twins
Asymmetric cell division is a mechanism for generating cell diversity as well as maintaining stem cell homeostasis in both Drosophila and mammals. In Drosophila, larval neuroblasts are stem cell-like progenitors that divide asymmetrically to generate neurons of the adult brain. Mitotic neuroblasts localize atypical protein kinase C (aPKC) to their apical cortex. Cortical aPKC excludes cortical localization of Miranda and its cargo proteins Prospero and Brain tumor, resulting in their partitioning into the differentiating, smaller ganglion mother cell (GMC) where they are required for neuronal differentiation. In addition to aPKC, the kinases Aurora-A and Polo also regulate neuroblast self-renewal, but the phosphatases involved in neuroblast self-renewal have not been identified. Thus study reports that aPKC is in a protein complex in vivo with Twins, a Drosophila B-type protein phosphatase 2A (PP2A) subunit, and that Twins and the catalytic subunit of PP2A, called Microtubule star (Mts), are detected in larval neuroblasts. Both Twins and Mts are required to exclude aPKC from the basal neuroblast cortex: twins mutant brains, twins mutant single neuroblast mutant clones, or mts dominant negative single neuroblast clones all show ectopic basal cortical localization of aPKC. Consistent with ectopic basal aPKC is the appearance of supernumerary neuroblasts in twins mutant brains or twins mutant clones. It is concluded that Twins/PP2A is required to maintain aPKC at the apical cortex of mitotic neuroblasts, keeping it out of the differentiating GMC, and thereby maintaining neuroblast homeostasis (Chabu, 2009).
Drosophila aPKC regulates neuroblast cell polarity and neuroblast self-renewal, however understanding of how aPKC is regulated is far from complete. Several kinases regulate neuroblast cell polarity and cell fate, but the identity of opposing phosphatases have remained elusive. This study identified Twins as part of a protein complex containing aPKC. Twins is a regulatory subunit of PP2A, and this study also shows that the catalytic subunit of PP2A, Mts, is immunoprecipitated by aPKC. Furthermore, mts and twins mutants have similar defects in neuroblast cell polarity and expansion in neuroblast numbers. This strongly suggests that the Twins/PP2A complex regulates neuroblast polarity and self-renewal (Chabu, 2009).
The primary defect in twins mutant neuroblasts is an expansion of aPKC from the apical cortex to the basal cortex, and this ectopic aPKC is active based on its ability to exclude Miranda from the basal cortex. Twins/PP2A may promote apical Baz localization, similar to the role of PP2A in promoting Baz/Par-3 apical localization in epithelia; a reduced level of apical Baz in neuroblasts may lead to failure to localize all cortical aPKC at the apical cortex and hence ectopic basal aPKC. Alternatively, PP2A may keep active aPKC from the basal cortex by directly dephosphorylating
aPKC at its N-terminus, consistent with the role of mammalian PP2A in dephosphorylating aPKCλ/ζ (Nunbhakdi-Craig, 2002). In support of this model, overexpression of aPKC lacking its N-terminus (aPKCΔN) displaces Miranda from the basal cortex into the cytoplasm, similar to twins mutant neuroblasts (Chabu, 2009).
How does Twins regulate neuroblast self-renewal? Ectopic active aPKC causes formation of supernumerary neuroblasts, as does reduced levels of the basal cortical protein Miranda. twins mutant neuroblasts have both ectopic basal cortical aPKC and a loss of basal cortical Miranda. It is likely that the primary defect causing supernumerary neuroblasts is ectopic aPKC, because reducing aPKC levels in
twins mutants can rescue both basal Miranda targeting and the formation of supernumerary neuroblasts. This is in contrast to the role of another phosphatase, PP4, in regulating Miranda localization independent of aPKC (Sousa-Nunes, 2009; Chabu, 2009 and references therein).
It has been shown that Dap160, a protein related to mammalian Intersectin, is apically localized and required to anchor aPKC at the apical cortex (Chabu, 2008). This study has shown that Twins is also required for tight apical localization of aPKC. A major difference, however, is that Dap160 directly stimulates the activity of aPKC, so that in dap160 mutant neuroblasts the ectopic basal aPKC is inactive and unable to exclude Miranda from the cortex. In contrast, twins mutants have ectopic basal aPKC that remains active and thus can drive Miranda off the cortex. This supports the conclusion, from biochemical experiments, that Twins does not stimulate aPKC activity. However, the possibility that
another regulatory subunit can target PP2A to aPKC in the absence of Twins cannot be excluded (Chabu, 2009).
Neuroectoderm cells of the optic lobe undergo a progressive differentiation to adopt a neuroblast fate. twins mutant optic lobes show a dramatic increase in optic lobe neuroblast numbers, suggesting that Twins normally functions to inhibit precocious neuroblast fate in the optic lobe neuroectoderm cells. How does Twins normally suppress
precocious neuroectodermal-to-neuroblast differentiation? This study has show that at least one pathway utilizes aPKC to regulate neuroectoderm differentiation;
twins mutant optic lobe with reduced active aPKC has a less severe phenotype compared to their twins mutant counter parts. Another pathway that has been implicated in the differentiation of neuroectoderm cells to neuroblast is the Janus Kinase/Signal transducer and activation of transcription (JAK/STAT) pathway. JAK/STAT signaling functions in
neuroectoderm cells inhibits expression of proneural genes, thereby blocking precocious neuroblast differentiation. Twins/PP2A could act positively at any point in the JAK/STAT–proneural pathway, or in an independent pathway in promoting the neuroectodermal-to-neuroblast transition in the optic lobe (Chabu, 2009).
The 55 kDa regulatory subunit of Drosophila protein phosphatase 2A is located in the cytoplasm at all cell cycle stages, by the criterion of immunofluorescence. No significant changes were detected in protein phosphatase activity during the nuclear division cycle of syncytial embryos. However, cell cycle function of the enzyme is suggested by the mitotic defects exhibited by two Drosophila mutants, aar1 and twinsP, defective in the gene encoding the 55 kDa subunit. The reduced levels of the 55 kDa subunit correlate with the loss of protein phosphatase 2A-like, okadaic acid-sensitive phosphatase activity of brain extracts against caldesmon and histone H1 phosphorylated by p34cdc2/cyclin B kinase, but not against phosphorylase a. Thus the mitotic defects of aar1 and twinsP are likely to result from the lack of dephosphorylation of specific substrates by protein phosphatase 2A (Mayer-Jaekel, 1994).
