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The 55 kDa regulatory subunit of Drosophila protein phosphatase 2A is located in the cytoplasm at all cell cycle stages, by the criterion of immunofluorescence. No significant changes were detected in protein phosphatase activity during the nuclear division cycle of syncytial embryos. However, cell cycle function of the enzyme is suggested by the mitotic defects exhibited by two Drosophila mutants, aar1 and twinsP, defective in the gene encoding the 55 kDa subunit. The reduced levels of the 55 kDa subunit correlate with the loss of protein phosphatase 2A-like, okadaic acid-sensitive phosphatase activity of brain extracts against caldesmon and histone H1 phosphorylated by p34cdc2/cyclin B kinase, but not against phosphorylase a. Thus the mitotic defects of aar1 and twinsP are likely to result from the lack of dephosphorylation of specific substrates by protein phosphatase 2A (Mayer-Jaekel, 1994).
Sex combs reduced (SCR) is a Drosophila Hox protein that determines the identity of the labial and prothoracic segments. In
search of factors that might associate with SCR to control its activity and/or specificity, a yeast two-hybrid screen was performed. A
Drosophila homolog of the regulatory subunit (B'/PR61) of serine-threonine protein phosphatase 2A (dPP2A,B') specifically
interacts with the SCR homeodomain. The N-terminal arm within the SCR homeodomain has been shown to be a target of
phosphorylation/dephosphorylation by cAMP-dependent protein kinase A and protein phosphatase 2A, respectively. In vivo
analyses reveal that mutant forms of SCR mimicking constitutively dephosphorylated or phosphorylated states of the
homeodomain are active or inactive, respectively. Inactivity of the phosphorylated mimic form is attributable to impaired DNA binding. Specific ablation of
dPP2A,B' gene activity by double-stranded RNA-mediated genetic interference results in embryos without salivary glands, an SCR null phenotype. These data
demonstrate an essential role for Drosophila PP2A,B' in positively modulating SCR function (Berry, 2000).
PP2A exists as a multisubunit enzyme complex in a variety of organisms and cell types. The enzyme complex is composed of a catalytic and a scaffold subunit,
which together form a core dimer that then associates with one of a number of regulatory subunits to constitute a trimeric enzyme complex. Regulatory subunits
of PP2A are encoded by at least three unrelated gene families: B (PR55), B' (PR61) and B" (PR72). Each family consists of several members, which in addition
can give rise to a number of splice variants, thereby greatly increasing the variety of distinct trimeric enzyme complexes. Several lines of
evidence suggest that the regulatory subunits of PP2A may serve as specific adaptors that confer substrate specificity to the core domain of PP2A. Specific interaction of dPP2A,B' with the SCR homeodomain, as documented here, therefore reflects its potential of reversibly recruiting SCR
into the PP2A complex (Berry, 2000).
The two phosphorylatable residues (T and S) within the N-terminal arm of the SCR homeodomain appear to be conserved, since at least one such site has been
found in all SCR homologs from other species, except for PS12-B of Atlantic salmon. The homeodomain of PS12-B in fact seems more closely related to ANTP than to SCR. In vivo results suggest that
in developing embryos, SCR is functionally inactive when the N-terminal arm of its homeodomain is phosphorylated and is active upon dephosphorylation. These
results may have important implications for the functional specificity of homeotic proteins in general: since ANTP has a glutamine instead of threonine at
position 6 (which is well conserved in all the SCR homologs), it is proposed that the differential modification of this residue plays an important role in determining
the functional specificity of these two homeotic proteins (Berry, 2000).
The data from the functional knockout of dPP2A,B' by dsRNA interference prove unequivocally that expression of dPP2A,B' is essential for the functional
activity of SCR. Genetic studies in Drosophila have shown that Ras-1 activity positively modulates the function of Hox proteins such as proboscipedia (PB) and SCR -- a finding that suggests that covalent modifications triggered by Ras-1-mediated signals might influence the activity of PB and SCR. The catalytic subunit of dPP2A has been identified as a component operating downstream of Ras-1. The
observation that the functional activity of SCR is dependent upon the presence of dPP2A,B' seems to provide a missing link, suggesting that Ras-1 might influence the activity of SCR via dPP2A (Berry, 2000).
