Twins/PP2A regulates aPKC to control neuroblast cell polarity and self-renewal

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).

Protein Interactions

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).

Planar cell polarization requires Widerborst, a B' regulatory subunit of protein phosphatase 2A

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).

A genome-wide RNA interference screen in Drosophila melanogaster cells for new components of the Hh signaling pathway

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).

The protein phosphatase PP2A-B' subunit Widerborst is a negative regulator of cytoplasmic activated Akt and lipid metabolism in Drosophila

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).

A screen for modifiers of Hedgehog signaling in Drosophila melanogaster identifies swm and mts

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).

PR130 is a modulator of the Wnt-signaling cascade that counters repression of the antagonist Naked cuticle

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).

Non-requirement of a regulatory subunit of Protein Phosphatase 2A, PP2A-B', for activation of Sex comb reduced activity in Drosophila melanogaster

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-BΔ, 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-BΔ 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).

Sequential phosphorylation of Smoothened transduces graded Hedgehog signaling

The correct interpretation of a gradient of the morphogen Hedgehog (Hh) during development requires phosphorylation of the Hh signaling activator Smoothened (Smo); however, the molecular mechanism by which Smo transduces graded Hh signaling is not well understood. This study shows that regulation of the phosphorylation status of Smo by distinct phosphatases at specific phosphorylated residues creates differential thresholds of Hh signaling. Phosphorylation of Smo was initiated by PKA and further enhanced by casein kinase I (CKI). Protein phosphatase 1 (PP1) directly dephosphorylates PKA-phosphorylated Smo to reduce signaling mediated by intermediate concentrations of Hh, whereas PP2A specifically dephosphorylates PKA-primed, CKI-phosphorylated Smo to restrict signaling by high concentrations of Hh. A functional link was established between sequentially phosphorylated Smo species and graded Hh activity. Thus, a sequential phosphorylation model is proposed in which precise interpretation of morphogen concentration can be achieved upon versatile phosphatase-mediated regulation of the phosphorylation status of an essential activator in developmental signaling (Su, 2011).

The conversion of a gradient of the morphogen Hh into distinct transcriptional responses is essential for cell-fate decisions and tissue patterning during development. This study has provided genetic and biochemical evidence to support a model in which sequential phosphorylation of Smo, which is established by distinct kinases and phosphatases on specific serines, transduces graded Hh signaling. A basal extent of Smo activity, regulated by as yet unknown kinases and phosphatases, was sufficient to transduce low-threshold Hh signaling. PKA and PP1 collaborate to sustain PKA-phosphorylated Smo to transduce intermediate-threshold Hh signaling, whereas CKI and PP2A facilitate high-threshold Hh signaling by maintaining PKA-primed, CKI-phosphorylated Smo (Su, 2011).

Wdb-PP2A directly and specifically acts on CKI-pSmo to restrict high-threshold Hh signaling. Apart from PP2A, another phosphatase, PP1, specifically dephosphorylated PKA-phosphorylated Smo. This collaborative regulation between different phosphatases on the same substrate also functions in other cellular processes. For example, PP1 and PP2A dephosphorylate Par-3 to regulate cell polarity in the specification of neuroblast cell fate. Similarly, PP2A and PP4 respond to different DNA damage signals to dephosphorylate γ-H2AX to facilitate the repair of DNA double-strand breaks (Su, 2011 and references therein).

The activity of several Hh signaling components, including Smo, Ci, and Cos2, is regulated by phosphorylation. For example, PKA- and CKI-mediated phosphorylation of Ci leads to its destabilization. PP2A is implicated in regulating Ci activity in flies. This study confirmed that the catalytic PP2A subunit Mts associates with both Smo and Ci in cl-8 cells. Consistent with the substrate specificity of PP2A being conferred by its obligate regulatory subunits, this study found that Wdb specifically regulates the signaling potential of Smo. Another regulatory subunit, Twins , may direct PP2A activity toward Ci, which may potentially promote the translocation of CiFL to the nucleus, thereby activating Hh signaling. The use of distinct PP2A regulatory subunits in the same developmental process was also observed in transforming growth factor-β (TGF-β) signaling. The effect of the regulatory Bα subunit on PP2A activity activates Smad2 signaling, whereas the Bδ subunit inhibits Smad2 activity. The elaborate regulation of these two signaling systems by PP2A highlights a potential paradigm in which differential PP2A activity plays an essential role in developmental signaling. PP2A is a strong tumor suppressor; thus, modulation of PP2A activity provides an additional route by which development and tumorigenesis might be controlled (Su, 2011).

Another phosphatase, PP4, may play a role in inhibiting Smo; however, inhibiting PP4 alone is not sufficient to promote constitutive cell surface localization of wild-type Smo, unless Hh protein is provided. The surface localization of Smo is tightly linked to PKA- and CKI-dependent enhanced phosphorylation of Smo. PP4-specific RNAi further increases the extent of constitutive surface localization of Smo mutants that mimic PKA- and CKI-mediated phosphorylation, which suggests that PP4 may act on sites other than those in the PKA-CKI clusters (Su, 2011).

