InteractiveFly: GeneBrief

happyhour: Biological Overview | References


Gene name - happyhour

Synonyms - map4k3

Cytological map position- 56C1-56C4

Function - signaling

Keywords - TOR pathway, nutrient-responsive regulation of cell growth and metabolism, Apoptosis, Ethanol-induced behaviors

Symbol - hppy

FlyBase ID: FBgn0263395

Genetic map position - 2R:15,066,259..15,114,705 [+]

Classification - Catalytic domain of Mitogen-activated protein kinase kinase kinase kinase-like Protein Serine/Threonine Kinases

Cellular location - cytoplasmic



NCBI links: Precomputed BLAST | EntrezGene
BIOLOGICAL OVERVIEW

The TOR pathway mediates nutrient-responsive regulation of cell growth and metabolism in animals. TOR Complex 1 activity depends, among other things, on amino acid availability. MAP4K3 (Drosophila Happyhour) has recently implicated in amino-acid signaling in cell culture. This study reports the physiological characterization of MAP4K3 mutant flies. Flies lacking MAP4K3 have reduced TORC1 activity detected by phosphorylation of S6K and 4EBP. Furthermore MAP4K3 mutants display phenotypes characteristic of low TORC1 activity and low nutrient availability, such as reduced growth rate, small body size, and low lipid reserves. The differences between control and MAP4K3 mutant animals diminish when animals are reared in low-nutrient conditions, suggesting that the ability of TOR to sense amino acids is most important when nutrients are abundant. Lastly, physical interaction is shown between MAP4K3 and the Rag GTPases raising the possibility they might be acting in one signaling pathway (Bryk, 2010).

The multiprotein complex TORC1, containing TOR kinase, is a central regulator of cellular growth and metabolism in animals. It is activated by a number of inputs relating to cellular energy and nutrient status. These include insulin, glucose, cellular energy levels and amino acid availability. In response, TORC1 activates protein synthesis via a number of mechanisms including activation of the ribosomal S6 kinase (S6K), repression of the translational inhibitor 4E-BP, and promotion of ribosome biogenesis via myc. In particular since TORC1 is a master regulator of protein biosynthesis, its regulation by amino acids, the building blocks of proteins, likely constitutes an important regulatory feedback mechanism. Furthermore, the importance of amino acid signaling to TOR is highlighted by the observation that circulating amino acids are elevated in humans with obesity, where they have been shown to activate TORC1 activity and modulate glucose metabolism. Despite this, understanding of the molecular mechanism by which amino acids regulate TOR remains fragmentary (Bryk, 2010).

Three protein complexes have recently been implicated in the activation of TORC1 in response to amino acids. The human class III PI3K (phosphoinositide 3-kinase) hVps34 is activated by amino acids via a calcium dependent mechanism (Gulati, 2008). This leads to accumulation of phosphatidylinositol 3-phosphate (PI(3)P) in cells, which is thought to cause the recruitment of proteins recognizing PI(3)P to early endosomes, forming an intracellular signaling platform that leads to TORC1 activation. This feature of the pathway may be specific for vertebrates, as flies mutant for Vps34 have been reported to not have TORC1 signaling defects (Juhasz, 2008). Recently, two groups discovered that Rag GTPases mediate amino acid signaling to TORC1 (Kim, 2008; Sancak, 2008). The emerging picture is that amino acids change the GDP/GTP loading of the Rag GTPases, thereby stimulating the binding of Rag heterodimeric complexes to TORC1. This in turn causes TORC1 to change its intracellular localization, perhaps relocalizing it to vesicles containing the activator Rheb. This mechanism appears to be evolutionarily conserved from flies to humans (Bryk, 2010).

The third protein recently identified as a mediator of amino acid signaling to TOR is MAP4K3 (Findlay, 2007); in HeLa cells the kinase activity of MAP4K3 is activated in the presence of amino acids. In turn, MAP4K3 is required for TOR to phosphorylate its targets S6K and 4E-BP1 in response to amino acid sufficiency. The cell-culture data also suggest this mechanism is conserved from flies to humans as knockdown of Drosophila MAP4K3 causes a reduction in TOR activity (Bryk, 2010).

Although considerable progress has been made, important questions remain unanswered. The role of TORC1 activity in vivo has been well studied in flies and mice, but fundamental issues regarding the regulation of TOR by amino acids have thus far only been explored in vitro in cell culture. To assess the functional significance of the ability of TORC1 to sense amino acids in the organismal context, use has been made of a Drosophila mutant for MAP4K3 (CG7097). dMAP4K3 mutant flies have reduced TOR activity, detected by phosphorylation of TOR targets. dMAP4K3 mutants are viable but display physiological aberrations emblematic of animals starved of nutrients: MAP4K3 mutant flies have retarded growth, reduced size, and low lipid reserves. Both the biochemical results and the phenotypes indicate that MAP4K3 modulates, but is not absolutely required, for TOR activity in vivo. This is similar to what is observed with other modulators of the pathway, such as Melted. Unexpectedly, the function of MAP4K3 is most required when nutrient conditions are rich (Bryk, 2010).

Recent reports have shown that not all components identified in cell culture as regulators of TORC1 activity also affect TORC1 in vivo in an animal model (Juhasz, 2008). The purpose of this study was two-fold: (1) to analyze whether MAP4K3 regulates TORC1 activity in vivo in the fly, and (2) to study the physiological consequences for the organism when the ability of TORC1 to sense amino acids is impaired (Bryk, 2010).

Biochemical evidence is presented that TORC1 activity is reduced in MAP4K3 mutant animals, consistent with published cell-culture data showing that MAP4K3 is required for full TORC1 activation (Findlay, 2007). Furthermore, MAP4K3 mutants have defects typical of reduced TORC1 activity. They are delayed in their development due to a reduced rate of growth. They eventually pupate leading to adults of reduced size and their tissues are comprised of cells that are smaller than normal. Furthermore, MAP4K3 mutants have significantly reduced triglyceride stores compared to controls. These physiological effects are similar to the phenotypes observed with mutants for other regulators of TOR, such as Melted. Melted mutant flies are also 10% smaller than controls and are significantly leaner (Bryk, 2010).