Sex combs reduced (SCR) is a Drosophila Hox protein that determines the identity of the labial and prothoracic segments. In
search of factors that might associate with SCR to control its activity and/or specificity, a yeast two-hybrid screen was performed. A
Drosophila homolog of the regulatory subunit (B'/PR61) of serine-threonine protein phosphatase 2A (dPP2A,B') specifically
interacts with the SCR homeodomain. The N-terminal arm within the SCR homeodomain has been shown to be a target of
phosphorylation/dephosphorylation by cAMP-dependent protein kinase A and protein phosphatase 2A, respectively. In vivo
analyses reveal that mutant forms of SCR mimicking constitutively dephosphorylated or phosphorylated states of the
homeodomain are active or inactive, respectively. Inactivity of the phosphorylated mimic form is attributable to impaired DNA binding. Specific ablation of
dPP2A,B' gene activity by double-stranded RNA-mediated genetic interference results in embryos without salivary glands, an SCR null phenotype. These data
demonstrate an essential role for Drosophila PP2A,B' in positively modulating SCR function (Berry, 2000).
PP2A exists as a multisubunit enzyme complex in a variety of organisms and cell types. The enzyme complex is composed of a catalytic and a scaffold subunit,
which together form a core dimer that then associates with one of a number of regulatory subunits to constitute a trimeric enzyme complex. Regulatory subunits
of PP2A are encoded by at least three unrelated gene families: B (PR55), B' (PR61) and B" (PR72). Each family consists of several members, which in addition
can give rise to a number of splice variants, thereby greatly increasing the variety of distinct trimeric enzyme complexes. Several lines of
evidence suggest that the regulatory subunits of PP2A may serve as specific adaptors that confer substrate specificity to the core domain of PP2A. Specific interaction of dPP2A,B' with the SCR homeodomain, as documented here, therefore reflects its potential of reversibly recruiting SCR
into the PP2A complex (Berry, 2000).
The two phosphorylatable residues (T and S) within the N-terminal arm of the SCR homeodomain appear to be conserved, since at least one such site has been
found in all SCR homologs from other species, except for PS12-B of Atlantic salmon. The homeodomain of PS12-B in fact seems more closely related to ANTP than to SCR. In vivo results suggest that
in developing embryos, SCR is functionally inactive when the N-terminal arm of its homeodomain is phosphorylated and is active upon dephosphorylation. These
results may have important implications for the functional specificity of homeotic proteins in general: since ANTP has a glutamine instead of threonine at
position 6 (which is well conserved in all the SCR homologs), it is proposed that the differential modification of this residue plays an important role in determining
the functional specificity of these two homeotic proteins (Berry, 2000).
The data from the functional knockout of dPP2A,B' by dsRNA interference prove unequivocally that expression of dPP2A,B' is essential for the functional
activity of SCR. Genetic studies in Drosophila have shown that Ras-1 activity positively modulates the function of Hox proteins such as proboscipedia (PB) and SCR -- a finding that suggests that covalent modifications triggered by Ras-1-mediated signals might influence the activity of PB and SCR. The catalytic subunit of dPP2A has been identified as a component operating downstream of Ras-1. The
observation that the functional activity of SCR is dependent upon the presence of dPP2A,B' seems to provide a missing link, suggesting that Ras-1 might influence the activity of SCR via dPP2A (Berry, 2000).
A model is proposed to describe the regulation of SCR activity: in a cell, where SCR function is not required
continuously, the protein is locked in an inactive state by phosphorylation of residues 6 and/or 7 within the N-terminal arm of the homeodomain. The fact that, in
older embryos, SCR is present but is no longer able to induce the expression of its target gene forkhead, may be a case in point. In
response to positive signals, SCR-specific protein phosphatase (dPP2A) becomes activated, possibly through a signaling cascade involving Ras-1. In the
absence of positive signals, or when negative signals, e.g. DPP and SP1 prevail, specific dPP2A activity is inhibited
and, as a result, SCR can no longer be maintained in its dephosphorylated state. PKA or PKA-like enzymes will phosphorylate residues 6/7 of the SCR
homeodomain, thus abrogating the ability of SCR to bind to its target genes. A delicate balance between the activities of SCR-specific PP2A and specific
protein kinases would thus allow a cell to fine-tune SCR activity (Berry, 2000).
widerborst (wdb), a B' regulatory subunit of PP2A, located at 98A6-8 and distinct from Protein phosphatase 2A at 85F (the B subunit of PP2A), has been identified as a conserved component of planar cell polarization mechanisms in both Drosophila and in zebrafish. The German name Widerborst means something stubborn or recalcitrant (derived from wider, meaning against, and borst, meaning bristle). PP2A is a holoenzyme that consists of a catalytic (C) subunit, an A regulatory subunit and one of a large family of B, B' or B'' subunits. The latter subunits are thought to regulate the activity of the C subunit and provide substrate specificity. In metazoans, the B' subunits have diverged into two related subclasses. The central regions of these proteins are strongly conserved, but they differ at their N and C termini. The protein encoded by widerborst is more closely related to the human alpha, ß and epsilon subunits (62%-66% identity) than to the ß or gamma subunits (52%-59% identity). Its sequence suggests that wdb might influence tissue polarization by regulating PP2A activity with respect to specific targets (Hannus, 2002).
In Drosophila, wdb acts at two steps during planar polarization of wing epithelial cells. It is required to organize tissue polarity proteins into proximal and distal cortical domains, thus determining wing hair orientation. It is also needed to generate the polarized membrane outgrowth that becomes the wing hair. Widerborst activates the catalytic subunit of PP2A and localizes to the distal side of a planar microtubule web that lies at the level of apical cell junctions. This suggests that polarized PP2A activation along the planar microtubule web is important for planar polarization. In zebrafish, two wdb homologs are required for convergent extension during gastrulation, supporting the conjecture that Drosophila planar cell polarization and vertebrate gastrulation movements are regulated by similar mechanisms (Hannus, 2002).