To delineate mechanisms whereby PP2A and PP4 might act on Smo, the expression of Hh signaling components as well as of Hh targets were systematically examined in wing discs expressing pp4 RNAi. In addition to the Smo stabilization, it was found that pp4 RNAi reduced the abundance of Cos2 protein. This might be as a consequence of the increased Smo abundance, because smo RNAi stabilized Cos2. Alternatively, PP4 might regulate Cos2 directly, because phosphorylated Cos2 is not stable. To distinguish between these two possibilities, the genetic relationship between smo and pp4 was examined by monitoring the stabilization of Cos2. Cos2 abundance was still reduced in wing discs containing both pp4 and smo RNAi. This effect is similar to the effect of pp4 RNAi alone, thus placing pp4 downstream of smo in regulating Cos2. Consistent with this, reduced expression of pp4 compromised Ptc and Collier/Knot (Col) expression at the AP boundary. The expanded area containing Ptc, albeit at a reduced abundance, away from the AP boundary has been observed previously. These experiments are consistent with a positive role of Cos2 in mediating maximal activation of Hh signaling in cl-8 cells as well as in wing discs; Ptc and Col expression are reduced in cos2 clones abutting the AP boundary. These data, together with the observation of a direct interaction between Cos2 and PP4 (Jia, 2009), argue that PP4 might also directly affect the extent of Cos2 phosphorylation (Su, 2011).

Smo contains three PKA-CKI phosphorylation clusters, with one PKA and two CKI consensus serines in each cluster. A previous study compared the signaling potential of phosphorylation-defective Smo by mutating PKA consensus serines to alanines in one, two, or three of the PKA-CKI clusters and concluded that at least six serines in Smo are required to fully induce the expression of ptc-lacZ, whereas only three serines are needed for the expression of dpp-lacZ. Another study further demonstrated that PKA- and CKI-mediated phosphorylation, which results in the generation of negatively charged residues, counteracts the positive charges conferred by nearby arginine clusters, thus enabling Smo to adopt a conformational change required to activate Hh signaling. These two studies support a model of collective Smo phosphorylation such that the identity of the phosphorylated serines in the PKA-CKI consensus clusters is probably not important; rather, the resulting negative charges collectively carried by these residues after phosphorylation are critical to determine the signaling strength of Smo (Su, 2011).

On the basis of this model, a variant Smo (Smo-CKI) in which the CKI, but not the PKA, consensus serines are mutated to alanines, thus rendering Smo-CKI resistant to CKI-mediated phosphorylation, would be anticipated to have the same signaling potential as Smo-PKA23, a variant containing a single intact PKA-CKI cluster, because both mutants contain three serines that can be phosphorylated. In smo loss-of-function clones, Smo-PKA23 is sufficient to drive expression of dpp-lacZ; however, Smo-CKI fails to rescue dpp-lacZ expression. The discrepancy between the effects of Smo-PKA23 and Smo-CKI on dpp-lacZ expression cannot be simply explained by the collective phosphorylation model. Moreover, these experiments reveal a functional distinction between different phosphorylated residues: Three PKA consensus serines in Smo-CKI may have less signaling activity than the single PKA and two CKI consensus serines in Smo-PKA23. The distinct signaling potentials of the two phosphorylation variants of Smo may be caused by different negative charge densities being carried by individual PKA-CKI clusters, or they may reflect intrinsic properties of sequential phosphorylation within each cluster (Su, 2011).

Indeed, the hierarchy of importance among individual PKA-CKI clusters in Smo has been revealed. Cluster 2 (also known as region V) is more prevalent than the other two clusters in activating ptc-luc reporter in smo-depleted cl-8 cells. Whether this functional distinction among PKA-CKI clusters also holds true in wing discs is unclear, because neither the hierarchical importance of individual clusters nor the relative importance of specific PKA and CKI phosphorylation events in neutralizing nearby arginine clusters has been directly studied. Nevertheless, when the relative importance of the three serines in cluster 2 was examined, the PKA-primed, CKI consensus sites (that is, sequential phosphorylation) were essential for Hh activation in cl-8 cells. The differential ability of SmoCKI-SA and SmoPKA-SA variants to activate dpp transcription in wing discs uncovered in this study is consistent with results obtained in cl-8 cells. Both observations challenge the model of collective Smo phosphorylation by arguing that the signaling potential of individual serines between each PKA-CKI cluster, as well as within a cluster, is most probably not equal. It is believed that regulated phosphorylation at specific serines may therefore contribute to graded Smo signaling (Su, 2011).