A model is proposed to describe the regulation of SCR activity: in a cell, where SCR function is not required
continuously, the protein is locked in an inactive state by phosphorylation of residues 6 and/or 7 within the N-terminal arm of the homeodomain. The fact that, in
older embryos, SCR is present but is no longer able to induce the expression of its target gene forkhead, may be a case in point. In
response to positive signals, SCR-specific protein phosphatase (dPP2A) becomes activated, possibly through a signaling cascade involving Ras-1. In the
absence of positive signals, or when negative signals, e.g. DPP and SP1 prevail, specific dPP2A activity is inhibited
and, as a result, SCR can no longer be maintained in its dephosphorylated state. PKA or PKA-like enzymes will phosphorylate residues 6/7 of the SCR
homeodomain, thus abrogating the ability of SCR to bind to its target genes. A delicate balance between the activities of SCR-specific PP2A and specific
protein kinases would thus allow a cell to fine-tune SCR activity (Berry, 2000).
widerborst (wdb), a B' regulatory subunit of PP2A, located at 98A6-8 and distinct from Protein phosphatase 2A at 85F (the B subunit of PP2A), has been identified as a conserved component of planar cell polarization mechanisms in both Drosophila and in zebrafish. The German name Widerborst means something stubborn or recalcitrant (derived from wider, meaning against, and borst, meaning bristle). PP2A is a holoenzyme that consists of a catalytic (C) subunit, an A regulatory subunit and one of a large family of B, B' or B'' subunits. The latter subunits are thought to regulate the activity of the C subunit and provide substrate specificity. In metazoans, the B' subunits have diverged into two related subclasses. The central regions of these proteins are strongly conserved, but they differ at their N and C termini. The protein encoded by widerborst is more closely related to the human alpha, þ and epsilon subunits (62%-66% identity) than to the þ 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 naÔvely expect an antagonist of Frizzled signaling to accumulate proximally instead of distally. Nevertheless, although the early distal localization of Wdb is suggestive, it has not been proven that distal localization is relevant to cortical polarization; for example, Wdb might have a role in transducing the Frizzled signal, for which distal localization is not required (Hannus, 2002).
What might be the importance of Wdb binding to the distal microtubule web? Binding to the cytoskeleton might simply allow stable distal localization of an otherwise diffusible cytosolic molecule. More interesting, this association raises the possibility that Widerborst directs the dephosphorylation of a microtubule-associated protein. Consistent with this idea, the structure of the planar microtubule web is disrupted by dnWdb expression. PP2A activity is important for the accumulation of stable microtubules, presumably through the effects of PP2A on the phosphorylation state of MAPs. Microtubule stability can affect the binding of microtubule motor proteins and can contribute to polarized protein delivery. In the wing, microtubules have been suggested to play important roles in hair polarity; depending on the time at which vinblastine is added, vinblastine treatment of pupal wings causes either failure of hair outgrowth or the formation of multiple wing hairs. Polarized dephosphorylation of MAPs within the planar microtubule web might bias the transport of vesicles containing components of the proximodistal cortical domains. At later stages, it might also help direct transport of components of the hair formation machinery to the distal side of the cell, or promote the stability of microtubules in the outgrowing hair. This model for Widerborst action could provide a single explanation for its effects on hair outgrowth and on cortical polarity. Identification of the relevant Widerborst substrate(s) should greatly advance understanding of the cell biology of tissue polarization (Hannus, 2002 and references therein).
The data also support other studies indicating that B' alpha/epsilon
regulatory subunits antagonize the classical Wnt signaling pathway. Experiments in Xenopus embryos and tissue culture cells have shown that increasing the level of a B' alpha subunit inhibits Wnt signaling and causes ventralization. Consistent with this, experiments in zebrafish show that reducing Wdb levels causes dorsalization of embryos. Although Wdb, like Frizzled and Dishevelled, is a shared component of both planar polarization and classical Wnt signaling pathways, it probably has different functions in each; during classical Wnt signaling, the B' alpha is thought to act downstream of Dishevelled, forming part of a ß-catenin degradation complex that plays no role in planar polarity signaling (Hannus, 2002).
The observation that widerborst is needed both for distal polarization of Drosophila wing hairs and for convergent extension movements during zebrafish gastrulation points to a conserved role for Wdb in regulating tissue polarity in development. Furthermore, it provides additional evidence supporting the conjecture that components of the planar polarization pathway in Drosophila are also used to control cell polarity and movement during vertebrate gastrulation. To date, the evidence for this is based on analysis of various dsh constructs and, more recently, on the analysis of vang/stbm and rhoA during vertebrate gastrulation. The identification of Wdb as another shared component provides further evidence that this signaling cascade is indeed conserved between Drosophila and vertebrates. Additional experiments will have to address the precise function(s) of vertebrate wdb homologs and where wdb acts in the genetic pathway regulating vertebrate gastrulation movements (Hannus, 2002).