As a whole, the MAP4K3- mutant phenotypes emulate the physiological effects observed when flies are grown on conditions of limiting food. When wildtype larvae are put on a low-nutrient diet, they are delayed in pupation and yield animals of small size that are lean. Thus loss of MAP4K3 activity phenocopies a reduced nutrient environment, consistent with MAP4K3 playing a role in the ability of animals to sense their nutrient conditions. This suggests the ability of TORC1 to sense amino acids is most important when nutrient conditions are rich, allowing animals to accelerate their growth accordingly. In contrast, on a low-nutrient diet, control and MAP4K3 mutant flies grow equally slowly consistent with TOR activity being low in both groups. This parallels nicely the results reported in cell culture by (Findlay, 2007): In the absence of amino acids, both control and MAP4K3 knockdown cells have low TOR activity whereas in the presence of amino acids, TOR is activated strongly in control cells but only weakly in MAP4K3 knockdown cells (Bryk, 2010).

Unexpectedly, it was found that MAP4K3 mutant animals are viable, although they have an elevated mortality rate compared to controls. This suggests that the amino acid sensing pathway might only modulate TORC1 activity. If TORC1 activity were completely blunted in MAP4K3 mutants, the animals would be dead, as is the case for TOR or Rheb mutants. Consistent with this, residual TORC1 activity was observed in MAP4K3 mutants, as detected by phosphorylation of the TORC1 targets S6K and 4EBP. This also parallels results from cell culture. The results presented in Findlay (2007) are obtained with cells starved of serum and consequently of insulin signaling. In the presence of insulin signaling, which resembles the physiological situation more closely, MAP4K3 mutant cells still retain residual TOR activity, similar to what was observed in vivo in this study. Consistent with these findings, it was observed that the Rheb expression is able to drive tissue growth also in the absence of MAP4K3 (Bryk, 2010).

Both MAP4K3 and the Rag GTPases have recently been shown to be required for amino acids to stimulate TORC1 activity. While studying dMAP4K3, it was noticed that dMAP4K3 binds physically to the Rag GTPases, suggesting they might act together as components of a single signaling pathway. This interaction is likely specific for several reasons: (1) no binding of MAP4K3 to another GTPase, Rheb, could be detected, (2) binding of MAP4K3 to the RagA/C complex was significantly stronger than binding of an unrelated HA-tagged protein, HA-medea (3) MAP4K3 bound FLAG-RagC significantly stronger than FLAG-RagA showing that MAP4K3 distinguishes between two Rag proteins and (4) binding of MAP4K3 to RagC depended on its GDP/GTP state (Bryk, 2010).

Further studies will be required to test whether this interaction is important for TORC1 to sense amino acids. The data suggest that MAP4K3 might be functioning upstream of the Rag GTPases, and not downstream since activated RagA does not require MAP4K3 to promote tissue growth in vivo. This raises the possibility that the Rag GTPases may be substrates for MAP4K3 phosphorylation. Indeed, RagC is phosphorylated in vivo in Kc167 cells on Ser388. If MAP4K3 were to phosphorylate RagC, this would provide a mechanism for regulation of the Rag GTPases, which to date is mysterious. Work in the near future should shed further light on this issue (Bryk, 2010).

In summary, this study has characterized the physiological function of MAP4K3 in Drosophila, and shown that it modulates TORC1 activity, tissue growth and lipid metabolism in the animal. Physical interaction data hints at a possible link between MAP4K3 and the Rag GTPases. The organismal function of amino acid sensing by TORC1 is mainly required to spur growth when nutrient conditions are rich (Bryk, 2010).

P4K3 is a component of the TORC1 signalling complex that modulates cell growth and viability in Drosophila melanogaster

MAP4K3 is a conserved Ser/Thr kinase that has being found in connection with several signalling pathways, including the Imd, EGFR, TORC1 and JNK modules, in different organisms and experimental assays. This study analyzed the consequences of changing the levels of MAP4K3 expression in the development of the Drosophila wing, a convenient model system to characterize gene function during epithelial development. Using loss-of-function mutants and over-expression conditions it was found that MAP4K3 activity affects cell growth and viability in the Drosophila wing. These requirements are related to the modulation of the TORC1 and JNK signalling pathways, and are best detected when the larvae grow in a medium with low protein concentration (TORC1) or are exposed to irradiation (JNK). MAP4K3 was also shown to display strong genetic interactions with different components of the InR/Tor signalling pathway, and can interact directly with the GTPases RagA and RagC and with the multi-domain kinase Tor. It is suggested that MAP4K3 has two independent functions during wing development, one related to the activation of the JNK pathway in response to stress and other in the assembling or activation of the TORC1 complex, being critical to modulate cellular responses to changes in nutrient availability (Resnik-Docampo, 2011).

This study has characterised the consequences of changing the amount of MAP4K3, encoded by hppy, in the development of the wing disc, focussing on its relationships with the TORC1 signalling pathway. Previous data suggested that MAP4K3 might be related with a variety of signalling pathways, including EGFR, ImD, JNK and TOR. For these reasons, the advantages of the wing model was used to analyse hppy, as in this system changes in the level of signalling by a variety of pathways lead to pathway-specific phenotypes. A reduction of hppy expression in the wing, using interference RNA or loss-of-function alleles, did not uncover a critical requirement of the gene for embryonic or larval viability. In hppy mutant wings, only a weak reduction was found in wing size and cell size, compatible with a moderate reduction of TORC1 activity. It has been recently reported (Bryk, 2010) that the developmental delay caused by protein starvation is similar in wild type and hppy mutant larvae, suggesting that MAP4K3 is required in vivo to activate TOR and promote growth mostly when amino acid conditions are rich. In contract, this study found a significant requirement for the gene when hppy mutant larvae grow under starvation conditions. Thus, these flies still develop smaller wings than controls, indicating a functional requirement of hppy when the availability of proteins is reduced. This difference could be due to the parameters measured (developmental delay vs. cell and wing size) or to the remnants of hppy function in the alleles used in each experiment (Resnik-Docampo, 2011).

