microtubule star: Biological Overview | References
Gene name - microtubule star
Cytological map position - 28D1-28D2
Function - signaling
Symbol - mts
FlyBase ID: FBgn0004177
Genetic map position - 2L: 7,827,743..7,832,745 [+]
Classification - Protein phosphatase 2A homologues; S/T kinase
Cellular location - cytoplasmic
Drosophila neural stem cells or neuroblasts undergo typical asymmetric cell division. An evolutionally conserved protein complex, comprising atypical protein kinase C (aPKC), Bazooka (Par-3) and Par-6, organizes cell polarity to direct these asymmetric divisions. Aurora-A (AurA) is a key molecule that links the divisions to the cell cycle. Upon its activation in metaphase, AurA phosphorylates Par-6 and activates aPKC signaling, triggering the asymmetric organization of neuroblasts. Little is known, however, about how such a positive regulatory cue is counteracted to coordinate aPKC signaling with other cellular processes. During a mutational screen using the Drosophila compound eye, microtubule star (mts), which encodes a catalytic subunit of protein phosphatase 2A (PP2A), was identified as a negative regulator for aPKC signaling. Impairment of mts function causes defects in neuroblast divisions, as observed in lethal (2) giant larvae (lgl) mutants. mts genetically interacts with par-6 and lgl in a cooperative manner in asymmetric neuroblast division. Furthermore, Mts tightly associates with Par-6 and dephosphorylates AurA-phosphorylated Par-6. This genetic and biochemical evidence indicates that PP2A suppresses aPKC signaling by promoting Par-6 dephosphorylation in neuroblasts, which uncovers a novel balancing mechanism for aPKC signaling in the regulation of asymmetric cell division (Ogawa, 2009).
Polarity is a fundamental characteristic of cells and underlies a variety of cellular processes involved in the development and homeostasis of living organisms. In epithelial cells, which consist of the apical and basolateral membrane domains, cell polarity creates distinct subcellular compartments to arrange the cells into a well-ordered structure. In asymmetric cell division, cell polarity is coupled with mitosis. Cell polarity creates two subcellular domains with distinct characteristics in the mitotic mother cell and coordinates the mitotic spindle with the polarity axis to allow the two daughter cells to be distinct. Because these cell polarity events are tightly linked to other elementary processes such as the cell cycle and mitotic events, cell polarity is finely controlled to coordinate with those cellular processes (Ogawa, 2009).
Drosophila neural-stem-like cells, or neuroblasts, undergo typical asymmetric divisions, providing an excellent model for the study of how cell polarity is controlled. Neuroblasts repeatedly divide into a large, self-renewing daughter (the neuroblast itself) and a smaller, differentiating daughter [the ganglion mother cell (GMC)]. Cell fate determinants, such as Prospero, Brain tumor (Brat) and Numb, are segregated to the GMC. The localization of these determinants and the coordination with mitotic spindle orientation are controlled by the apically localized protein complexes -- the aPKC-Par complex and the Pins complex -- which are mutually linked by Inscuteable (Insc). The aPKC-Par complex consists of atypical protein kinase C (aPKC), Bazooka (Baz) and Par-6 and is primarily involved in organizing cell polarity and the asymmetric distribution of the cell fate determinants along the axis of polarity. The Pins complex, which consists of Partner of Inscuteable (Pins), Locomotion defects (Loco) and Gαi, determines the orientation of the mitotic spindle relative to the cell polarity axis (Ogawa, 2009).
aPKC is a key enzyme involved in establishment of neuroblast polarity and definition of the apical cortex. A tumor suppressor protein, Lethal (2) giant larvae (Lgl), is thought to antagonize aPKC as an inhibitory substrate. Although aPKC binds to non-phosphorylated Lgl, Lgl that is phosphorylated by aPKC dissociates from it and is released from the cell cortex. In the absence of aPKC, the entire cortex becomes basal, and Miranda, an adaptor protein for Prospero and Brat, distributes uniformly throughout the cortex. However, loss of Lgl results in uniform activation of aPKC in the cortex just as if the entire cortex were apical. Consequently, Miranda misdistributes into the cytoplasm and concentrates on mitotic spindles (Ogawa, 2009).
The apical complex and the basal determinants dynamically change their localization as the cell cycle progresses. The apical complex accumulates at the apical cortex during late interphase, retains its apical localization during metaphase, and then initiates expansion through the cortex in anaphase. Miranda and its cargos are temporally found in the apical cortex in late interphase and, after spreading into the cytoplasm at the onset of mitosis, form the basal crescent that is complementary to the localization of the apical complex during metaphase. At late anaphase onwards, they are restricted to the GMC compartment, which is separated by the contractile ring from the neuroblast compartment (Ogawa, 2009).