The collective phosphorylation model does not distinguish between the contributions of individual phosphorylated residues in PKA-CKI clusters. This study of phosphorylation-defective Smo variants revealed a Smo activity gradient in which phosphorylation at the PKA consensus sites and phosphorylation at the PKA-primed, CKI consensus sites were required for intermediate- and high-threshold Hh signaling, respectively. This activity gradient of Smo was directly visualized with the α-Smo-pS667 antibody. The abundance of PKA-phosphorylated Smo species, which is uncovered in Hh-stimulated fly cells by mass spectrometric analysis, increased initially but then declined sharply in response to Hh. As predicted from the model, phosphorylated Smo in response to intermediate-threshold Hh signaling was sensitive to dephosphorylation by PP1 but much less so to PP2A. Together, these data highlight the importance of sequential Smo phosphorylation to the transduction of graded Hh signaling. Sequential phosphorylation may be required to initialize graded Smo signaling activity. In addition, collective phosphorylation between different clusters may reinforce and maximize the Smo signaling potential to ensure the appropriate Hh signaling outcome (Su, 2011).

The presence of up to 26 serine or threonine residues in Smo that can be phosphorylated in response to Hh resembles the composition of residues found in the Kv2.1 potassium channel. Variable calcineurin-dependent dephosphorylation of Kv2.1 at 16 phosphorylated residues generates an activity gradient for channel gating and neuronal firing. The opposing actions of kinases and phosphatases on a multisite substrate are known through mathematical modeling to efficiently generate a range of stable phosphorylated forms. The spectrum of such distributions can be further increased with the number of phosphorylated sites. Two additional kinases, CK2 and G protein (heterotrimeric guanosine 5'-triphosphate-binding protein)-coupled receptor kinase 2 (GRK2), phosphorylate sites in Smo other than those targeted by PKA and CKI. Thus, the complex composition of phosphorylated residues in the cytoplasmic tail of Smo, coupled with versatile dephosphorylation by distinct phosphatases, provides an efficient and reliable mechanism to precisely convert the concentration thresholds of Hh into a graded signaling activity (Su, 2011).

The Protein Phosphatase 2A regulatory subunit Twins stabilizes Plk4 to induce centriole amplification

Centriole duplication is a tightly regulated process that must occur only once per cell cycle; otherwise, supernumerary centrioles can induce aneuploidy and tumorigenesis. Plk4 (Polo-like kinase 4) activity initiates centriole duplication and is regulated by ubiquitin-mediated proteolysis. Throughout interphase, Plk4 autophosphorylation triggers its degradation, thus preventing centriole amplification. However, Plk4 activity is required during mitosis for proper centriole duplication, but the mechanism stabilizing mitotic Plk4 is unknown. This paper shows that PP2A [Protein Phosphatase 2A(Twins)] counteracts Plk4 autophosphorylation, thus stabilizing Plk4 and promoting centriole duplication. Like Plk4, the protein level of PP2A's regulatory subunit, Twins (Tws), peaks during mitosis and is required for centriole duplication. However, untimely Tws expression stabilizes Plk4 inappropriately, inducing centriole amplification. Paradoxically, expression of tumor-promoting simian virus 40 small tumor antigen (ST), a reported PP2A inhibitor, promotes centrosome amplification by an unknown mechanism. ST actually mimics Tws function in stabilizing Plk4 and inducing centriole amplification (Brownlee, 2011).

Plk4 protein is maintained at near-undetectable levels for the majority of the cell cycle by ubiquitin-mediated proteolysis. The ubiquitin ligase SCFSlimb is responsible for Plk4 degradation and recognizes an extensively phosphorylated degron situated immediately downstream of the kinase domain (KD; ~50 amino acids containing the Slimb-binding domain)KD. Slimb is appropriately positioned on centrioles throughout the cell cycle to promote rapid Plk4 destruction, but centrioles are not required for its activity. In any case, Plk4 degradation is critical in blocking all pathways of centriole amplification. Unlike other Polo kinase members, Plk4 is a homodimer capable of autophosphorylating its downstream regulatory element (DRE), a serine-rich region surrounding its SBD, in trans to promote Slimb binding. Autoregulation is a conserved feature of Plk4. Moreover, a RNAi screen of the fly kinome suggests that no other kinase is required for Plk4 degradation. The continuous and efficient degradation of Plk4 indicates that Plk4 is immediately active when expressed and that control of Plk4’s protein level is key to regulating its activity (Brownlee, 2011).