It was also found that, loss of hppy does not affect cell viability or JNK signalling, but that in a hppy loss-of-function genetic background the activation of JNK signalling in response to irradiation is reduced. Thus, the function of hppy might become significant mostly when the organism is challenged by stress signals induced for example by irradiation, indicating a role for the gene in the modulation of JNK signalling in vivo (Resnik-Docampo, 2011).

The increase in happyhour expression does have more dramatic consequences than its loss, causing a severe reduction in the size of the wing independently of environmental conditions. Wing size reduction is associated with both apoptosis and a smaller than normal cell size. The overall morphology and pattern of these wings is normal, with only a weak phenotype of extra-veins in the strongest combinations. Cell death induction and reduced cell size are the diagnostic phenotypes of increased JNK and reduced InR/Tor signalling, respectively. The same processes are affected by loss of MAP4K3 expression in the wing, and therefore, from this analysis it is concluded that MAP4K3 has the potential to activate cell death through the JNK signalling pathway, and also that it can interfere with some component/s of the InR/Tor cascade. The effects of loss- and gain of MAP4K3 on JNK activity are opposite, which is expected from a protein with kinase activity. In contrast both loss and gain of MAP4K3 seem to reduce the function of TORC1. It is likely that in this case MAP4K3 acts as part of a protein complex that can be made non-functional by changes in the stochiometry of its components. What seems clear is that the effects of MAP4K3 on JNK and TORC1 are exerted through independent mechanisms, because the contribution of cell death to the wing phenotype of MAP4K3 over-expression is very modest, and Tor reductions only lead to cell death when cells with different levels of Tor activity are confronted (Resnik-Docampo, 2011).

The phenotype of MAP4K3 over-expression is very sensitive to changes in the levels or activity of several members of the InR/Tor pathway. Thus, strong synergistic interactions were observed when Akt, raptor and Tor are reduced in the background of MAP4K3 over-expression, and the presence of the dominant-negative form TorTED in this background entirely eliminates the wing. Conversely, loss of hppy expression rescues the effects of TorTED expression. These results suggest that MAP4K3 could act at the level of TORC1. This possibility is compatible with the suppression by MAP4K3 over-expression of phenotypes caused by increased levels of InR/Tor signalling generated by lower than normal levels of PTEN and TSC1/2. In addition to genetic data in the wing, experiments in cell culture with both the fly and human MAP4K3 homologue proteins indicated that MAP4K3 is required to generate maximal activity of TORC1 in response to aminoacids. Therefore, it is suggested that although MAP4K3 is normally required to promote TORC1 signalling, when the protein is over-expressed the balance between TORC1 components required for its normal function in vivo is modified. This effect appears to depend exclusively on the kinase domain of MAP4K3, because the over-expression of this domain causes a strong reduction in wing and cell size. This sstudy has shown that MAP4K3 can interact with RagA, RagC and Tor in pull-down experiments in vitro, and therefore it is speculated that the excess of MAP4K3 alters the phosphorylation levels of TORC1 components and this leads to the assembly of inactive complexes. A similar mechanism might explain the dominant-negative effect of Tor, as it was suggested that Tor over-expression leads to the sequestering of TORC1 components in non-functional complexes. In summary, it is suggested that MAP4K3 normally potentiate TORC1 and JNK functions in response to environmental challenges, without being strictly required to generate some levels of TORC1 or JNK activity, and that MAP4K3 hyper-activity leads to high levels of JNK signalling and to reduced TORC1 function, in this case due to the formation of inactive TORC1 complexes (Resnik-Docampo, 2011).

Identification of Happyhour/MAP4K as alternative Hpo/Mst-like kinases in the Hippo kinase cascade

In Drosophila and mammals, the canonical Hippo kinase cascade is mediated by Hpo/Mst acting through the intermediary kinase Wts/Lats to phosphorylate the transcriptional coactivator Yki/YAP/TAZ. Despite recent reports linking Yki/YAP/TAZ activity to the actin cytoskeleton, the underlying mechanisms are poorly understood and/or controversial. Using Drosophila imaginal discs as an in vivo model, this study shows that Wts, but not Hpo, is genetically indispensable for cytoskeleton-mediated subcellular localization of Yki. Through a systematic screen, the Ste-20 kinase Happyhour (Hppy) and its mammalian counterpart MAP4K1/2/3/5 were identified as an alternative kinase that phosphorylates the hydrophobic motif of Wts/Lats in a similar manner as Hpo/Mst. Consistent with their redundant function as activating kinases of Wts/Lats, combined loss of Hpo/Mst and Hppy/MAP4K abolishes cytoskeleton-mediated regulation of Yki/YAP subcellular localization, as well as YAP cytoplasmic translocation induced by contact inhibition. These Hpo/Mst-like kinases provide an expanded view of the Hippo kinase cascade in development and physiology (Zheng, 2015).