Widerborst is unique in that it does not colocalize with other tissue polarity proteins at the cell cortex. Instead, as cortical polarization is beginning (18-24 hours apf), it is found on microtubules on the distal side of each wing epithelial cell. Furthermore, it localizes there before obvious organization of proximodistal cortical domains, and its polarization is independent of them. Strikingly, at earlier developmental stages (7-9 hours apf), Wdb polarity is not distal but proximal. These dynamic shifts in Wdb polarity and their independence from previously described tissue polarity genes suggest the existence of a novel polarization mechanism (Hannus, 2002).
How might Wdb operate to specify cortical polarity? When Wdb activity is reduced, components of the cortical domains like Dsh and Fmi accumulate uniformly around the cell cortex at high levels. By contrast, disruption of Frizzled signaling interferes with the accumulation of Dsh and Fmi at the cell cortex. This suggests that Wdb is not required to activate Frizzled signaling, but rather is important for making it asymmetric (Hannus, 2002).
The genetic data indicate that Wdb exerts its activity by activating the catalytic subunit of PP2A with respect to specific substrates, and the localization of Wdb suggests that it does so on the distal side of the planar microtubule web. Which proteins might be targeted for dephosphorylation by Widerborst? One possibility is Dishevelled. Heterozygosity for wdb strongly suppresses the mwh phenotype of dsh1 suggesting that, during tissue polarization, these two proteins act antagonistically. Dishevelled cortical localization correlates with hyperphosphorylation, and the cortical localization of Dsh is certainly expanded in Wdb dominant-negative expressing cells. Supporting this possibility, two-hybrid experiments have indicated that Dishevelled can physically interact with a Xenopus B' regulatory subunit. If Wdb normally acted by antagonizing Dsh, then the dominant-negative might overactivate Frizzled signaling and cause defects in tissue polarity. This model is not easily reconcilable with a role for the distal localization of Wdb; one might naïvely expect an antagonist of Frizzled signaling to accumulate proximally instead of distally. Nevertheless, although the early distal localization of Wdb is suggestive, it has not been proven that distal localization is relevant to cortical polarization; for example, Wdb might have a role in transducing the Frizzled signal, for which distal localization is not required (Hannus, 2002).
What might be the importance of Wdb binding to the distal microtubule web? Binding to the cytoskeleton might simply allow stable distal localization of an otherwise diffusible cytosolic molecule. More interesting, this association raises the possibility that Widerborst directs the dephosphorylation of a microtubule-associated protein. Consistent with this idea, the structure of the planar microtubule web is disrupted by dnWdb expression. PP2A activity is important for the accumulation of stable microtubules, presumably through the effects of PP2A on the phosphorylation state of MAPs. Microtubule stability can affect the binding of microtubule motor proteins and can contribute to polarized protein delivery. In the wing, microtubules have been suggested to play important roles in hair polarity; depending on the time at which vinblastine is added, vinblastine treatment of pupal wings causes either failure of hair outgrowth or the formation of multiple wing hairs. Polarized dephosphorylation of MAPs within the planar microtubule web might bias the transport of vesicles containing components of the proximodistal cortical domains. At later stages, it might also help direct transport of components of the hair formation machinery to the distal side of the cell, or promote the stability of microtubules in the outgrowing hair. This model for Widerborst action could provide a single explanation for its effects on hair outgrowth and on cortical polarity. Identification of the relevant Widerborst substrate(s) should greatly advance understanding of the cell biology of tissue polarization (Hannus, 2002 and references therein).
The data also support other studies indicating that B' alpha/epsilon
regulatory subunits antagonize the classical Wnt signaling pathway. Experiments in Xenopus embryos and tissue culture cells have shown that increasing the level of a B' alpha subunit inhibits Wnt signaling and causes ventralization. Consistent with this, experiments in zebrafish show that reducing Wdb levels causes dorsalization of embryos. Although Wdb, like Frizzled and Dishevelled, is a shared component of both planar polarization and classical Wnt signaling pathways, it probably has different functions in each; during classical Wnt signaling, the B' alpha is thought to act downstream of Dishevelled, forming part of a ß-catenin degradation complex that plays no role in planar polarity signaling (Hannus, 2002).
The observation that widerborst is needed both for distal polarization of Drosophila wing hairs and for convergent extension movements during zebrafish gastrulation points to a conserved role for Wdb in regulating tissue polarity in development. Furthermore, it provides additional evidence supporting the conjecture that components of the planar polarization pathway in Drosophila are also used to control cell polarity and movement during vertebrate gastrulation. To date, the evidence for this is based on analysis of various dsh constructs and, more recently, on the analysis of vang/stbm and rhoA during vertebrate gastrulation. The identification of Wdb as another shared component provides further evidence that this signaling cascade is indeed conserved between Drosophila and vertebrates. Additional experiments will have to address the precise function(s) of vertebrate wdb homologs and where wdb acts in the genetic pathway regulating vertebrate gastrulation movements (Hannus, 2002).
Members of the Hedgehog (Hh) family of signaling proteins are powerful regulators of developmental processes in many organisms and have been implicated in many human disease states. This study reports the results of a genome-wide RNA interference screen in Drosophila cells for new components of the Hh signaling pathway. The screen identified hundreds of potential new regulators of Hh signaling, including many large protein complexes with pleiotropic effects, such as the coat protein complex I (COPI), the ribosome and the proteasome. The multimeric protein phosphatase 2A (PP2A) and two new kinases, the D. melanogaster orthologs of the vertebrate PITSLRE and cyclin-dependent kinase-9 (CDK9) kinases, were identified as Hh regulators. A large group of constitutive and alternative splicing factors, two nucleoporins involved in mRNA export and several RNA-regulatory proteins were identified as potent regulators of Hh signal transduction, indicating that splicing regulation and mRNA transport have a previously unrecognized role in Hh signaling. Finally, it was shown that several of these genes have conserved roles in mammalian Hh signaling (Nybakken, 2005).