However, surprisingly little is known about the converse event: how Plk4 is activated. The results reveal the existence of a previously unknown facet of the regulation of centriole duplication, a process which transiently stabilizes and activates Plk4 specifically during mitosis. Serine/threonine phosphatases were investigated as possible effectors to counteract Plk4 autophosphorylation. PP2A is an excellent candidate to fulfill this role as it has important functions in mitosis and localizes to mitotic centrioles in cultured fly cells and centrosomes in dividing Caenorhabditis elegans embryos. A previous study found that the number of γ-tubulin foci in mitotic S2 cells was diminished after PP2A RNAi, but whether this resulted from a bona fide loss of centrioles or instead reflects a requirement for PP2A for centrosome maturation was not determined. Subsequently, a role for PP2A in centrosome maturation was identified in a genome-wide RNAi screen. The current results indicate that PP2A and the regulatory subunit Tws are required for centriole duplication by dephosphorylating and stabilizing Plk4. Without PP2ATws, Plk4 cannot be stabilized, and centrioles fail to duplicate. PP2A is also required for centriole assembly in C. elegans embryos but functions downstream in the centriole assembly process (Kitagawa, 2011; Song, 2011). Although the catalytic and structural PP2A subunits are abundant, regulatory subunits are needed for intracellular targeting and recognition of a myriad of substrates. Tws overexpression is sufficient to stabilize Plk4 in a dose-dependent manner, causing centriole amplification and multipolar spindle formation. Like Plk4, Tws protein levels are low during interphase but rise and peak during mitosis. Accordingly, the results suggest that PP2ATws stabilizes mitotic Plk4 by counteracting Plk4 autophosphorylation, enabling cells to switch Plk4 activity (and thus centriole duplication) on and off. This mechanism is inherently highly sensitive to the presence of Tws, a rate-limiting component. Moreover, this is likely a conserved mechanism because overexpression of human Tws also stabilizes fly Plk4 in S2 cells. Clearly, an important goal for future studies is to establish whether the regulation of Tws levels and cell cycle control are linked. In addition, the results suggest that up-regulation of Tws could be a means to amplify centrioles in multiciliated cells and that increased Tws activity could be a condition found in cancerous cells (Brownlee, 2011).

Centrosome amplification is a hallmark of cancer and is also observed upon expression of DNA tumor virus proteins, which include SV40 ST, human papillomavirus E7, human T cell leukemia virus type-1 Tax, hepatitis B virus oncoprotein X, and human adenovirus E1A. However, mechanisms for centrosome amplification by viral oncoproteins are not known. SV40 ST has been found to directly bind the highly conserved Drosophila catalytic and structural PP2A subunits and to induce centrosome overduplication in cultured fly cells (Kotadia, 2008). Notably, ST is a well-established PP2A inhibitor and is known to bind structural PP2A subunits, forcing endogenous PP2A regulatory subunits to be displaced and inhibiting PP2A activity. However, the current results demonstrate that ST expression does not inhibit all PP2A activities but, instead, stimulates PP2A stabilization of Plk4. This represents the first evidence that ST mimics the function of a PP2A regulatory subunit in cells. It will be important to determine whether ST targets additional PP2A substrates during tumorigenesis and whether other tumorigenic viruses (e.g., human papillomavirus and hepatitis B) known to promote centrosome amplification exploit this same mechanism. Intriguingly, human papillomavirus E7 oncoprotein binds PP2A catalytic and structural subunits and prevents PP2A from dephosphorylating Akt. Although a previous study has suggested that PP2A may function as a tumor suppressor, these findings indicate that unregulated PP2A activity leads to centriole amplification and chromosomal instability and should therefore be considered as a potential oncogenic factor (Brownlee, 2011).

Essential roles of the Tap42-regulated protein phosphatase 2A (PP2A) family in wing imaginal disc development of Drosophila melanogaster

Protein ser/thr phosphatase 2A family members (PP2A, PP4, and PP6) are implicated in the control of numerous biological processes, but understanding of the in vivo function and regulation of these enzymes is limited. This study investigated the role of Tap42, a common regulatory subunit for all three PP2A family members, in the development of Drosophila wing imaginal discs. RNAi-mediated silencing of Tap42 using the binary Gal4/UAS system and two disc drivers, pnr- and ap-Gal4, not only decreased survival rates but also hampered the development of wing discs, resulting in a remarkable thorax cleft and defective wings in adults. Silencing of Tap42 also altered multiple signaling pathways (HH, JNK and DPP) and triggered apoptosis in wing imaginal discs. The Tap42RNAi-induced defects were the direct result of loss of regulation of Drosophila PP2A family members (MTS, PP4, and PPV), as enforced expression of wild type Tap42, but not a phosphatase binding defective Tap42 mutant, rescued fly survivorship and defects. The experimental platform described in this study identifies crucial roles for Tap42 phosphatase complexes in governing imaginal disc and fly development (Wang, 2012).

Understanding about the in vivo function of α4/Tap42, especially in development, is limited in part because global knockout of this gene in mice and flies leads to early embryonic death (see Cygnar, 2005 and Kong, 2004). Cellular studies have also revealed that depletion of α4/Tap42 causes death in embryonic stem cells, mouse embryonic fibroblasts, adipocytes, hepatocytes, B and T cells of the spleen and thymus, and Drosophila S2 cells (Bielinski, 2007; Kong, 2004; Yamashita, 2006). Although studies of a conditional (Cre-LoxP) α4 knockout in mouse hepatocytes and a mosaic assay of Tap42 in Drosophila wing disc have provided insights into the cellular biology of α4 and Tap42 (Cygnar, 2005; Kong, 2004), the impact of these gene products on the development of tissues and host have not yet been described. This report utilized Tap42-targeted RNAi and the Gal4/UAS system to investigate the biological effects of silencing Tap42 expression in specific Drosophila tissues. Suppressing the Tap42 gene using two tissue-specific drivers (pnr-Gal4 and ap-Gal4) led to a pleiotropic fly phenotype, which included major deformities in the adult thorax and wings as well as decreased survival rates. The experimental platform described in this study has allowed exploration of the role of Tap42 and Tap42-regulated phosphatases in the control of cellular signaling, tissue development, and Drosophila viability (Wang, 2012).