Understanding of the core kinase cascade of the Hippo pathway has been aided by multiple lines of investigation. First, genetic screens for tumor suppressors using mosaic flies have identified Hpo, Sav, Wts, and Mats as main constituents of the core kinase cassette. Second, biochemical studies of the activation mechanism of NDR family kinases, which include Lats1/2 and NDR1/2, demonstrate the importance of regulatory phosphorylation sites on the activation loop and the hydrophobic motif. The realization that the hydrophobic motif of Wts is phosphorylated by Hpo provides a fitting molecular explanation for the linear genetic pathway uncovered by in vivo studies. The simplicity of this linear pathway begun to be challenged based on the observation that Mst1/2 null cells still showed high levels of Lats phosphorylation on the hydrophobic motif. Indeed, in many subsequent reports, various signals have been reported to still regulate YAP/TAZ activity in Mst1/2 null cells. While semantically these observations were implied to support the existence of 'Mst1/2-independent' mechanisms, the molecular underpinning of this phenomenon has been elusive. It was also unclear whether this represents a mammalian-specific phenomenon as there has been no evidence to date that a similar mechanism operates in Drosophila (Zheng, 2015).

The current study addresses these issues in several significant ways. Definitive evidence is provided supporting an alternative Hpo-independent mechanism of Hippo pathway activation in Drosophila by demonstrating the genetic requirement of Wts, but not Hpo, in LatB-induced nuclear exclusion of Yki. Through a systematic screen, the Ste-20 family kinase Hppy/MAP4K was identified as a plausible molecular explanation for Mst1/2-independent regulation of Hippo signaling. Not only does Hppy/MAP4K directly phosphorylate the hydrophobic motif of Wts/Lats in vitro and in cell cultures, but loss of Hppy/MAP4K also abolishes LatB-induced Yki/YAP cytoplasmic translocation in Hpo/Mst null cells in both Drosophila tissues and mammalian cell cultures. These findings support the view that Hpo/Mst and Hppy/MAP4K act as redundant kinases targeting the hydrophobic motif of Wts/Lats. It is also noted that analysis of Hpo/Mst and Hppy/MAP4K in F-actin-mediated Hippo signaling was largely based on LatB treatment. Thus, it remains to be determined how these kinases cooperate with each other in a more physiological setting of cytoskeleton modulation. Nevertheless, the fact that MAP4K mediates Mst-independent regulation of YAP target gene expression and contact inhibition of YAP nuclear localization suggests that these kinases co-regulate Wts/Lats in multiple contexts beyond LatB-induced F-actin disruption. Since the hydrophobic motif of NDR1/2 can be phosphorylated by both Mst1 and Mst3, it is suggested that phosphorylation of hydrophobic motif by multiple Ste-20 kinases may be a common feature of the NDR family kinases. It is noted that two other kinases, CK2 and MSN/MAP4K4, were recently reported to promote Wts/Lats activity toward Yki. However, neither kinase was shown to directly phosphorylate the hydrophobic motif of Wts/Lats. Furthermore, although MSN was shown to promote Yki phosphorylation when Wts was co-expressed in S2 cells, MSN alone did not affect Yki phosphorylation, as was observed in this study. Thus, the mechanisms by which these kinases promote Hippo signaling remain to be determined (Zheng, 2015).

Recent studies have implicated cellular mechanical force as a regulator of Yki/YAP/TAZ activity. Reorganization of F-actin cytoskeleton has been suggested as the common mediator of mechanical forces arising from cell-cell and cell-matrix interactions. However, the underlying mechanism by which F-actin controls Yki/YAP/TAZ activity remains poorly understood and/or controversial. While some studies suggested that cytoskeleton-mediated regulation of YAP/TAZ is independent of the Hippo kinase cascade, others suggested that it requires the Hippo kinase cascade. The observation that LatB-induced Yki cytoplasmic localization is Wts dependent is more consistent with a Hippo signaling-dependent mechanism. An important modification brought by the current study is that the canonical Hippo kinase cascade should be expanded to include Hppy/MAP4K at the level of Hpo. This expanded Hippo kinase cascade may also include NDR1/2 at the level of Lats1/2, given the recent report of NDR1/2 as Lats1/2-like kinases capable of phosphorylating YAP (Zheng, 2015).

Finally, it is suggested that the Hippo-signaling-dependent and -independent regulation of YAP/TAZ by F-actin may be potentially reconciled with each other. A major discrepancy between the two models came from the analysis of mutant YAP/TAZ that lacks all the Lats phosphorylation sites (YAP5SA or TAZ4SA). It is noted, however, that a different readout was used to assay the regulation of these YAP/TAZ mutants in the different studies. A luciferase reporter assay was used to show that the transcriptional activity of YAP5SA or TAZ4SA still responded to F-actin reorganization, whereas subcellular localization was used to show that YAP5SA no longer responded to cytoplasmic localization of YAP induced by F-actin disruption. These results may reflect the functionality of different subcellular pools of F-actin ; inasmuch as YAP/TAZ localization is regulated by F-actin through the Hippo pathway, F-actin may also play a separate role in regulating the transcriptional activity of YAP/TAZ in the nucleus, especially given the increasing appreciation of a more direct role of nuclear F-actin in transcriptional regulation (Zheng, 2015).

EGFR signaling modulates synaptic connectivity via Gurken

Synaptic target selection is critical for establishing functional neuronal circuits. The mechanisms regulating target selection remain incompletely understood. This study describes a role for the EGF receptor and its ligand Gurken in target selection of octopaminergic Type II neurons in the Drosophila neuromuscular system. Mutants in happyhour, a regulator of EGFR signaling, form ectopic Type II neuromuscular junctions. These ectopic innervations are due to inappropriate target selection. It was demonstrated that EGFR signaling is necessary and sufficient to inhibit synaptic target selection by these octopaminergic Type II neurons, and that the EGFR ligand Gurken is the post-synaptic, muscle-derived repulsive cue. These results identify a new pathway mediating cell-type and branch-specific synaptic repulsion, a novel role for EGFR signaling in synaptic target selection, and an unexpected role for Gurken as a muscle-secreted repulsive ligand (Naylor, 2011).