Phosphorylation is associated with the activities of at least five components of the Hh pathway: Fu, Cos, Smo, Su(fu) and Ci. Little is known about the kinases that phosphorylate Su(fu) and Fu, but at least two sites in Cos are phosphorylated by Fu, and several kinases are involved in phosphorylating Ci and Smo, including PKA-C1, CkIalpha and Sgg. But no phosphatase has been implicated in Hh signaling, and a previous RNAi screen did not identify any phosphatases involved in Hh signaling. The screen identified microtubule star (mts), which encodes the D. melanogaster PP2A catalytic subunit, as a gene that substantially reduced Hh signaling when targeted by RNAi. PP2A is a multimeric enzyme that consists at minimum of the catalytic subunit, a regulatory A subunit (encoded by CG33297 in D. melanogaster) and a B subunit principally involved in substrate selection. The B-subunit family in D. melanogaster is represented by the gene twins (tws), the B' family by the genes widerborst (wdb) and PP2A-B', and the B" family by CG4733. All the PP2A component dsRNAs were obtained and tested from a dsRNA library and additional, distinct dsRNAs to these components were generated and tested. In addition to confirming the mts result, it was found that both the original-library dsRNA and three new, unique dsRNAs targeting wdb all reduced Hh signaling.
This indicates that Wdb is likely to be the B subunit that targets Mts to its substrate in the Hh signaling pathway. This hypothesis is in agreement with recent findings from Xenopus laevis, where the wdb ortholog encoding B56e has been found to regulate Hh signaling. In addition, some PP2A-B' amplicons cause a reduction in reporter activity averaging ~30%, indicating that they may have a partially redundant role in targeting PP2A to its Hh pathway substrate (Nybakken, 2005).
To determine whether PP2A acts on Cos, whether overexpression of cos and mts results in similar phenotypes was examined. When overexpressed in Hh-stimulated clone 8 cells, cos completely abrogates Hh signaling, reducing it to near uninduced levels, whereas overexpression of mts reduces Hh signaling by 40%. Thus, Mts and Cos have different overexpression profiles and do not seem to regulate Hh signaling in the same way. The overexpression phenotype of mts was compared with those of cos and 14 other hits from the screen, including the fu, Cdk9 and Pka-C1 kinases. Overexpressing cos in uninduced cells further reduces background signaling, whereas mts overexpression doubled reporter activity, although these levels are still very low compared with the Hh-activated state. Of the 18 other genes tested, only Pka-C1 overexpression had an effect on Hh reporter activity similar to that of mts: doubling of reporter activity in the Hh-uninduced state and a 50% reduction of activity in the Hh-stimulated state. It is therefore possible that PKA-C1 and Mts act on similar substrates. Because several studies have identified Ci as a substrate of PKA-C1, Mts could also be acting on Ci, perhaps removing inhibitory phosphates in response to Hh stimulation (Nybakken, 2005).
This screen allowed the grouping of the ribosome, proteasome, COPI complex and PP2A phosphatase as important regulators of Hh signaling, none of which had been identified as Hh regulators in vivo. Notably, some of the components identified in the screen had already been implicated in aspects of Hh signaling. For instance, the gene encoding eRF1, a translational regulator, was identified in a screen for modifiers of a gain-of-function smo allele, and polyhomeotic and additional sex combs have both been shown to modify ectopic hh expression phenotypes. These results open many new avenues for investigation of Hh signaling. In particular, elucidation of the Hh pathway substrates affected by PP2A will be important in defining the role of dephosphorylation in Hh signaling. Finally, the paradigm of Hh signaling would change substantially if further investigation determines that alternative splicing and mRNA regulation do have vital roles in Hh signaling (Nybakken, 2005).
Inappropriate regulation of the PI3-kinase/PTEN/Akt kinase-signalling cassette, a key downstream target of insulin/insulin-like growth factor signalling (IIS), is associated with several major human diseases such as diabetes, obesity and cancer. In Drosophila, studies have recently revealed that different subcellular pools of activated, phosphorylated Akt can modulate different IIS-dependent processes. For example, a specific pool of activated Akt within the cytoplasm alters aspects of lipid metabolism, a process that is misregulated in both obesity and diabetes. However, it remains unclear how this pool is regulated. The protein phosphatase PP2A-B' regulatory subunit Widerborst (Wdb), which coimmunoprecipitates with Akt in vivo, selectively modulates levels of activated Akt in the cytoplasm. It alters lipid droplet size and expression of the lipid storage perilipin-like protein LSD2 in the Drosophila ovary, but not in epithelial cells of the eye imaginal discs. It is concluded that isoforms of PP2A-B' can act as subcellular-compartment-specific regulators of PI3-kinase/PTEN/Akt kinase signalling and IIS, potentially providing new targets for modulating individual subcellular pools of activated Akt in insulin-linked disease (Vereshchagina, 2008).
The signalling cassette involving Class I phosphatidylinositol 3-kinase (PI3K), phosphatase and tensin homologue on chromosome 10 (PTEN) and Akt (also known as protein kinase B or PKB) is part of a major intracellular kinase cascade that regulates multiple cellular functions including metabolism, growth, proliferation and survival. It responds to a variety of stimuli, such as insulin, other growth factors including PDGF and FGF, and attachment to the extracellular matrix. Upon activation, PI3K catalyses the formation of phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P3] from phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2]. PtdIns(3,4,5)P3 is a lipid second messenger, which in turn recruits the PH-domain-containing Akt protein kinase from the cytosol to the plasma membrane. Here it is activated through phosphorylation at Thr308 by 3-phosphoinositide-dependent protein kinase 1 (PDK1) and at Ser473 (or Ser505 in the unique Drosophila Akt kinase, Akt1) by PDK2, which is thought to be the rictor-mTOR complex. Once activated, Akt subsequently phosphorylates multiple targets, leading to its numerous downstream effects (Vereshchagina, 2008).
Misregulation of Akt and its cellular targets is linked to several major human diseases. For example, cellular insulin resistance is associated with reduced signalling by the PI3K/PTEN/Akt cassette and is an important defect in individuals suffering from Type 2 diabetes. By contrast, hyperactivation of this cassette, most notably through loss-of-function mutations in the tumour suppressor PTEN, which converts PtdIns(3,4,5)P3 back to PtdIns(4,5)P2, is strongly associated with many forms of human cancer (Vereshchagina, 2008 and references therein).