It was recently shown that the mitotic kinase Aurora-A (AurA) has an important role in linking the cell cycle to the asymmetric cell division of neuroblasts and sensory organ precursors (SOPs) by phosphorylating Par-6 (Wirtz-Peitz, 2008). When AurA is inactive, aPKC binds to unphosphorylated Par-6 and Lgl and remains inactive. Phosphorylation of Par-6 by AurA blocks the interaction of Par-6 with aPKC, which in turn leads to activation of aPKC. Activated aPKC then phosphorylates Lgl to replace it with Baz. The Par complex that has recruited Baz is able to phosphorylate Numb, leading to an exclusion of Numb from the apical domain. Because AurA becomes active in the mitotic phase, it is able to synchronize aPKC activation with the entry into mitosis. Given the role of AurA as a positive regulator of aPKC signaling, it is also likely that dephosphorylation negatively regulates this signaling pathway (Ogawa, 2009).
The serine/threonine phosphatases are grouped into four major classes based on their sensitivity to inhibitors and requirement for divalent cations: protein phosphatase 1 (PP1), protein phosphatase 2A (PP2A), protein phosphatase 2B (PP2B) and protein phosphatase 2C (PP2C) (Sontag, 2001). PP2A holoenzyme functions as a heterotrimeric complex comprising a catalytic C subunit [Microtubule star (Mts) in Drosophila (Snaith, 1996)], a scaffolding A subunit [PP2A-29B in Drosophila (Mayer-Jaekel, 1992)] and a regulatory B-subunit [Twins (Tws) (Shiomi, 1994; Uemura, 1993), Widerborst (Wdb) (Hannus, 2002) and PP2A-B' in Drosophila]. The A-subunit can serve as a linker between the C- and B-subunits, and the B-subunit can influence the enzymatic activity and substrate specificity of the holoenzyme. In a genetic screen using the Drosophila compound eye, mts was identified as an enhancer of the aPKC-induced eye phenotype. The genetic and biochemical evidence indicating that Mts suppresses aPKC activity by enhancing dephosphorylation of Par-6 in neuroblasts uncovered an antagonistic role of PP2A in the aPKC signaling pathway (Ogawa, 2009).
The Drosophila compound eye is composed of repetitive ommatidia that contain epithelial retinal cells. Because of the crystal-like arrangement of ommatidia in the compound eye, it is sensitive to defects in epithelial polarity and therefore ideal for use in mutational screens for components involved in epithelial polarity. A modifier screen was undertaken under a sensitized background to look for mutations that affected epithelial polarity. When the membrane-tethered aPKC (aPKCCAAX) is expressed by GMR-GAL4, it becomes expressed in all differentiated retinal cells, the apicobasal polarity of the retinal cells is severely impaired, and the compound eye becomes small and rough. Kinase activity of aPKCCAAX is essential for inducing this eye phenotype, because the kinase-dead version, in which Lys293 is mutated to Trp (K293W), does not alter the eye morphology. Using this system, mutants were screened that modify the aPKCCAAX-induced eye phenotype among lethal mutants available from stock centers, and mts was identified as a phenotypic enhancer. When the GMR-GAL4/UAS-aPKCCAAX fly was crossed to the mts02496 or mtsXE-2258 mutant fly, a smaller and rougher eye was observed, suggesting that Mts acts as an antagonist for the aPKC signaling pathway (Ogawa, 2009).
The mts gene is expressed ubiquitously during embryogenesis and its protein product localizes to the cytoplasm in neuroblasts as well as in epithelial cells. Because a large amount of mts mRNA is maternally supplied, zygotic mts mutant embryos do not show significant defects with regard to cell polarity, and germline clones do not produce an egg. Therefore loss-of-function phenotypes were examined in neuroblasts by overexpressing a dominant-negative mutant of Mts (dnMts) (Hannus, 2002), which lacks the N-terminal region of the phosphatase domain. In wild-type neuroblasts, the protein complex containing aPKC, Par-6 and Baz localizes to the apical cortex and directs Miranda to the basal cortex at metaphase. In the dnMts-expressing embryos, the apical complex localizes to the apical cortex, and its distribution is broader than normal. By contrast, localization of Miranda is severely affected, and it is distributed less asymmetrically along the cell cortex and into the cytoplasm, where it is concentrated on the mitotic spindles. This phenotype resembles that of lgl, raising the possibility that Mts functions in the same pathway as Lgl (Ogawa, 2009).