Synaptic target selection is a critical step in establishing functional neural circuits. The molecular mechanisms governing this selection have not yet been fully explored. The observation that hppy mutants have an increased frequency of ectopic octopaminergic Type II NMJs has resulted in identification of a pathway critical for synaptic target recognition in these neurons. EGFR signaling pathway was found to be required to prevent the development of inappropriate synaptic contacts. This inhibitory signal is mediated by muscle-derived EGFR ligand, Gurken, working through the EGF receptor in type II motoneurons. This mechanism sculpts the neuronal wiring pattern in a cell-type and branch-specific manner (Naylor, 2011).

There are many signaling pathways that influence the innervation pattern of Drosophila motor neurons. These findings identify a novel role for EGFR signaling in mediating a repulsive guidance cue to Type II neurons. EGF has previously been demonstrated to regulate axon growth and guidance. For example, EGF positively regulates Sema-3a levels in the cornea and interacts with NCAM-180 to promote neuritogenic activity. However, these data are the first to demonstrate that an EGF receptor and ligand provide a synaptic targeting signal (Naylor, 2011).

Ectopic Type II NMJs were found in hppy mutants, as well as when a dominant negative EGFR was expressed in Type II neurons or its ligand Gurken was knocked down in the muscle targets. This presents an apparent contradiction. hppy is described as a negative regulator of EGFR (Corl, 2009), and its phenotype in this system is suppressed by a hypomorphic mutation in rolled (ERK), consistent with hppy functioning as a negative regulator of EGFR signaling. Why then does loss of hppy have the same phenotype as inhibition of the receptor or ligand? It was posited that the well-described strong negative feedback induced by EGFR signaling may be the explanation. A model is proposed in which activation of the EGFR pathway mediates a signal that inhibits the formation or stabilization of Type II NMJs. In hppy mutants, however, loss of negative regulation would allow for excess activation of the EGFR that would induce a quick, strong and long-lasting negative feedback activity early in development, essentially turning off EGFR signaling in cells expressing happyhour. The result would be loss of the synaptic inhibitory signal mediated by EGFR at the stage when these Type II neurons are extending to their targets and the promotion of ectopic synapse formation. It is appreciated that this model is speculative, and that an alternative is that hppy and rolled are regulating a pathway that is distinct from the EGFR/gurken pathway (Naylor, 2011).

By what mechanism does EGFR signaling affect synapse formation in Type II neurons? Presumably, there is a molecular program downstream of EGFR activation that modifies the Type II neuron such that it does not form and/or maintain an NMJ with an inappropriate target. These changes could occur at the level of the cell body or locally within individual branches. It is unlikely that cell body changes are central to the phenotype because such neuron-wide mechanisms could not easily be translated into branch-specific behavior. In contrast, local effects of EGFR signaling within neurites could explain such specificity. The cell biological mechanism mediating the branch-specific inhibition is not known. Possibilities include alterations in the local translation or membrane insertion of synaptogenic molecules, local modulation of cytoskeletal dynamics, or failure to properly prune Type II connections (Naylor, 2011).

Not only is the EGFR mediating a branch-specific effect, but it is also cell-type specific. The Type II motoneuron MNSNb/d-II and the Type Is motoneuron MNSNb/d-Is travel together and presumably encounter the same cues across the hemisegment, however they generate different innervation patterns. This implies that these two types of neurons have developed cell-type specific repertoires of receptors or signaling pathways that shape their target choices (Naylor, 2011).

While the data indicate a role for EGFR signaling in Type II synaptic target selection it is also likely that Type II target selection has multiple components. The phenotypes occur at a relatively low penetrance, so it is likely that complementary and combinatorial guidance cues function with the EGFR pathway to shape target selection of Type II neurons (Naylor, 2011).

Gurken has been studied exclusively in the developing oocyte and has no known roles in other tissues. Hence, it is surprising that Gurken conveys the repulsive signal from muscle to the octopaminergic Type II neurons. In support of a function in muscle, the modEncode RNA-seq project has found that Gurken transcript is enriched 3.5 fold in larval body wall muscles. While Gurken may be secreted from all muscles, a model is preferred in which localized expression in a muscle subset shapes the branching pattern of the innervating motoneuron. In this model, Gurken released from muscles 6 and 7, as well as other targets that should not be innervated, would locally inhibit synaptogenesis, blocking the formation of ectopic connections while allowing for the normal innervation at muscles 12 and 13. This model is consistent with findings that knockdown of Gurken in muscle results in ectopic NMJs while localized overexpression in the normal target cell inhibits formation of appropriate NMJs. While these functional data are strong, the model must remain speculative because it has not been possible to determine the localization of Gurken using currently available reagents. Future studies will investigate how this Gurken/EGFR pathway is integrated with the recently defined semaphorin- and activity-dependent mechanisms that also play an important role in shaping synaptic target selection in these neurons (Naylor, 2011).

Drosophila happyhour modulates JNK-dependent apoptosis

Mitogen-activated protein kinase kinase kinase kinase-3 (MAP4K3) is a Ste20 kinase family member that modulates multiple signal transduction pathways. MAP4K3 has been identified as proapoptotic kinase using an RNA interference screening approach. In mammalian cells, MAP4K3 enhances the mitochondrial apoptosis pathway through the post-transcriptional modulation of selected proapoptotic Bcl-2 homology domain 3-only proteins. Recent data suggest that MAP4K3 mutations contribute to pancreatic cancer, which highlights the importance of studying the in vivo function of this kinase. To determine whether the cell death function is conserved in vivo and which downstream signalling pathways are involved, transgenic flies were generated expressing happyhour (hppy), the Drosophila MAP4K3 orthologue. This study shows that the overexpression of hppy promotes caspase-dependent apoptosis and that the hypothetical kinase domain is essential for inducing cell death. In addition, it was shown that hppy expression triggers the activation of both the c-Jun N-terminal kinase (JNK) and target of rapamycin (TOR) signalling pathways; however, only JNK signalling is required for apoptosis. Together, these results show that hppy has a JNK-dependent proapoptotic function in Drosophila, which reinforces the hypothesis that MAP4K3 might act as tumour suppressor by regulating apoptosis in higher eukaryotes (Lam, 2010).