Molecular genetic studies in Drosophila have resulted in several fundamental insights into the regulation and functions of the PI3K/PTEN/Akt-signalling cassette. Not only has this work highlighted the central importance of nutrient-regulated insulin/insulin-like growth factor signalling (IIS) in controlling the activity of this cassette and cell growth, but it has also revealed a critical downstream link with the nutrient-sensitive mTOR-signalling cascade, which regulates several cellular processes including protein translation and autophagy. Furthermore, studies in invertebrates have indicated roles for PI3K/PTEN/Akt and mTOR in ageing, cell polarity and neurodegeneration, functions that all appear to be conserved in mammals and which might involve a combination of cellular and metabolic defects (Vereshchagina, 2008).
If the role of PI3K/PTEN/Akt in insulin-linked diseases is to be fully understood, it is essential to determine how this single signalling cassette regulates so many different cellular functions. One important part of the explanation is presumably the existence of cell-type-specific downstream-signalling targets that perform different roles. However, recent work, much of it again initiated in flies, has indicated that Akt activity can also be differentially regulated in specific subcellular domains and that these subcellular pools of activated Akt can control different processes. For example, precise regulation of Akt activity at the apical membrane of epithelial cells by localised PTEN is required for normal apical morphology in higher eukaryotes. By contrast, cytoplasmic activated Akt appears to be required for transcription of specific IIS target genes and regulation of lipid metabolism and droplet size in nurse cells of the Drosophila female germ line (Vereshchagina, 2006). These observations have highlighted the importance of finding the molecules that regulate different pools of activated Akt in vivo, because their modulation might alter specific functions of IIS in health and disease more selectively (Vereshchagina, 2008).
In a screen for novel phosphatase regulators of IIS, Widerborst (Wdb), one of the B' regulatory subunits of the protein phosphatase PP2A, was identified as a negative regulator of the PI3K/PTEN/Akt-signalling cassette. Although wdb is essential for cell viability in some tissues, wdb mutant cells in the germ line and follicular epithelium of the ovary are viable and display phenotypes that are similar to those seen in PTEN mutant ovaries. This study shows that Wdb and Drosophila Akt1 physically interact in the ovary, and that within this tissue, Wdb regulates the subcellular pool of activated Akt1 in the cytoplasm. This study therefore highlights an important new function for PP2A-B' subunits in selectively modulating certain IIS-dependent processes by controlling signalling in a specific subcompartment of the cell (Vereshchagina, 2008).
Several lines of evidence confirm that Wdb controls IIS activity and Akt1 phosphorylation state. First, when overexpressed, wdb genetically modifies phenotypes produced by altered IIS signalling, rescuing a lethal PTEN mutant combination and modifying the effects of FOXO in the eye. Second, loss-of-function wdb mutations produce very similar phenotypes to PTEN mutations in nurse cells, elevating levels of cytoplasmic pAkt1 and LSD2 [a Perilipin/ADRP homologue that regulates lipid metabolism, and inducing an abnormal accumulation of lipid droplets. Third, although wdb mutations do not independently appear to have strong effects on growth, they do suppress growth phenotypes produced by reduced Akt1 signalling both in mutant follicle cells homozygous for the Akt11 allele and in animals carrying a hypomorphic viable combination of Akt1 alleles. Genetic interactions with the PP2A catalytic subunit Mts in the eye indicate that these effects are dependent on the PP2A regulatory activity of Wdb (Vereshchagina, 2008).
Coimmunoprecipitation experiments revealed that Akt1 and Wdb form a complex in ovaries, the tissue in which the most obvious effects of wdb on pAkt1 levels are seen. The data suggest that one isoform of Wdb affects IIS within a complex containing Akt1, presumably by directly modulating the phosphorylation state of this molecule. This regulatory interaction appears to be evolutionarily conserved, because several studies in mammalian cell culture have shown that a PP2A-type activity controls Akt phosphorylation at Ser473, the equivalent position to Ser505 in Drosophila Akt1. PP2A-B' activity has been implicated in this process. Furthermore, mammalian PP2A can dephosphorylate Akt in vitro. The phosphorylation state of Thr308 might also be affected by PP2A. However, current tools do not allow determination of the phosphorylation state of Thr342 (the equivalent position to Thr308 in mammalian Akt) in wdb mutant cells in ovaries. Nevertheless, this study adds to the current understanding of the effects of PP2A on Akt by showing for the first time that at least one PP2A-B' isoform can act as a pool-specific suppressor of activated Akt. It is thought that that this property is likely to be shared by some mammalian PP2A-B' isoforms (Vereshchagina, 2008).
Unlike several other previously characterised components of the IIS cascade, the effects of wdb mutations on IIS appear to be tissue specific. Although pAkt1 levels are strongly upregulated in wdb mutant nurse cells and follicle cells, they appear unaffected in clones within the eye. PP2A is a broad-specificity protein phosphatase, which is selectively targeted to specific signalling molecules by regulatory subunits such as Wdb. Wdb has already been shown to be involved in several signalling events, including those regulating apoptosis and the Hedgehog (Hh) pathway, pathways that might be implicated in the wdb mutant phenotype observed in the eye imaginal disc (Vereshchagina, 2008).
How can Wdb have such a central IIS-regulatory role in the ovary, but show no detectable effect on this pathway in the developing eye? It seems unlikely that wdb mutant cells in the eye die too rapidly to observe changes in Akt1 phosphorylation, because wdb clones are seen in posterior positions within eye imaginal discs, which must have formed many hours previously. The IIS cascade is active in this tissue, because mutations altering IIS produce significant effects on growth in the eye disc. However, unlike in nurse cells, activation of IIS in the developing eye primarily leads to cell surface accumulation of pAkt1, at least in pupae. Surface-localised activated Akt1 may normally be sufficient to promote eye growth, since a myristoylated membrane-anchored form of Akt1 dominantly induces overgrowth in this and other tissues. One possible explanation for these data is therefore that cytoplasmic pAkt1 levels in the eye are restricted by other unknown molecules in addition to Wdb in this tissue, so loss of wdb here has little effect, whereas increased expression can still modify the FOXO phenotype (Vereshchagina, 2008).