This study shows that PP2A functions as a negative regulator of the aPKC signaling pathway in Drosophila neuroblasts. Although several studies have suggested that PP2A negatively regulates aPKC signaling in mammalian culture cells (Nunbhakdi-Craig, 2002; Zhang, 2008), the critical target(s) of PP2A is unknown in these studies. The substrates of aPKC, which include Lgl and aPKC itself, can also be substrates for PP2A. However, none of them has been molecularly identified as a target to be dephosphorylated by PP2A. This study has identified Par-6 as a direct target of PP2A, which has its catalytic subunit encoded by the mts gene. Par-6 is known to be phosphorylated by AurA to trigger aPKC activation when neuroblasts and SOPs enter mitosis. Biochemical and genetic evidence reveals that Mts dephosphorylates Par-6 to suppress the aPKC pathway, suggesting an antagonistic role for Mts against AurA in the regulation of cell polarity that is governed by aPKC signaling (Ogawa, 2009).
Co-immunoprecipitation assays of overexpressed Par-6, aPKC or Lgl with Mts in S2 cells indicated that all these molecules can form a complex either directly or indirectly. Among these, the association of Mts with aPKC and Lgl is relatively weaker than the association with Par-6, although a previous study suggested that PP2A associates with aPKC to suppress its kinase activity in mammalian cultured cells (Nunbhakdi-Craig, 2002). In the current study results indicate that, in S2 cells, Par-6 most efficiently forms a complex with Mts. Consistently, the in vitro dephosphorylation assay showed that PP2A effectively dephosphorylates AurA-phosphorylated Par-6 but not the auto-phosphorylated PKCzeta or the PKCzeta-phosphorylated Lgl. It is inferred from these results that Par-6 is a direct target of Mts. Substrate specificity of PP2A is greatly influenced by the B-subunit incorporated into the holoenzyme (Sontag, 2001). Thus, differences in the affinity with Mts among the three tested molecules in co-immunoprecipitation assays might, therefore, partly reflect the B-subunit(s) that is expressed in S2 cells endogenously. The Drosophila genome contains three genes for the B-subunit: tws, wdb and PP2A-B', all of which are ubiquitously expressed during embryogenesis, as is mts (Ogawa, 2009).
At present, it is not clear which of these three is used for targeting Par-6. Among them, tws mutants often show bristle duplications that are due to defective cell fate decisions of the SOP, as lgl4/lglts3 flies show. Since this lgl4/lglts3 phenotype is enhanced by mts, mts is also likely to be involved in the same pathway. Furthermore, a recent study demonstrated that Tws, together with Mts, is included in the aPKC complex to regulate the asymmetric cell division of larval neuroblasts (Chabu, 2009). These results suggest that Mts uses Tws to target Par-6 in the asymmetric cell divisions of SOPs as well as of neuroblasts (Ogawa, 2009).
Par-6 is an essential cofactor for aPKC activity, and it is known to keep aPKC inactive in the absence of AurA-dependent phosphorylation of Par-6 in neuroblasts. The complete deprivation of Par-6 results in the uniform distribution of Miranda into the cell cortex, which is reminiscent of the aPKC mutant phenotype. Thus, Par-6 is required to produce functional aPKC, and its kinase activity becomes active only when Par-6 is phosphorylated by AurA. par-6 heterozygotes (par-6δ226/+) do not show any defect in Miranda localization during the embryonic stage, which indicates that one copy of par-6 in addition to the maternal supply is sufficient to support normal aPKC function. When mts is further inactivated under this condition (par-6δ226/+, mtsXE-2258/mts02496), some neuroblasts exhibit an lgl-like phenotype in Miranda localization, which is indicative of aPKC hyperactivation and unlike the par-6 loss-of-function mutant. This result suggests that Par-6, because of its hyper-phosphorylation, becomes unable to restrict aPKC activity within the normal range. Thus, a probable normal function of Mts is to promote the inhibitory function of Par-6 on aPKC without affecting its function as an essential subunit of the aPKC complex (Ogawa, 2009).