The work reported here identifies Hppy, the Drosophila orthologue of human MAP4K3, as an in vivo modulator of apoptosis. In mammalian cells, MAP4K3 promotes apoptosis by inducing the post-transcriptional activation of a subset of BH3-only Bcl-2 family proteins. Expression of MAP4K3 leads to cell death through the mitochondrial (intrinsic) pathway; this action is suppressed by JNK and TOR inhibition. This study provides the first in vivo evidence that the fly orthologue of MAP4K3, Hppy, is a death-inducing kinase that promotes caspase-dependent apoptosis. Mitochondria have a crucial role in the intrinsic apoptosis pathway in vertebrates. In Drosophila, however, the role of mitochondria in apoptosis is unclear, as mutants of the Drosophila bcl-2 homologues debcl and buffy show no obvious defects in developmental apoptosis and this form of cell death is not blocked in cells with depleted Cyt-C (Lam, 2010).

The data suggest that Hppy-mediated cell death occurs independently of the mitochondrial pathway; buffy expression failed to block Hppy-dependent apoptosis, and negative modulation of the TOR pathway in flies failed to suppress Hppy-dependent cell death. Given that the human orthologue of Hppy employs the TOR pathway to stimulate the mitochondrial pathway of apoptosis, it is proposed that suppression of this pathway in flies fails to affect Hppy-dependent cell death because the mitochondrial apoptosis pathway may be of limited importance in flies. Genetic analysis suggests that the JNK pathway positively regulates Hppy-dependent cell death. It is unlikely that the JNK pathway induces cell death by activating the mitochondrial pathway in Drosophila. In flies, the genes reaper, grim and hid (RGH) promote caspase activation by antagonising the Drosophila inhibitor of apoptosis proteins. Although reaper and grim are only expressed in cells that are destined for death, hid expression is controlled at both the transcriptional and post-transcriptional levels. Hid has been shown to be transcriptionally activated in a JNK- and Foxo-dependent manner. Additionally, the proapoptotic activity of Foxo is opposed by the action of receptor tyrosine kinases (RTKs) such as EGFR and insulin-like growth factor receptor. Given the recent observation that Hppy is a potent negative modulator of EGFR signalling, it is tempting to propose that this kinase might promote apoptosis by modulating signalling pathways that affect the levels of RGH proteins. This idea is consistent with the observation that another Ste20 family member, Hippo, promotes hid expression (Lam, 2010).

A single amino-acid substitution in MAP4K3 has been associated with pancreatic cancer, suggesting that this kinase might be an important modulator of tumourigenesis (Lam, 2010).

It was also previously found that the levels of MAP4K3 are significantly reduced in pancreatic tumour samples (Lam, 2010).

Given that in Drosophila, Hppy acts to both stimulate JNK signalling it is tempting to propose that mammalian MAP4K3 might act as a tumour suppressor, not only by promoting JNK-dependent apoptosis but also by blocking the prosurvival effects of EGFR signalling mechanisms (Lam, 2010).

Happyhour, a Ste20 family kinase, implicates EGFR signaling in ethanol-induced behaviors
The consequences of alcohol use disorders (AUDs) are devastating to individuals and society, yet few treatments are currently available. To identify genes regulating the behavioral effects of ethanol, a genetic screen was conducted in Drosophila and a mutant, happyhour (hppy), was identified due to its increased resistance to the sedative effects of ethanol. Hppy protein shows strong homology to mammalian Ste20 family kinases of the GCK-1 subfamily. Genetic and biochemical experiments revealed that the epidermal growth factor (EGF)-signaling pathway regulates ethanol sensitivity in Drosophila and that Hppy functions as an inhibitor of the pathway. Acute pharmacological inhibition of the EGF receptor (EGFR) in adult animals altered acute ethanol sensitivity in both flies and mice and reduced ethanol consumption in a preclinical rat model of alcoholism. Inhibitors of the EGFR or components of its signaling pathway are thus potential pharmacotherapies for AUDs (Corl, 2009).

Two P element mutants in the CG7097/happyhour (hppy) gene region were identified and characterized and it was found that reduced hppy expression resulted in decreased sensitivity to ethanol-induced sedation, whereas neuronal overexpression of hppy caused the opposite effect. By in situ hybridization and QPCR, evidence was found for hppy expression in adult brain, and behavioral rescue experiments demonstrated that neuronal expression of hppy was sufficient to rescue the hppy sedation resistance phenotype (Corl, 2009).

Like its mammalian homologs, the GCK-1 subfamily of Ste20 family kinases, the predicted hppy products contain N-terminal serine/threonine kinase domains and C-terminal regulatory domains known as citron homology domains. In vitro studies of these homologs of Hppy, including GCK, GCK-like kinase, kinase homologous to SPS1/STE20, and hematopoietic progenitor kinase (HPK), have revealed that they activate JNK signaling, but not ERK or p38 signaling. HPK1 and GLK have both been shown to phosphorylate MAP3Ks in the JNK pathway, implying that GCK-1 kinases are MAP4Ks acting upstream of JNK signaling (Corl, 2009 and references therein).