Members of the Hedgehog (Hh) family of signaling proteins are powerful regulators of developmental processes in many organisms and have been implicated in many human disease states. This study reports the results of a genome-wide RNA interference screen in Drosophila cells for new components of the Hh signaling pathway. The screen identified hundreds of potential new regulators of Hh signaling, including many large protein complexes with pleiotropic effects, such as the coat protein complex I (COPI), the ribosome and the proteasome. The multimeric protein phosphatase 2A (PP2A) and two new kinases, the D. melanogaster orthologs of the vertebrate PITSLRE and cyclin-dependent kinase-9 (CDK9) kinases, were identified as Hh regulators. A large group of constitutive and alternative splicing factors, two nucleoporins involved in mRNA export and several RNA-regulatory proteins were identified as potent regulators of Hh signal transduction, indicating that splicing regulation and mRNA transport have a previously unrecognized role in Hh signaling. Finally, it was shown that several of these genes have conserved roles in mammalian Hh signaling (Nybakken, 2005).
Phosphorylation is associated with the activities of at least five components of the Hh pathway: Fu, Cos, Smo, Su(fu) and Ci. Little is known about the kinases that phosphorylate Su(fu) and Fu, but at least two sites in Cos are phosphorylated by Fu, and several kinases are involved in phosphorylating Ci and Smo, including PKA-C1, CkIalpha and Sgg. But no phosphatase has been implicated in Hh signaling, and a previous RNAi screen did not identify any phosphatases involved in Hh signaling. The screen identified microtubule star (mts), which encodes the D. melanogaster PP2A catalytic subunit, as a gene that substantially reduced Hh signaling when targeted by RNAi. PP2A is a multimeric enzyme that consists at minimum of the catalytic subunit, a regulatory A subunit (encoded by CG33297 in D. melanogaster) and a B subunit principally involved in substrate selection. The B-subunit family in D. melanogaster is represented by the gene twins (tws), the B' family by the genes widerborst (wdb) and PP2A-B', and the B" family by CG4733. All the PP2A component dsRNAs were obtained and tested from a dsRNA library and additional, distinct dsRNAs to these components were generated and tested. In addition to confirming the mts result, it was found that both the original-library dsRNA and three new, unique dsRNAs targeting wdb all reduced Hh signaling.
This indicates that Wdb is likely to be the B subunit that targets Mts to its substrate in the Hh signaling pathway. This hypothesis is in agreement with recent findings from Xenopus laevis, where the wdb ortholog encoding B56e has been found to regulate Hh signaling. In addition, some PP2A-B' amplicons cause a reduction in reporter activity averaging ~30%, indicating that they may have a partially redundant role in targeting PP2A to its Hh pathway substrate (Nybakken, 2005).
To determine whether PP2A acts on Cos, whether overexpression of cos and mts results in similar phenotypes was examined. When overexpressed in Hh-stimulated clone 8 cells, cos completely abrogates Hh signaling, reducing it to near uninduced levels, whereas overexpression of mts reduces Hh signaling by 40%. Thus, Mts and Cos have different overexpression profiles and do not seem to regulate Hh signaling in the same way. The overexpression phenotype of mts was compared with those of cos and 14 other hits from the screen, including the fu, Cdk9 and Pka-C1 kinases. Overexpressing cos in uninduced cells further reduces background signaling, whereas mts overexpression doubled reporter activity, although these levels are still very low compared with the Hh-activated state. Of the 18 other genes tested, only Pka-C1 overexpression had an effect on Hh reporter activity similar to that of mts: doubling of reporter activity in the Hh-uninduced state and a 50% reduction of activity in the Hh-stimulated state. It is therefore possible that PKA-C1 and Mts act on similar substrates. Because several studies have identified Ci as a substrate of PKA-C1, Mts could also be acting on Ci, perhaps removing inhibitory phosphates in response to Hh stimulation (Nybakken, 2005).
This screen allowed the grouping of the ribosome, proteasome, COPI complex and PP2A phosphatase as important regulators of Hh signaling, none of which had been identified as Hh regulators in vivo. Notably, some of the components identified in the screen had already been implicated in aspects of Hh signaling. For instance, the gene encoding eRF1, a translational regulator, was identified in a screen for modifiers of a gain-of-function smo allele, and polyhomeotic and additional sex combs have both been shown to modify ectopic hh expression phenotypes. These results open many new avenues for investigation of Hh signaling. In particular, elucidation of the Hh pathway substrates affected by PP2A will be important in defining the role of dephosphorylation in Hh signaling. Finally, the paradigm of Hh signaling would change substantially if further investigation determines that alternative splicing and mRNA regulation do have vital roles in Hh signaling (Nybakken, 2005).
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).
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