Whereas AurA seems to be active only during the mitotic phase in cell-cycling cells, mitotically inactive or interphase epithelial cells exhibit concrete apicobasal polarity. How do those cells activate aPKC signaling even though AurA is inactive? This apparent paradox raises several possibilities. Par-6 phosphorylation is required for aPKC activation in epithelial cells but might be mediated by kinase(s) other than AurA. Alternatively, aPKC might be activated by mechanisms other than the phosphorylation of Par-6. Indeed, it has been reported that the active form of Cdc42 binds to the CRIB domain of Par-6 to relieve its inhibitory effect on aPKC, leading to the activation of aPKC (Ogawa, 2009).
Although obvious defects are not detected in the epithelial cells of zygotic mts mutant embryos, Oogenesis clones of mts show dramatic defects in their epithelial polarity. This follicle cell phenotype is different from that caused by hyperactivation of aPKC, as observed in the lgl mutant, suggesting that the action of Mts is mechanistically different in the maintenance of follicle cell polarity from that observed in neuroblasts. In photoreceptor cells, Mts operates antagonistically against Par-1 kinase, which restricts Baz to the adherens junctions. It is also known that Par-1 phosphorylates Baz directly to inhibit its incorporation into the apical aPKC complex, thereby restricting Baz to the apical domain in follicle cells. These data raise the possibility that Mts antagonizes Par-1 in Baz localization in follicle cells by inhibiting Par-1-dependent Baz phosphorylation. If this is the case, Mts positively regulates the aPKC pathway in follicle cells and photoreceptor cells, unlike the situation in neuroblasts. To date, there is no report for Par-1 function in Drosophila neuroblasts. Further study is necessary to test whether a similar antagonism between Mts and Par-1 has a role in the regulation of neuroblast polarity (Ogawa, 2009).
AurA-mediated Par-6 phosphorylation is a key step in initiating the asymmetric segregation of the cell fate determinants in the neuroblast cell cycle. Once Par-6 is phosphorylated, aPKC will be continuously activated during the mitotic phase. The apical domain would overwhelm the entire cortex unless an antagonistic reaction occurred. PP2A will be able to balance AurA in Par-6 phosphorylation during mitosis. Thus, PP2A, together with the antagonistic ligand Lgl, might have a role in maintaining aPKC activity at an appropriate level to create both apical and basal domains in the cortex during mitosis. Although both Mts and Lgl negatively regulate aPKC signaling, Mts operates on aPKC activity by directly regulating the cell-signaling cascade, whereas Lgl does so through the direct physical association as a substrate. Therefore, they are different in their mechanisms of action (Ogawa, 2009).
When neuroblasts complete cell cleavage, the basal membrane is largely segregated into the GMC, and the entire cell cortex of neuroblasts appears to become apical. It is therefore necessary to repolarize in order to make the apical and basal domains in the cell cortex for the onset of the next cell cycle. To do so, Par-6 phosphorylation must be removed before entering the next cell cycle, to reset the configuration of the apical complex. A model is proposed in which Mts actively dephosphorylates Par-6 to reset the membrane polarity after the completion of each division cycle. In this context, it will be important to examine whether Mts function depends on the cell-cycle stage in neuroblasts (Ogawa, 2009).
In eukaryotes, serine/threonine phosphatases are categorized into four major groups: PP1, PP2A, PP2B and PP2C. Recent studies have shown that PP1α affects Par-3 activity through the regulation of a phosphorylation-dependent interaction of Par-3 with 14-3-3 or PKCzeta (Traweger, 2008). This study also identified a Pp1-87B mutation as an enhancer of the aPKCCAAX-induced eye phenotype in the genetic screen and found defects in localization of Miranda as well as in epithelial cell polarity in the Pp1-87B mutant. Furthermore, Sousa-Nunes and colleagues reported recently that protein phosphatase 4 (PP4), which is a PP2A family member, regulates Miranda localization in Drosophila neuroblasts, although the direct substrate of PP4 is not yet clear (Sousa-Nunes, 2009). Thus, other classes of phosphatases in addition to PP2A are involved in the regulation of cell polarity in various cellular contexts. Further delineation of phosphatase functions and the crosstalk between phosphatases should help in the understanding of the global control of cellular processes regulated by cell polarity (Ogawa, 2009).