This study provides evidence that Hppy, a presumed MAP4K in the GCK-1 subfamily of Ste20 kinases, can modulate EGFR/ERK signaling in a manner that is consistent with it acting as an inhibitor of the pathway. First, retinal hppy overexpression respectively enhanced and suppressed the rough-eye phenotypes brought about by EGFR/ERK pathway inhibition and activation. Second, increased hppy expression enhanced the semilethality caused by ectopic expression of transgene EGFR pathway inhibitors. Third, decreasing levels of hppy completely suppressed the enhanced ethanol sensitivity brought about by neuronal EGFR downregulation. Finally, ethanol induced robust phosphorylation of ERK/Rolled in a hppy mutant, but not in control flies. What, then, is the biochemical mechanism through which Hppy inhibits EGFR/ERK signaling? The answer to this question is still unknown. However, an in vitro study of another GCK-1 subfamily kinase, HPK1, offers an intriguing possibility (Anafi, 1997). This study showed that HPK1 physically associates with the EGFR adaptor protein Grb2 and that EGF stimulation recruits the Grb2/HPK1 complex to the autophosphorylated EGFR. This recruitment leads to the tyrosine phosphorylation of HPK1. It will be interesting to determine whether such a physical association exists between Hppy and components of the EGFR/ERK signaling cascade and, if so, what the consequences may be on signaling (Corl, 2009).

The experiments cannot completely rule out a role for hppy in regulating JNK signaling, although JNK signaling perturbation did not affect ethanol-induced sedation. In addition, hppy mutant flies responded normally to a variety of stress stimuli known to activate the JNK and p38 pathways, including oxidative stress, heat stress, and starvation. Indeed, studies in HeLa cells show a lack of involvement of hppy in JNK activation in response to osmotic stress and the protein synthesis inhibitor anisomycin (Findlay, 2007; Corl, 2009).

In recent years, studies in vitro and in vivo have revealed an intriguing link between ethanol and the mammalian EGFR/ERK pathway, demonstrating that EGFR autophosphorylation and ERK phosphorylation are both inhibited by pharmacologically relevant concentrations of ethanol (Chandler, 2005; Ma, 2005). In addition, elevated expression of several MAPKs and their regulators has been reported in the brains of mice and rats selected for high ethanol preference. This study uncover a role for the EGFR/ERK pathway in mediating the behavioral responses to ethanol in Drosophila. Neuronal manipulations that activate the EGFR/ERK pathway resulted in enhanced resistance to the sedative effects of ethanol, whereas neuronal inhibition of the pathway caused increased sensitivity. These effects were seen upon manipulations of several different components of the EGFR/ERK pathway. In contrast, no evidence was found for the other two major MAPK pathways, JNK and p38, in mediating the sedative response to ethanol. The finding that EGFR activation specifically in either insulin-producing cells (IPCs) or dopaminergic cells affects ethanol sensitivity is consistent with previous studies implicating both the IPCs and dopaminergic systems in the behavioral response to ethanol in Drosophila and suggests that the EGFR/ERK pathway may interact with the insulin- and dopamine-signaling pathways to control drug responses. Equally interesting is the observation that EGFR activation in many other brain regions, including those previously shown to play a role in ethanol-related behaviors, such as the ellipsoid body and the cells defined by the 201Y GAL4 line, had no effect on ethanol-induced sedation. Thus, the EGFR pathway appears to play a role in only a subset of brain regions that regulate flies' response to ethanol (Corl, 2009).

The mechanisms through which the EGFR/ERK cascade detects ethanol and how it might transduce those signals into a behavioral response remain unknown. It was found that acute ethanol exposure, at concentrations that are behaviorally relevant, led to a rapid and transient increase in ERK/Rolled phosphorylation in the heads of hppy mutants, an effect that was not observed in wild-type flies. Although this finding supports a role for Hppy as an inhibitor of the pathway, it is unclear whether it explains the increased resistance to ethanol-induced sedation observed in hppy flies or in flies in which the EGFR/ERK pathway was chronically upregulated. It is curious that the data do not reveal an inhibitory effect of ethanol on the EGFR/ERK pathway, as has been reported in rodents. While this may reflect a fundamental dissimilarity in the way that the pathway operates in flies and mammals, this discrepancy is more likely due to the fact that mammalian experiments used chronic ethanol exposure paradigms, whereas the current experiments relied on acute exposure (Corl, 2009).

This study shows that the EGFR has a role in regulating ethanol behaviors in adult flies and mammals. Administration of two well-studied EGFR inhibitors, erlotinib (Tarceva) and gefitinib (Iressa), to adult flies resulted in enhanced sensitivity in the LOR assay. Though the current results do not rule out a developmental role for the EGFR/ERK pathway, they do show that this pathway can function in the adult fly to regulate the sedative effects of ethanol. Similarly, acute administration of erlotinib enhanced sensitivity of mice to the sedating effects of ethanol, implying that the role of the EGFR in this behavior is conserved among flies and rodents. Most importantly, it was found that treatment of adult rats with erlotinib significantly decreased ethanol preference in a two-bottle-choice drinking paradigm. This effect appears to be ethanol specific, as preference for a second rewarding substrate, sucrose, was not altered. Together, these data reveal a potentially conserved role for the EGFR pathway in regulating ethanol behaviors in both flies and rodents. Because both erlotinib (Tarceva) and gefitinib (Iressa) (as well as many other small molecule EGFR inhibitors) are FDA-approved drugs, are known to cross the blood-brain barrier, and are well-tolerated, they offer a possible therapeutic avenue for the treatment of AUDs in humans (Corl, 2009).

A MAP4 kinase related to Ste20 is a nutrient-sensitive regulator of mTOR signalling

The mTOR (mammalian target of rapamycin) signalling pathway is a key regulator of cell growth and is controlled by growth factors and nutrients such as amino acids. Although signalling pathways from growth factor receptors to mTOR have been elucidated, the pathways mediating signalling by nutrients are poorly characterized. Through a screen for protein kinases active in the mTOR signalling pathway in Drosophila a Ste20 family member (MAP4K3) was identified that is required for maximal S6K (S6 kinase)/4E-BP1 [eIF4E (eukaryotic initiation factor 4E)-binding protein 1] phosphorylation and regulates cell growth. Importantly, MAP4K3 activity is regulated by amino acids, but not the growth factor insulin and is not regulated by the mTORC1 inhibitor rapamycin. These results therefore suggest a model whereby nutrients signal to mTORC1 via activation of MAP4K3 (Findley, 2007).