In this context, at least two other phosphatases might be involved in Akt1 regulation. First, there is a second isoform of PP2A-B' in flies [called PP2A-B', CG7913 or Well-rounded (Wrd); that is most closely related to mammalian PP2A-B'γ isoforms. Simian virus 40 small t antigen acts as a specific inhibitor of mammalian PP2A-B'γ, stimulating phosphorylation of Akt and other targets, and thereby promoting growth. Reduced PP2A-B'γ activity has also been linked to the establishment and progression of melanomas (Vereshchagina, 2008).
Surprisingly, a recent report suggests Wrd is nonessential. Unless it acts redundantly with Wdb, it cannot therefore play a significant role in growth regulation). Analysis of the PP2A catalytic subunit Mts, using a dominant-negative construct, indicates that this enzyme enhances the effects of FOXO and is important in normal growth regulation in the eye, perhaps consistent with the idea that the two PP2A-B' isoforms do act redundantly. Alternatively, Mts may perform some of its growth regulatory functions independently of PP2A-B' (Vereshchagina, 2008 and references therein).
A second candidate negative regulator of Akt is the novel phosphatase PHLPP, which directly dephosphorylates human Akt at Ser473 and Drosophila Akt1 at Ser505 in cell culture, a function that may be disrupted in some tumours. Drosophila PHLPP could therefore control pAkt1 accumulation at the cell surface and perhaps reduce the amount of pAkt1 that can diffuse into the cytoplasm in tissues such as the eye. Since loss of wdb in either follicle cells or nurse cells is sufficient to elevate levels of cytoplasmic pAkt1, PHLPP presumably does not play such an important role in these cell types (microarray data suggest that PHLLP is not expressed at detectable levels in the adult ovary) (Vereshchagina, 2008).
Interestingly, the data in the ovary suggest further variable tiers of pAkt1 control. In nurse cells, loss of PTEN leads to accumulation of pAkt1 and LSD2 in the cytoplasm, but most PTEN mutant follicle cell clones do not show these phenotypes, presumably because other pAkt1 regulators such as Wdb play a more dominant role in these cells. No good explanation is available for how genetically identical clones can show such phenotypic variability. There is no obvious correlation with clone size or position in the small minority of PTEN-mutant follicular clones where pAkt1 and LSD2 upregulation is observed (Vereshchagina, 2008).
Because perilipin, the mammalian LSD2 orthologue, is thought to be regulated via insulin-dependent transcriptional and post-translational mechanisms, it is proposed that the increased LSD2 expression seen in PTEN mutant nurse cell clones results from similar effects of IIS on this molecule in flies. An alternative explanation is that increased IIS promotes excess triacylglyceride (TAG) synthesis and that LSD2 is only indirectly upregulated to permit proper packaging of these triacylglycerides into lipid droplets. Analysis of wdb mutant follicle cell clones does not support this latter model, since these clones strongly upregulate LSD2 expression, but do not show obvious changes in lipid droplet accumulation (Vereshchagina, 2008).
When wdb is overexpressed in the differentiating eye, the external structure of the eye becomes more disorganised and there is a slight reduction in overall eye size. Since this effect is not noticeably suppressed by co-overexpressing Akt1, it seems unlikely to be caused by reduced IIS. Unlike PTEN mutant follicle cells, wdb mutant follicle cells are not noticeably larger than their wild-type neighbours. Furthermore, although low level constitutive expression of Wdb in a pupal-lethal PTEN mutant background can rescue these flies to viability, the rescue may be explained by altered metabolism, because the rescued flies are still larger than normal. All these observations are consistent with the model that Wdb modulates cytoplasmic pAkt1 and has less of an effect on cell surface pAkt1, which is thought to be the primary regulator of normal growth. Wdb shows a relatively strong genetic interaction with the IIS-regulated transcription factor FOXO and this is completely suppressed by Akt1, raising the possibility that low levels of pAkt1 in the cytoplasm may play an important part in controlling FOXO activity (Vereshchagina, 2008).
Although wdb does not appear to modulate growth significantly under normal IIS-signalling conditions, mutations in wdb do enhance growth when Akt1 activity is reduced. Viable Akt1 mutant animals are larger in the presence of a heterozygous wdb mutation, while the Akt11 recessive growth phenotype in follicle cells is strongly suppressed by wdb. Interestingly, it has been reported that mutations in foxo have no effect on growth in otherwise normal animals, but that when IIS is reduced in chico mutants, which produce small adults, this phenotype is partially suppressed by loss of foxo function. The current data are consistent with this result, and may indicate that growth regulation in chico flies relies more on cytoplasmic pAkt1 and its effects on downstream targets like FOXO than it does in normal flies (Vereshchagina, 2008).
In conclusion, the identification of a PP2A-B' subunit as a novel cell-type-specific regulator of IIS within a specific subcellular compartment highlights the importance of studying the subcellular control of this signalling pathway in multiple cell types in vivo. Akt activation also promotes lipid synthesis and droplet formation in many mammalian cell types. This is likely to involve similar regulatory control mechanisms for cytoplasmic pAkt to those uncovered in flies. This work therefore raises new issues concerning the underlying causes of IIS-associated disease. For example, excess accumulation of lipid and obesity could be linked to selective changes in cytoplasmic pAkt control and might therefore be modulated by specific PP2A-B' subunits. Developing a better understanding of this form of regulation could therefore suggest new strategies for disease-specific treatments of IIS-linked disorders in the future (Vereshchagina, 2008).
Signaling by Hedgehog (Hh) proteins shapes most tissues and organs in both vertebrates and invertebrates, and its misregulation has been implicated in many human diseases. Although components of the signaling pathway have been identified, key aspects of the signaling mechanism and downstream targets remain to be elucidated. An enhancer/suppressor screen was performed in Drosophila to identify novel components of the pathway and 26 autosomal regions were identified that modify a phenotypic readout of Hh signaling. Three of the regions include genes that contribute constituents to the pathway: patched, engrailed, and hh. One of the other regions includes the gene microtubule star (mts) that encodes a subunit of protein phosphatase 2A. mts is necessary for full activation of Hh signaling. A second region includes the gene second mitotic wave missing (swm). swm is recessive lethal and is predicted to encode an evolutionarily conserved protein with RNA binding and Zn+ finger domains. Characterization of newly isolated alleles indicates that swm is a negative regulator of Hh signaling and is essential for cell polarity (Casso, 2008).