Drosophila larval brain neuroblasts divide asymmetrically to generate a self-renewing neuroblast and a ganglion mother cell (GMC) that divides terminally to produce two differentiated neurons or glia. Failure of asymmetric cell division can result in hyperproliferation of neuroblasts, a phenotype resembling brain tumors. This study has identified Drosophila Protein phosphatase 2A (PP2A) as a brain tumor-suppressor that can inhibit self-renewal of neuroblasts. Supernumerary larval brain neuroblasts are generated at the expense of differentiated neurons in PP2A mutants. Neuroblast overgrowth was observed in both dorsomedial (DM)/posterior Asense-negative (PAN) neuroblast lineages and non-DM neuroblast lineages. The PP2A heterotrimeric complex, composed of the catalytic subunit (Mts), scaffold subunit (PP2A-29B) and a B-regulatory subunit (Tws), is required for the asymmetric cell division of neuroblasts. The PP2A complex regulates asymmetric localization of Numb, Pon and Atypical protein kinase C, as well as proper mitotic spindle orientation. Interestingly, PP2A and Polo kinase enhance Numb and Pon phosphorylation. PP2A, like Polo, acts to prevent excess neuroblast self-renewal primarily by regulating asymmetric localization and activation of Numb. Reduction of PP2A function in larval brains or S2 cells causes a marked decrease in Polo transcript and protein abundance. Overexpression of Polo or Numb significantly suppresses neuroblast overgrowth in PP2A mutants, suggesting that PP2A inhibits excess neuroblast self-renewal in the Polo/Numb pathway (Wang, 2009).
Mammalian PP2A is a tumor suppressor that participates in malignant transformation by regulating multiple pathways (Westermarck, 2008). However, it is unknown whether PP2A controls neural stem cell self-renewal. These data explicitly show that the Drosophila PP2A trimeric complex confers brain tumor-suppressor activity and controls the balance of self-renewal and differentiation of neural stem cells. This study shows that PP2A mutation leads to neural stem cell overproliferation in Drosophila larval brains, which is associated with dramatically reduced neuronal differentiation. Cell cycle genes including CycE, and phospho-Histone H3 and growth factor Myc are upregulated in PP2A mutants, consistent with the neuroblast overgrowth phenotype. Neuroblasts overproliferate in PP2A mutant MARCM clones. When these mutant clones that were generated at larval stages are kept until adulthood, neural stem cells continue to proliferate in adult brains, which is never observed for wild-type clones. Therefore, PP2A can inhibit excess self-renewal and promote neuronal differentiation of neural stem cells (Wang, 2009).
This overgrowth of neural stem cells in PP2A mutants is a consequence of defects in the asymmetric division of neural stem cells. PP2A regulates asymmetric protein localization as well as mitotic spindle orientation. It has been shown that Polo kinase is a brain tumor-suppressor that regulates Numb/Pon and aPKC asymmetric localization, as well as mitotic spindle orientation. Although polo mutants displayed pleiotropic phenotypes during asymmetric divisions, Polo primarily regulates asymmetric division of neural stem cells by regulating Numb asymmetry. Polo directly phosphorylates Pon on Ser611, which leads to the asymmetric localization of Pon and subsequently Numb (Wang, 2007). Strikingly similar to Polo, PP2A also regulates the asymmetric localization of aPKC, Pon and Numb, and is required for Pon phosphorylation on Ser611. Interestingly, both PP2A and Polo are required for Numb phosphorylation, which may be important for Numb asymmetric localization or activity on the cortex. Thus, Numb is a major downstream factor for both PP2A and Polo in regulating neural stem cell self-renewal. Consistent with this, overexpression of Numb, but not PonS611D, a phospho-mimetic form of Pon, in polo mutants significantly rescues the neuroblast overgrowth phenotype (Wang, 2007; Wang, 2009).
It was further discovered that PP2A functions upstream of Polo/Numb in the same pathway to control self-renewal of neuroblasts. Polo transcript and protein abundance is dependent on PP2A function. The expression of several other genes, including numb, baz and lgl, are not affected by PP2A knockdown, suggesting that the downregulation of polo in PP2A mutants appears to be specific. Moreover, overexpression of GFP-Polo or Numb can largely suppress neuroblast overgrowth in PP2A mutants, suggesting that PP2A primarily acts in the Polo/Numb pathway to inhibit neuroblast overgrowth. This discovery suggests that PP2A and Polo, both of which are crucial brain tumor-suppressors and cell cycle regulators, can function in the same pathway to regulate stem cell self-renewal and tumorigenesis. Currently, it is not clear how PP2A, which is a protein phosphatase, promotes polo expression. It is conceivable that PP2A dephosphorylates a transcription factor and consequently activates it to allow polo transcription. Alternatively, PP2A may dephosphorylate a protein that is required for polo mRNA stabilization (Wang, 2009).