Analyses of Tap42RNAi wing discs revealed significant alterations in multiple signal transduction pathways including JNK, DPP, and HH. Marked increases in p-JNK signals were found in ap-Gal4>Tap42RNAi wing discs. This observation, together with previous studies showing increased c-Jun phosphorylation in α4-null mouse embryonic fibroblasts (Kong, 2004) and activated JNK in Tap42-depleted clones of fly wing discs (Cygnar, 2005), indicate that α4/Tap42 likely plays a negative role in regulation of JNK signaling. Silencing the Tap42 gene in the ap gene domain also changed DPP and HH signaling in the wing discs. Although ap-Gal4-mediated silencing of Tap42 had a profound effect on JNK, DPP, and HH signaling, these pathways were unaffected in pnr-Gal4>Tap42RNAi wing discs, thus demonstrating that the thorax cleft phenotype seen in the pnr-Gal4>Tap42RNAi flies is not due to alterations in these pathways. Collectively, these findings indicate that Tap42 plays a crucial role in the modulation of JNK, DPP, and HH signaling, but the effects of Tap42 on these pathways appear to play a minimal role in normal thorax development (Wang, 2012).

The HH pathway is one of the major guiding signals for imaginal disc development. Recent investigations have revealed that the phosphorylation state of Ci and Smo, two components of the HH signaling pathway, are controlled by Drosophila PP2A (Mts) and PP4 (Jia, 2009). Additional studies implicate a role for specific Mts complexes in the control of HH signaling, whereby holoenzyme forms of Mts containing the Wdb and Tws regulatory B subunits act at the level of Smo and Ci, respectively (Su, 2011). Together, these findings point to key roles for Mts and PP4 in HH signaling and suggest that a common subunit of these phosphatases, namely Tap42, may also be involved in HH signaling. Indeed, the current data clearly show that Tap42 plays an important regulatory role in this pathway as silencing of Tap42 within the wing discs leads to an elimination of both Smo and Ci expression. Although the precise role(s) of Tap42 in the control of HH signaling remains unclear, it likely involves Tap42-dependent regulation of one or more phosphatase catalytic subunits (e.g., Mts, PP4, and possibly PPV) or specific holoenzymes forms of these phosphatases (e.g., Wdb/Mts, Tws/Mts). The pleiotropic effects of Tap42RNAi on JNK, DPP, and HH signaling could be due to loss of Tap42's regulation of phosphatase activity, cellular levels, holoenzyme assembly, or subcellular localization (Wang, 2012).

Depletion of α4 in mouse embryonic fibroblasts caused an increase in phosphorylation of a variety of established PP2A substrates, which was attributed to a 'generalized defect in PP2A activity.' Instead of the expected unidirectional increase in protein phosphorylation, the current findings demonstrate a dual role for Tap42 in the control of JNK activation as hyperphosphorylation and hypophosphorylation of JNK were observed in the dorsal and ventral sides of the Tap42RNAi wing disc, respectively, relative to control wing discs. Silencing of Tap42 in the ap domain also impacted DPP in a bi-directional fashion; these flies exhibited significantly decreased DPP expression in the scutellum but augmented expression around the wing blade. Consistent with previous studies showing that PP2A functions at different levels within the Ras1 and HH pathways, the current data indicate that Tap42-regulated phosphatases likely target multiple substrates within the JNK and DPP pathways in different regions of wing discs (Wang, 2012).

Close examination of the PE cells in the wing disc revealed that Tap42 expression occurs in only a fraction of these cells. It is noteworthy that the majority of Tap42 localized in rows of cells delineating the PE/DP (peripodial epithelium/disc proper) boundary. These cells are commonly referred to as 'medial edge' cells, which represent a subpopulation of PE cells that play a crucial role in thorax closure during metamorphosis. Interestingly, α4-PP2A complexes appear to play a major role in the control of cell spreading, migration, and cytoskeletal architecture, presumably via their ability to modulate the activity of the small G-protein Rac. Yeast Tap42 has also been implicated in the cell cycle-dependent and polarized distribution of actin via a Rho GTPase-dependent mechanism. Therefore, it is hypothesized that the wing disc structural deformities and thorax cleft phenotype of Tap42RNAi flies are a result of unregulated phosphatases leading to defective spreading and migration of the medial edge cells during metamorphosis. The thorax cleft phenotype provides an opportunity to delineate the precise roles of Tap42-phosphatase complexes in processes controlling thoracic closure (e.g., cell spreading and migration) (Wang, 2012).