In metazoans both nutrient amino acids and mitogens are required to promote mTOR signalling and cell growth. Activation of mTOR signalling by mitogens involves activation of protein kinases such as PKB, p90RSK and ERK that have been reported to inhibit the function of the TSC1-2 complex which is then thought to lead to increased GTP loading of the Ras-like GTPase Rheb. In contrast, amino acids do not appear to regulate Rheb GTP loading, and, although a class III PI3K has been implicated upstream of mTORC1, no analogous protein kinases have been identified that respond to amino acids and activate mTOR signalling. This study found, both in Drosophila and in mammalian cells, that suppression of MAP4K3 inhibited mTOR signalling to S6K in the context of activation of the pathway induced by deficiency in TSC1-2. Since a variety of oncogenic signalling pathways inhibit TSC1-2, and TSC1-2 is predicted also to be inactivated by loss of function of the tumour suppressor PTEN (phosphatase and tensin homologue deleted on chromosome 10) via activation of PKB, MAP4K3 may represent a promising new candidate for inhibition of the pathway in diseases of TSC1-2 or PTEN loss-of-function. Although the mechanism of MAP4K3 action on mTOR signalling has not been defined, the data on the positive regulation of both S6K1 activity and phosphorylation of 4E-BP1 by MAP4K3 overexpression points to the mTORC1 complex as the likely target of MAP4K3 action. Interestingly, addition of excess amino acids (relative to the normal concentrations found in DMEM) has been reported to fully activate S6K in the absence of growth factors, which is similar to current findings with overexpressed MAP4K3, suggesting that MAP4K3 may be activated further by amino acid excess. Future studies will be required to clarify these points. However, having defined the action of a new protein kinase in the mTOR pathway, delineating the signals downstream of amino acids that activate MAP4K3, and determining its mechanism of action will likely shed further light on nutrient regulation of cell growth (Findley, 2007).

In conclusion, these data indicate that amino acids stimulate the activity of a Ste20-family kinase, MAP4K3, with maximal kinase activation occurring concordant with phosphorylation of the Thr389 site of S6K1. In HeLa cells MAP4K3 activity is required for amino acid-induced activation of S6K1 and mediates rapamycin-senstive signalling to two effectors of mTORC1, but is not itself stimulated by insulin or inhibited by rapamycin. Lastly, MAP4K3 promotes cell growth in human HeLa cells in culture in a similar manner to Rheb and mTORC1 (Findley, 2007).


REFERENCES

Search PubMed for articles about Drosophila

Anafi, M., et al. (1997), SH2/SH3 adaptor proteins can link tyrosine kinases to a Ste20-related protein kinase, HPK1. J. Biol. Chem. 272: 27804-27811. PubMed ID: 9346925

Bryk, B., Hahn, K., Cohen, S. M. and Teleman, A. A. (2010). MAP4K3 regulates body size and metabolism in Drosophila. Dev. Biol. 344(1): 150-7. PubMed ID: 20457147

Chandler L. J. and Sutton, G. (2005), Acute ethanol inhibits extracellular signal-regulated kinase, protein kinase B, and adenosine 3':5'-cyclic monophosphate response element binding protein activity in an age- and brain region-specific manner. Alcohol. Clin. Exp. Res. 29: 672-682. PubMed ID: 15834234

Corl, A. B., et al. (2009). Happyhour, a Ste20 family kinase, implicates EGFR signaling in ethanol-induced behaviors. Cell 137(5): 949-60. PubMed ID: 19464045

Findlay, G. M., Yan, L., Procter, J., Mieulet, V. and Lamb, R. F. (2007). A MAP4 kinase related to Ste20 is a nutrient-sensitive regulator of mTOR signalling. Biochem. J. 403(1): 13-20. PubMed ID: 17253963

Gulati, P., et al., (2008). Amino acids activate mTOR complex 1 via Ca2+/CaM signaling to hVps34. Cell Metab. 7: 456-465. PubMed ID: 1846033

Juhasz, G., et al., (2008). The class III PI(3)K Vps34 promotes autophagy and endocytosis but not TOR signaling in Drosophila. J. Cell Biol. 181: 655-666. PubMed ID: 18474623

Kim, E., et al. (2008) Regulation of TORC1 by Rag GTPases in nutrient response, Nat. Cell Biol. 10: 935-945. PubMed ID: 18604198

Lam, D., et al. (2010). Drosophila happyhour modulates JNK-dependent apoptosis. Cell Death Dis. 1(8): e66. PubMed ID: 21364671

Ma, C., et al., (2005). The role of epidermal growth factor receptor in ethanol-mediated inhibition of activator protein-1 transactivation. Biochem. Pharmacol. 69: 1785-1794. PubMed ID: 15878157

Naylor, S. A. and Diantonio, A. (2011). EGFR signaling modulates synaptic connectivity via Gurken. Dev. Neurobiol. [72(9): 1229-42. PubMed ID: 22021126

Resnik-Docampo, M. and de Celis, J. F. (2011). MAP4K3 is a component of the TORC1 signalling complex that modulates cell growth and viability in Drosophila melanogaster. PLoS One. 6(1): e14528. PubMed ID: 21267071

Sancak, Y., et al (2008).The Rag GTPases bind raptor and mediate amino acid signaling to mTORC1, Science 320: 1496-1501. PubMed ID: 18497260

Zheng, Y., Wang, W., Liu, B., Deng, H., Uster, E. and Pan, D. (2015). Identification of Happyhour/MAP4K as alternative Hpo/Mst-like kinases in the Hippo kinase cascade. Dev Cell 34(6):642-55. PubMed ID: 26364751


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date revised: 20 October 2015

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