This screen identified twenty-six autosomal regions that modified a smo hypomorphic phenotype in a dosage-sensitive manner. Two aspects of its design were key to its success. First, its two-generation crossing scheme eliminated background effects by homogenizing the genetic backgrounds of both experimental and control flies. It also generated reasonably large numbers of both classes of progeny so that a good estimate of an average phenotype could be obtained. These features allowed monitoring of subtle variations in wing vein morphology, despite the significant strain differences among the many lines tested. Second, its high scoring threshold rendered it relatively insensitive to changes in Hh signaling strength, thereby helping to submerge weak influences. Key to this property was the ptcGAL4 driver that was used to express smo RNAi; it functioned in part as a 'genetic buffer.' Since ptcGAL4 is itself responsive to Hh, a modifier that increased Hh signaling would also be predicted to increase the expression of ptcGAL4 and smo RNAi, while a modifier that decreased Hh signaling might be expected to decrease the expression of the ptcGAL4 and smo RNAi. ptcGAL4 therefore buffered against changes in signaling strength and decreased the effects of genetic factors that enhance or suppress signaling; as a consequence, only highly penetrant and consistent phenotypes were scored (Casso, 2008).
The screen netted many of the known core components of the Hh signaling pathway, including smo, ptc, hh, and en. mts and swm were two genes whose haplo-insufficiency phenotypes were sufficiently strong to score above the threshold set by the genetic tests. Many other known regulators of Hh signaling were not identified in this screen. There are perhaps multiple reasons, including the high scoring threshold of the smo RNAi screen, or the possibility that not all pathway regulators have haplo-insufficiency phenotypes. skinny hedgehog or suppressor of fused were not included among those identified in the screen, despite the fact that deficiencies that removed them interacted with smo RNAi. The reason is that mutant alleles of these genes that were tested did not yield similar interaction phenotypes. Since many examples were observed of interaction between null alleles of Hh pathway regulators and smo RNAi but consistent failure of hypomorphic alleles to interact, lack of interaction is not viewed as evidence against a gene being a smo RNAi enhancer/suppressor. The possibility that stronger alleles might interact cannot be discounted. It was surprising that hemizygosity of cos2 did not show an interaction with smo RNAi. This could be because it is not haplo-insufficient in the particular assay or because of the complex positive and negative roles cos2 plays in Hh signaling. Finally, there was no apparent overlap between the regions identified and the mutant lines that were identified in previous screens for modifiers of Hh phenotypes; the smo RNAi assay may be less sensitive but more specific (Casso, 2008).
mts lies within 1 of 16 regions that enhanced the smo RNAi phenotype, suggesting that its wild-type function augments the Hh response. mts encodes the catalytic (C) subunit of PP2A, a heterotrimeric phosphatase that has two regulatory subunits, B and B'. It was previously identified as a Hh pathway regulator in CL8 cells (Nybakken, 2005); the current study provides in vivo evidence for a role in Hh signaling during development. Three proteins in Hh signal transduction have been shown to be functionally phosphorylated. Phosphorylation of the Smo C terminus is induced by Hh and is required for surface accumulation of Smo and normal activation of the pathway. Thus, reduction of PP2A activity and increased phosphorylation of Smo would not be expected to decrease Hh signaling and enhance the smo RNAi phenotype. Other possible targets of Mts are Ci and Cos2. Phosphorylation of Ci by PKA, casein kinase 1α, and GSK3β is required to convert Ci from its full-length form to its transcriptional repressor form, Ci-75. Hh signaling blocks this proteolytic transformation and also promotes conversion of Ci to an activator form. A decrease in phosphatase activity might increase levels of phosphorylated Ci to effect enhanced conversion to Ci-75 and reduced levels of Ci activator. Levels of Hh signaling would be predicted to decrease. Alternatively, Mts might control phosphorylation of Cos2 by Fu. Phosphorylation of Cos2 prevents its binding to Smo and release of Smo from Cos2 increases the cell surface accumulation of Smo that is necessary for pathway activation. Therefore, a reduction of Smo on the plasma membrane due to loss of PP2A activity might attenuate Hh pathway activation (Casso, 2008).
While the catalytic subunit of PP2A carries enzymatic phosphatase activity, the substrate specificity of PP2A is directed by its regulatory subunits. The phenotypes of mutants in genes that encode the B and B' regulatory subunits of PP2A, twins and widerborst (wdb), respectively, are interesting to consider in the context of Hh signaling. Wing discs in the twinsP mutant have mirror symmetrical posterior compartment duplications that are associated with ectopic compartment borders. Symmetric wing duplications have also been observed after ectopic expression of Hh or Dpp, or after loss of en/inv induces an ectopic compartment border. Since loss of PP2A function should reduce Hh signaling, it is not obvious how loss of the B twins regulatory subunit leads to an ectopic signaling center. Understanding this interesting aspect of the twins phenotype warrants further investigation (Casso, 2008).
Misexpression of PP2A can cause cell planar polarity defects in the wing. Misexpression of mts, wdb, or mutant alleles of these genes disrupted wing hair polarity. Like mts, reducing wdb expression with RNAi reduced Hh signaling in CL8 cells (Nybakken, 2005). This evidence, as well as the wing hair polarity phenotype of swm mutants, raises the possibility that PP2A links Hh signaling with cell polarity. The PCP and Hh pathways may be parallel and independent if PP2A activity is simply common to both, but evidence that Hh is required to establish PCP in the Drosophila embryonic and adult epidermis has recently been described. The phenotype of swm mutants provides additional evidence for an association of Hh signaling with cell polarity (Casso, 2008).
swm was first identified as l(2)37Dh in a screen for recessive lethal alleles within Df(2L)E55 (37D2-38A1). It was shown to exhibit synthetic lethality as an enhancer of Minutes. Among the mutant chromosomes from the current screen that failed to complement swm, one had a Minute-like phenotype. No changes in the swm coding sequence were found in this mutant; rare escapers that eclosed as heterozygotes with the verified swm alleles had a variety of phenotypes including loss of ocelli, thin macrochetae, and deformed legs. In contrast to swm mutant escapers, however, both their eyes and wings were phenotypically normal (Casso, 2008).
swm was identified as a suppressor of the roughex eye phenotype. Alleles of ptc were also isolated in this screen. These interactions between rux, ptc, and swm were confirmed. Since Ptc is a negative regulator of the Hh pathway and ptc mutations are therefore likely to elevate Hh signaling, and since Hh plays a key role in eye morphogenesis, the rux phenotype is apparently sensitive to Hh levels. Therefore the identification of both ptc and swm mutants as rux suppressors is interpreted as a consequence of the same mechanism -- an increase in Hh signaling caused by a decrease in the level of a negative regulator (Casso, 2008).
