways that regulate the immune and inflammation response in mammals. Recent studies also suggest a critical role of IRAKs in tumor development, though the underlying mechanism remains elusive. Pelle is the sole Drosophila IRAK homolog implicated in the conserved Toll pathway that regulates Dorsal/Ventral patterning, innate immune response, muscle development and axon guidance. This study reports a novel function of pll in modulating apoptotic cell death, which is independent of the Toll pathway. It was found that loss of pll results in reduced size in wing tissue, which is caused by a reduction in cell number but not cell size. Depletion of pll up-regulates the transcription of pro-apoptotic genes, and triggers caspase activation and cell death. The transcription factor dFoxO is required for loss-of-pll induced cell death. Furthermore, loss of pll activates dFoxO, promotes its translocation from cytoplasm to nucleus, and up-regulates the transcription of its target gene Thor/4E-BP. Finally, Pll physically interacts with dFoxO and phosphorylates dFoxO directly. This study not only identifies a previously unknown physiological function of pll in cell death, but also sheds light on the mechanism of IRAKs in cell survival/death during tumorigenesis (Wu, 2015).

Drosophila melanogaster has emerged as an excellent model organism to study apoptotic cell death and has made significant contribution to understand cell death regulation and its role in development. While mammalian IRAKs function as the mediator of IL-1Rs/TLRs signal transduction in the immune and inflammatory responses, Pll, the sole Drosophila orthologue of IRAKs, has been implicated as a central regulator of Toll pathway involved in embryonic dorsal/ventral patterning, innate immune response, muscle development and axon guidance. This work identified a Toll pathway independent function of Pll in modulating caspase-mediated cell death in animal development (Wu, 2015).

Previous studies have suggested that Drosophila wing vein formation is a result of cell fate specification regulated by multiple signaling pathways including Notch, Hedgehog, EGF (epidermal growth factor) and BMP (bone morphogenetic proteins) pathways. The present study found that knock-down pll along the A/P compartment boundary of the developing wing (ptc>pll-IR) resulted in extensive cell death in the wing disc and a loss-of-ACV phenotype in the adult wing, implying a potential role of cell death in vein patterning. Consistent with this notion, the loss-of-ACV phenotype is rescued by blocking apoptotic cell death, suggesting cell death is responsible for the loss of ACV. To investigate whether cell death is able to impede vein patterning, apoptosis was initiated by expressing the pro-apoptotic protein Grim under the control of ptc-Gal4. ptc>Grim caused extensive cell death in tissue ablation between L3 and L4 in the adult wing. In most cases, L3 and L4 were fused in the proximal area where ACV is located. To adjust Grim expression and cell death, Tub-Gal80^ts that represses Gal4 activity was added in a temperature sensitive manner. At 25°C, Tub-Gal80^ts partially blocks ptc-Gal4 activity and allows limited Grim expression and therefore, cell death, between L3 and L4. Intriguingly, under this condition, the loss-of-ACV phenotype was observed to be accompanied by a slight reduction of area between L3 and L4, suggesting that both reduced area and loss-of-ACV phenotypes are consequences of cell death. The loss-of-ACV phenotype is more sensitive to cell death, since weak cell death is sufficient to generate the phenotype, whereas stronger cell death is required to delete tissue between L3 and L4. Consistent with this notion, while ptc>pll-IR flies reared at 25°C only displayed the loss-of-ACV phenotype , those raised at 29°C also showed reduced area between L3 and L4, which was caused by a reduction in cell number, but not cell size (Wu, 2015).

Pll regulates caspase activation and cell death through dFoxO. Mechanistically, loss of pll promotes the nuclear translocation of dFoxO, which otherwise is retained in the cytoplasm by phosphorylation. A number of kinases, including AKT, IκK and JNK, have been reported to phosphorylate FoxO and regulate its nuclear-cytoplasmic trafficking. This study provides evidence that Pll is another dFoxO kinase that phosphorylates dFoxO and inhibits its nuclear localization. Thus, it would be very interesting to check whether a similar interaction is conserved between IRAKs and FoxOs in mammal (Wu, 2015).

The FoxO family proteins have been implicated in multiple important biological processes, including cell death and tumor suppression. It has been reported that conditional deletion of FoxO1, FoxO3 and FoxO4 simultaneously results in the development of hemangiomas and thymic lymphomas, and IκB kinase represses FoxO3a activity to promote human breast tumorigenesis and acute myeloid leukemia (AML). IRAKs also show altered expression level in tumors and surrounding stroma, and participate in tumor initiation and progression, yet the underlying mechanisms remain poorly understood. Thus, the inhibitory effect of Pll on dFoxO activity in Drosophila provides a beneficial framework for a better understanding of mammalian IRAKs’ crucial roles in tumor development (Wu, 2015).