α4/Tap42 appears to function as an essential anti-apoptotic factor as cells lacking this common regulatory subunit of PP2A family members undergo rapid death. These studies implicate a role for α4/Tap42-dependent regulation of PP2A-like enzymes, and presumably the phosphorylation state of multiple pro- and anti-apoptotic proteins, in the maintenance of cell survival. The current findings reveal that silencing Tap42 in wing discs triggers apoptosis, thus providing supportive in vivo evidence that depletion of Tap42 (α4) leads to deregulated phosphatase action, which switches these enzymes from pro-survival to pro-apoptotic mediators. Because JNK activation is a hallmark feature of apoptosis, the overlap of apoptotic cells and hyperphosphorylated JNK indicates that the Tap42RNAi-induced apoptosis may be dependent on JNK activation (Wang, 2012).

Since α4 is required for maintaining the normal function of PP2A, PP4, and PP6, it is suspected that misregulation of these phosphatases could be responsible for the pleiotrophic phenotypes observed in Tap42RNAi flies. Consistent with this idea, introduction of the mtsXE2258 heterozygous allele into ap-Gal4>UAS-Tap42RNAi flies partially rescued the thorax and wing defects, and significantly improved fly survival rates. The partial rescue by mtsXE2258 suggests that the defects seen in the Tap42RNAi flies are due, in part, to unregulated Mts activity, possibly as a result of increased Mts levels or enzymatic activity. Indeed, previous studies have demonstrated an accumulation of Mts in Tap42-depleted clones of the fly wing disc. Thus, mtsXE2258 appears to function as a mild mutant that partially restores misregulated Mts function following depletion of Tap42. However, given the biochemical findings showing that Tap42 also interacts with PP4 and PPV, additional studies will be needed to determine the relative contribution of these phosphatases to the Tap42RNAi-induced defects (Wang, 2012).

The phenotypes observed in flies expressing Tap42RNAi could also be attributed to loss of a phosphatase-independent function(s) of Tap42 that controls normal fly development. However, introduction of a phosphatase binding-defective mutant of Tap42 (Tap42ED) into the Tap42RNAi background failed to rescue the phenotypes and lethality associated with Tap42 depletion. In contrast to Tap42ED, introduction of Tap42WT fully rescued the phenotypes and lethality of the Tap42RNAi flies. These findings indicate that the Tap42RNAi-induced phenotypes are entirely due to the impaired interactions between Tap42 and PP2A family members, and provide compelling support for the hypothesis that Tap42-dependent regulation of the functions of these enzymes is crucial for normal wing disc development and Drosophila viability (Wang, 2012).

Although understanding the exact molecular mechanisms underlying Tap42's regulation of PP2A family members is still incomplete, these studies clearly demonstrate that Tap42-phosphatase interactions play crucial roles in the control of multiple signaling pathways governing cell growth and survival. The experimental platform described in this report will undoubtedly serve as a valuable system to further explore the in vivo function and regulation of Tap42-phosphatase complexes. Furthermore, given the remarkable phenotypes seen in the Tap42RNAi flies (e.g., thorax cleft and deformed wings), it is anticipated that this model system will drive future studies (e.g., phenotype-based suppressor/enhancer screens) aimed at identifying direct targets of Tap42-regulated phosphatases, as well as additional pathways under the control of these phosphatase complexes (Wang, 2012).

Histone Chaperone NAP1 Mediates Sister Chromatid Resolution by Counteracting Protein Phosphatase 2A

Chromosome duplication and transmission into daughter cells requires the precisely orchestrated binding and release of cohesin. This study found that the Drosophila histone chaperone NAP1 is required for cohesin release and sister chromatid resolution during mitosis. Genome-wide surveys revealed that NAP1 and cohesin co-localize at multiple genomic loci. Proteomic and biochemical analysis established that NAP1 associates with the full cohesin complex, but it also forms a separate complex with the cohesin subunit stromalin (SA). NAP1 binding to cohesin is cell-cycle regulated and increases during G2/M phase. This causes the dissociation of protein phosphatase 2A (PP2A) from cohesin, increased phosphorylation of SA and cohesin removal in early mitosis. PP2A depletion led to a loss of centromeric cohesion. The distinct mitotic phenotypes caused by the loss of either PP2A or NAP1, were both rescued by their concomitant depletion. In is concluded that the balanced antagonism between NAP1 and PP2A controls cohesin dissociation during mitosis (Moshkin, 2013).

As reflected by their name, a major activity of histone chaperones is to prevent illicit liaisons and guide newly synthesized histones to sites of chromatin assembly. This study describes a mitotic function for the canonical histone chaperone NAP1 that is unrelated to nucleosome assembly. NAP1 was found to bind cohesin and block dephosphorylation of SA by PP2A, thereby promoting cohesin dissociation from the chromosome arms. Consequently, chromosomal binding of cohesin during mitosis is controlled by the balance between the opposing activities of NAP1 and PP2A (Moshkin, 2013).