The results provide several additional lines of evidence that swm negatively regulates Hh signaling. swm mutants dominantly suppress smo hypomorphic phenotypes (smo RNAi and smo5A, enhance a Hh gain-of-function phenotype (hhMrt), and increase targets of Hh signaling such as Ptc and Ci. These effects on Hh signaling seem to occur through swm activity in the anterior compartment since swm RNAi expressed in these cells is sufficient to suppress smo RNAi. Although these interactions implicate Swm, it has not been determined how and where Swm impacts signal transduction or what its molecular function might be. Swm protein has features suggestive of a function in nucleic acid metabolism -- it has a putative RRM RNA binding domain and a CCCH Zn+ finger, and a GFP-Swm fusion that was examined localized to nuclei in cultured cells. Presumably, Swm affects expression, production, or presentation of proteins involved in Hh signaling or signal transduction. However, swm function is not specific to Hh signaling, since many aspects of the phenotype (e.g., ectopic venation, wing hair polarity, cell size, and interaction with Minutes) are not attributable to defects in Hh signaling (Casso, 2008).
swm is expressed broadly in both embryos and larvae, and in wing discs, it appears to be required in all cells. Null alleles, which are cell lethal in a swm/+ background, share some, but not all characteristics of Minute ribosomal protein mutants. Although swm mutants do not have thin bristles as is characteristic of Minutes, they are recessive lethal and developmentally delayed, and they interact genetically with Minutes and Minute-like loci. The wings of the Minute locus RpL38 have defects which are similar to swm wings -- extra venation, expanded distance between veins 3 and 4, wing hair polarity abnormalities, and increased cell size. Although RpL3845-72, Df(2R)M41A10, and M41A4 suppressed hhMrt, they did not interact with smo RNAi or smo5A (Casso, 2008).
While the interactions between swm and Minutes, as well as the similar phenotypes of swm and the RpL38 genes, might indicate a direct role in ribosome function, both Drosophila Swm and one of its two vertebrate homologs (RBM-27) are nuclear. The presence of RRM sequences in Swm and its homologs might suggest a role in RNA binding or metabolism, and the RRM of RBM-27 binds RNA. However, RRMs can have a structural role in protein-protein interactions independent of RNA binding, so the molecular function of Swm and its homologs cannot be determined by genetic methods alone. The fact is intriguing that the other vertebrate homolog, RBM-26, was identified as se70-2, an autoantigen that is recognized by sera of cutaneous T-cell lymphoma patients and has been used as a diagnostic marker for this tumor. In addition, the mouse RBM-26/se70-2 locus was identified as one of four genes deleted in a region required for normal murine skeletal, cartilage, and craniofacial development. Perhaps the roles of Hh that extend beyond pattern formation to cell cycle regulation, growth control, and cell polarity signify that Hh signal transduction integrates inputs from all three pathways. The pleiotropy of swm and mts may reflect these multiple inputs (Casso, 2008).
The Wnt-signaling cascade is required for several crucial steps during early embryogenesis, and its activity is modulated by various agonists and antagonists to provide spatiotemporal-specific signaling. Naked cuticle is a Wnt antagonist that itself is induced by Wnt signaling to keep Wnt signaling in check. Little is known about the regulation of this antagonist. It has been shown that the protein phosphatase 2A regulatory subunit PR72 is required for the inhibitory effect of Naked cuticle on Wnt signaling. The present study shows that PR130, which has an N terminus that differs from that of PR72 but shares the same C terminus, also interacts with Naked cuticle but instead functions as an activator of the Wnt-signaling pathway, both in cell culture and during development. PR130 modulates Wnt signal transduction by restricting the ability of Naked cuticle to function as a Wnt inhibitor. These data establish PR130 as a modulator of the Wnt-signaling pathway and suggest a mechanism of Wnt signal regulation in which the inhibitory activity of Naked cuticle is determined by the relative level of expression of two transcripts of the same protein phosphatase 2A regulatory subunit (Creyghton, 2006).
The Drosophila HOX transcription factor, Sex combs reduced (SCR), is required for determining labial and the first thoracic segmental identity. A Protein Phosphatase 2A holoenzyme assembled with the PP2A-B′ regulatory subunit is proposed to specifically interact with, and dephosphorylate, the SCR homeodomain activating SCR protein activity. To test this hypothesis further, a null mutation was created in the PP2A-B′ gene, PP2A-B′Δ, using Flip-mediated, site-specific recombination. The number of sex comb bristles, salivary gland nuclei and pseudotracheal rows are SCR-dependent and were counted as a measure of SCR activity in vivo. Adults and larvae homozygous for PP2A-B′Δ showed no decrease in SCR activity. In addition, no evidence of functional redundancy of PP2A-B′ with other regulatory subunits, Twins (TWS) and Widerborst (WDB), for dephosphorylation and activation of SCR activity was observed. In conclusion, a PP2A holoenzyme containing the PP2A-B′ regulatory subunit has no role in the dephosphorylation and activation of SCR, and analysis of functional redundancy of PP2A regulatory subunits uncovered no evidence supporting a role of PP2A activity in dephosphorylation and activation of SCR (Moazzen, 2009).
Although the gene that encodes PP2A-B' is dispensable for viability, PP2A-B' is functional. The analysis of functional redundancy between PP2A-B' and TWS/WDB showed that removal of PP2A-B' in a genetic background deficient for one or both of the tws and wdb loci significantly increased the number of sex comb bristles. This suggests that the PP2A holoenzyme containing either TWS, the B regulatory subunit, or WDB, a B' regulatory subunit, may functionally substitute for the loss of PP2A-B'. Although PP2A-B' is dispensable for development, it may have an essential and specific role in biological processes not assayed in this study like the immune response, mating behaviour or circadian rhythm (Moazzen, 2009).
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