As the Toll/NF-κB pathway is not implicated in loss-of-pll triggered dFoxO-dependent cell death, it was of interest to learn about what are the pathways or factors act upstream of Pll to regulate its role in cell death. Since dFoxO has been reported as a downstream transcription factor in the JNK and Insulin pathways in Drosophila, it was asked whether Pll is also involved in these pathways. Activation of JNK signaling between L3 and L4 by expressing Egr (Drosophila TNF) or Hep (Drosophila JNK Kinase), or depleting puc (encoding a JNK inhibitor), produced similar loss-of-ACV and reduced area phenotypes as that of pll depletion, yet the phenotypes were not affected by gain or loss of pll. Inactivation of the Insulin pathway by expressing a dominant negative form of PI3K, or knocking-down PI3K or Akt, resulted in diminished area between L3 and L4, which remained unaffected by gain or loss of pll. The Hippo pathway, known to play a crucial role in regulating cell death and organ size, was also examined. Up-regulation of Hippo pathway in distinct wing areas by different Gal4 drivers led to various small wing or wing tissue ablation phenotypes, which were not altered by changing Pll level. Finally, dMyc, the fly homolog of c-Myc that regulates cell growth and cell death in Drosophila, and the cell polarity gene scribble (scrib), whose depletion promotes cell death were also examined. Depletion of dMyc triggered wing phenotype and loss of scrib induced cell death are both independent of Pll. Thus, while Pll directly regulates dFoxO-mediated caspase-dependent cell death in development, the upstream factors modulating Pll activity remain unknown, which deserve further investigation (Wu, 2015)

GENE STRUCTURE

Bases in 5' UTR - 172

Bases in 3' UTR - 202

PROTEIN STRUCTURE

Amino Acids - 501

Structural Domains

DNA sequence analysis revealed that pelle encodes a protein of 501 amino acids, the last 292 comprising a protein kinase catalytic domain. Microinjection of in vitro synthesized transcripts containing site-directed mutations indicates that the kinase catalytic domain is required for biological activity. This domain is most similar to that of the RAB and MOS protein kinases and is predicted to have a serine and threonine specificity. The identity between Pelle and Drosophila Raf, a kinase acting in the ras pathway, is equivalent to that between Pelle and human Raf (27% and 26.6% respectively) (Shelton, 1993).

The interaction between the death domains (DDs) of Tube and the protein kinase Pelle is an important component of the Toll pathway. Published crystallographic data suggests that the Pelle-Tube DD interface is plastic and implies that in addition to the two predominant Pelle-Tube interfaces, a third interaction is possible. The NMR solution structure of the isolated death domain of Pelle is presented along with a study of the interaction between the DDs of Pelle and Tube. The data suggests the solution structure of the isolated Pelle DD is similar to that of Pelle DD in complex with Tube. Additionally, they suggest that the plasticity observed in the crystal structure may not be relevant in the functioning death domain complex (Moncrieffe, 2005).

The crystal structure suggests that the PelleÐTube DD complex can exist as a tetramer comprised of Pelle and Tube heterodimers arranged in a linear sequence P1:T1:P2:T2 and whether this persists in solution needs to be determined as it may have implications for the function of the DD complex. To address these issues, the structure of the isolated Pelle-DD in solution and the interaction between the DDs of Pelle and Tube have been solved using nuclear magnetic resonance (NMR) spectroscopy. The results suggest that in the beta-helical regions, the structure of Pelle-DD is similar to that of Pelle-DD in complex with the DD of Tube, and of the two types of Pelle-Tube dimer interfaces observed in the crystal structure (P1:T1 and P2:T2) only one is likely to persist in solution (Moncrieffe, 2005).

The NMR data presented confirm the extensive nature of the Pelle-Tube death domain interface because all twenty seven residues that constitute the core interaction surface on Pelle-DD show significant chemical shift differences between the ¹H-¹⁵N HSQC spectrum of Pelle-DD and the ¹H-¹⁵N HSQC-TROSY spectrum of the Pelle-Tube DD complex. The NMR data also suggest that the Tube1:Pelle2 interaction observed in the crystals, may persist in solution. This implies that the death domains of Pelle and Tube are capable of forming a tetramer and this is corroborated by the sedimentation velocity data which reveals that the concentration of the tetrameric complex is very small (0.4%) relative to that of the dimer. The dominant species formed by the interaction of Pelle-DD and Tube-DD is a dimer and whether the very small amount of the tetrameric species plays a role in signaling is unknown; for example, a mutant of Pelle bearing a mutation in the Tube1:Pelle2 interface (D50K) fails to express protein. It is conceivable that the residues in Pelle that are part of the Tube1:Pelle2 interface are sites of interaction for other members of the signaling complex. A recent report argues against that partner being dMyD88 since this component is thought to bind predominantly to Tube. A more likely scenario however, given that dMyD88 appears to bind predominantly to Tube and not Pelle, is that in the functioning complex, P2 is occupied by dMyD88. Thus, the residues in Tube-DD that contribute to the T1:P2 interface are likely to be the scaffold that recruits dMyD88 (Moncrieffe, 2005).

In summary, the structure of the isolated Pelle DD is similar to that of the Pelle DD in complex with the DD of Tube. While the death domains of Pelle and Tube may form a tetramer, consistent with the crystallographic observation the concentration of this species is extremely low. Consequently, inferences regarding the “plasticity” of the Pelle-Tube DD complex in vivo should be reevaluated (Moncrieffe, 2005).

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