NAP1 is part of a large assemblage including the full cohesin complex and PP2A. In addition, NAP1 and SA form a subcomplex, which lacks the other cohesin subunits and PP2A. An attractive scenario is that the NAP1-SA module or NAP1 alone competes with PP2A-bound SA within the full cohesion complex. PP2A displacement by NAP1 allows stable phosphorylation of cohesin and its dissociation during early mitosis. NAP1 might also act as a direct inhibitor of PP2A catalytic activity, because a mammalian NAP1 homolog, SET, has been identified as a potent PP2A inhibitor, which promotes sister chromatid segregation during mouse oocyte miosis (Qi, 2013; Chambon, 2012). In addition, NAP1 might help cohesin phosphorylation by tethering Polo kinase to cohesin. In fact, a potential association between NAP1 and Polo kinase was detected. However, the dramatic chromosome condensation defects after Polo kinase depletion precluded a functional evaluation of a possible role of NAP1 in its function. Nevertheless, although additional NAP1 activities cannot be excluded, functional experiments established that blockage of PP2A suffices to explain the crucial role of NAP1 during sister chromatid resolution (Moshkin, 2013).

NAP1 not only regulates the chromosomal distribution of cohesin and PP2A, but also that of MeiS332, a fly homolog of Sgo. The function of MeiS332 and PP2A appears to be largely conserved from mammals to flies because they bind each other and depletion of either factor causes a loss of centromeric cohesion. Either knockdown of NAP1 or over-expression of PP2A caused spreading of MeiS332 onto the arms of mitotic chromosomes, accompanying the loss of sister chromatid resolution. Thus, the balanced antagonism between NAP1 and PP2A controls chromosomal association of both cohesin and MeiS332 during mitosis (Moshkin, 2013).

One level of regulation involves changes in NAP1's subcellular localization and chromatin binding through the cell cycle. At prophase there is a strong increase in the level of nuclear NAP1, but by metaphase, NAP1 and cohesin have dissociated from the chromosomes. Thus, the dynamic behavior of NAP1 correlates well with its function in promoting cohesin release at early mitosis. Regulation of NAP1 localization may involve cyclin B-cdc2/cdk1 kinase complexes. Previously it was found that yeast and vertebrate NAP1 are phosphorylated by cyclin B-cdc2 and that yeast cyclin B requires NAP1 for its full range of mitotic functions (Moshkin, 2013).

It is suggested that histone chaperones are at the hubs of specialized protein networks that perform a wide variety of tasks in chromosome biology. Through association with distinct partners, NAP1 is able to perform different functions. By acting as a histone chaperone, NAP1 mediates chromatin assembly. Through recruitment of the histone H3 deacetylase and H3K4 demethylase complex RLAF, NAP1 controls gene-selective silencing at developmental loci. Finally, by binding cohesin and blocking SA dephosphorylation by PP2A, NAP1 mediates sister chromatid resolution during mitosis. These results emphasize the surprisingly diverse- and specific regulatory functions of histone chaperones in chromosome biology (Moshkin, 2013).

Contribution of Orb2A stability in regulated amyloid-like oligomerization of Drosophila Orb2

How learned experiences persist as memory for a long time is an important question. In Drosophila the persistence of memory is dependent upon amyloid-like oligomers of the Orb2 protein. However, it is not clear how the conversion of Orb2 to the amyloid-like oligomeric state is regulated. The Orb2 has two protein isoforms, and the rare Orb2A isoform is critical for oligomerization of the ubiquitous Orb2B isoform. This study reports the discovery of a protein network comprised of protein phosphatase 2A (PP2A), Transducer of Erb-B2 (Tob), and Lim Kinase (LimK) that controls the abundance of Orb2A. PP2A maintains Orb2A in an unphosphorylated and unstable state, whereas Tob-LimK phosphorylates and stabilizes Orb2A. Mutation of LimK abolishes activity-dependent Orb2 oligomerization in the adult brain. Moreover, Tob-Orb2 association is modulated by neuronal activity and Tob activity in the mushroom body is required for stable memory formation. These observations suggest that the interplay between PP2A and Tob-LimK activity may dynamically regulate Orb2 amyloid-like oligomer formation and the stabilization of memories (White-Grindley, 2014).

Previous work suggested that conversion of neuronal CPEB to an amyloid-like oligomeric state provides a molecular mechanism for the persistence of memory. However, it is not known how Orb2 oligomerization is regulated so that it will occur in a neuron/synapse-specific and activity-dependent manner. This study reports that factors that influence Orb2A stability and thereby abundance regulate Orb2 oligomerization (White-Grindley, 2014)

Tob, a previously known regulator of SMAD-dependent transcription and CPEB-mediated translation, associates with both forms of Orb2, but increases the half-life of only Orb2A. Stimulation with tyramine or activation of mushroom body neurons enhances the association of Tob with Orb2, and overexpression of Tob enhances Orb2 oligomerization. Both Orb2 and Tob are phosphoproteins. Phosphorylation destabilizes Orb2-associated Tob, whereas it stabilizes Orb2A. Tob promotes Orb2 phosphorylation by recruiting LimK, and PP2A controls the phosphorylation status of Orb2A and Orb2B (White-Grindley, 2014).

PP2A, an autocatalytic phosphatase, is known to act as a bidirectional switch in activity-dependent changes in synaptic activity. PP2A activity is down-regulated upon induction of long-term potentiation of hippocampal CA1 synapses (LTP) and up-regulated during long-term depression (LTD). Similarly, Lim Kinase, which is synthesized locally at the synapse in response to synaptic activation, is also critical for long-term changes in synaptic activity and synaptic growth (White-Grindley, 2014).

Based on these observations a model is proposed for activity-dependent and synapse-specific regulation of amyloid-like oligomerization of Orb2. It is postulated that in the basal state synaptic PP2A keeps the available Orb2A in an unphosphorylated and thereby unstable state. Neuronal stimulation results in synthesis of Orb2A by a yet unknown mechanism. The Tob protein that is constitutively present at the synapse binds to and stabilizes the unphosphorylated Orb2A and recruits the activated LimK to the Tob-Orb2 complex, allowing Orb2 phosphorylation. Concomitant decreases in PP2A activity and phosphorylation by other kinases enhances and increases Orb2A half-life. The increase in Orb2A level as well as phosphorylation may induce conformational change in Orb2A, which allows Orb2A to act as a seed. Alternatively, accumulation and oligomerization of Orb2A may create an environment that is conducive to overall Orb2 oligomerization. In the absence of an in vitro Orb2A-Orb2B oligomerization assay, it is not possible to distinguish between these two possibilities (White-Grindley, 2014).

For Tob, initial Orb2 association stabilizes Tob. However, association with Orb2 as well as suppression of PP2A activity leads to additional phosphorylation, which results in dissociation of Tob from the Orb2-Tob complex and destabilization. The destabilization of Orb2-associated Tob provides a temporal restriction to the Orb2 oligomerization process. The coincident inactivation of PP2A and activation of LimK may also provide a mechanism for stimulus specificity and synaptic restriction (White-Grindley, 2014).

Orb2A and Orb2B are phosphorylated at multiple sites, including serine/threonine and presumably tyrosine residues. These phosphorylation events are likely mediated by multiple kinases because overexpression of LimK did not affect Orb2 phosphorylation to the extent observed with the inhibition or activation of PP2A, raising several interesting questions. In what order do these phosphorylations occur? What function do they serve individually and in combination? What kinases are involved? Moreover, similar to mammalian CPEB family members, in addition to changing stability, phosphorylation may also influence the function of Orb2A and Orb2B (White-Grindley, 2014).

Does Tob regulate Orb2 function? In mammals Tob has been shown to recruit Caf1 to CPEB3 target mRNA, resulting in deadenylation, and CPEB3 is known to act as a translation repressor when ectopically expressed. This study found Drosophila Tob also interacts with Pop2/Caf1 and Orb2A and Orb2B can repress translation of some mRNA. Orb2 has also been identified as a modifier of Fragile-X Mental Retardation Protein (FMRP)-dependent translation, and Fragile-X is believed to act in translation repression (Cziko, 2009). Therefore, the Tob-Orb2 association may contribute to Orb2-dependent translation repression, and the degradation of Orb2-associated Tob may relieve translation repression. Additionally, if the oligomeric Orb2 has an altered affinity for either mRNA or other translation regulators, Tob can affect Orb2 function by inducing oligomerization. However, the relationship between Tob phosphorylation and its function is unclear at this point (White-Grindley, 2014).

Does involvement of Tob both in transcription and translation serve a specific purpose in the nervous system? Tob inhibits BMP-mediated activation of the Smad-family transcription activators (Smad 1/5/8) by promoting association of inhibitory Smads (Smad 6/7) with the activated receptor. In Drosophila BMP induces synaptic growth via activation of the Smad-family of transcriptional activators, and subsequent stabilization of these newly formed synapses via activation of LimK. These studies suggest Tob and LimK also regulate Orb2-dependent translation, raising the possibility Tob may coordinate transcriptional activation in the cell body to translational regulation in the synapse (White-Grindley, 2014).

Feedback control of chromosome separation by a midzone Aurora B gradient

Accurate chromosome segregation during mitosis requires the physical separation of sister chromatids before nuclear envelope reassembly (NER). However, how these two processes are coordinated remains unknown. This study, carried out in Drosophila S2 cells, identified a conserved feedback control mechanism that delays chromosome decondensation and NER in response to incomplete chromosome separation during anaphase. A midzone-associated Aurora B gradient was found to monitor chromosome position along the division axis and to prevent premature chromosome decondensation by retaining Condensin I. PP1/PP2A phosphatases counteracted this gradient and promoted chromosome decondensation and NER. Thus, an Aurora B gradient appears to mediate a surveillance mechanism that prevents chromosome decondensation and NER until effective separation of sister chromatids is achieved. This allows the correction and reintegration of lagging chromosomes in the main nuclei before completion of NER (Afonso, 2014).

twins: Biological Overview | Developmental Biology | Effects of Mutation | References

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