PP2A is involved in a broad range of cellular processes including signal transduction, transcriptional regulation and cell cycle control (Westermarck, 2008). PP2A regulates the Wnt/Wingless signaling pathway and affects the degradation of β-catenin, a transcription factor and the central molecule of this pathway (Eichhorn, 2009). Two of the components of Wnt/Wingless signaling pathway, Adenomatous polyposis coli (APC) and Shaggy (also known as GSK3), do not regulate neuroblast polarity. So it remains to be determined whether Wnt/Wingless signaling plays a role in neuroblast polarity. Mammalian PP2A directly dephosphorylates oncogene cMyc and tumor suppressor p53, both of which are transcription factors (Eichhorn, 2009; Junttila, 2007). Future studies should identify potential substrate(s) of PP2A that can promote polo expression and control neural stem cell self-renewal (Wang, 2009).
Interestingly, it was also observed that cMyc protein levels are increased in PP2A mutants, suggesting that PP2A may have a conserved role in modulating cMyc protein and suppressing its function. However, ectopic expression of cMyc alone does not induce brain tumor formation in Drosophila, suggesting that PP2A can regulate multiple pathways to affect neural stem cell self-renewal. However, the PP2A/Numb pathway appears to be one of the major pathways by which PP2A controls the balance of self-renewal and differentiation in Drosophila, as overexpression of Polo or Numb can largely suppress neural stem cell overgrowth in PP2A mutants. Furthermore, PP2A may regulate Numb function and activity by both promoting polo expression and antagonizing aPKC phosphorylation of Numb. Whether mammalian PP2A also regulates stem cell polarity will be of great interest for future study (Wang, 2009).
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 (Yasugi, 2008). 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).
Bazooka/Par-3 (Baz) is a key regulator of cell polarity in epithelial cells and neuroblasts (NBs). Phosphorylation of Baz by PAR-1 and aPKC is required for its function in epithelia, but little is known about the dephosphorylation mechanisms that antagonize the activities of these kinases or about the relevance of Baz phosphorylation for NB polarity. This study found that protein phosphatase 2A (PP2A) binds to Baz via its structural A subunit. By using phospho-specific antibodies, it was shown that PP2A dephosphorylates Baz at the conserved serine residue 1085 and thereby antagonizes the kinase activity of PAR-1. Loss of PP2A function leads to complete reversal of polarity in NBs, giving rise to an 'upside-down' polarity phenotype. Overexpression of PAR-1 or Baz, or mutation of 14-3-3 proteins that bind phosphorylated Baz, causes essentially the same phenotype, indicating that the balance of PAR-1 and PP2A effects on Baz phosphorylation determines NB polarity (Krahn, 2009).
Apical-basal polarity of NBs is controlled by a relatively small number of proteins which assemble into protein complexes localized to the NB cortex in an asymmetric fashion. These cortical proteins interact with each other in a functional hierarchy. At the top of the hierarchy is Baz, because it can localize to the apical NB cortex in loss-of-function mutants for any of the other factors, including PAR-6, aPKC, Insc, Pins, and others (Krahn, 2009).
This study shows that Baz gets frequently mislocalized to the basal NB cortex when it is moderately overexpressed or when it is excessively phosphorylated at S1085, either by overexpression of PAR-1 or by loss-of-function of PP2A. It is expected that similar antagonistic activities of kinases and phosphatases regulate the phosphorylation state of additional sites of Baz/PAR-3 that are relevant in different cellular contexts. Loss of function of 14-3-3ζ and to a lesser extent of 14-3-3epsilon causes mislocalization of endogenous Baz in NBs, whereas overexpression of 14-3-3ζ and 14-3-3epsilon suppresses the mislocalization of overexpressed Baz. It is therefore suggested that the ratio of Baz phosphorylated at S1085 to the amount of available 14-3-3 determines whether Baz gets mislocalized to the basal cortex. In this model, the 14-3-3 proteins function as a buffer to inactivate mislocalized, phosphorylated Baz. This inactivation could be explained by the inhibition of aPKC binding to Baz upon association of 14-3-3 with Baz. If the amount of overexpressed Baz exceeds the buffering capacity of 14-3-3, this would lead to the formation of active Baz/aPKC complexes at the basal cortex. These basally localized, active Baz/aPKC complexes may in turn affect the localization of PAR-1. The mammalian aPKC homolog PKCζ can phosphorylate PAR-1 at a conserved serine residue, and this phosphorylation causes a strong reduction of PAR-1 kinase activity and the release of PAR-1 from the plasma membrane. If the same was true in Drosophila, it would explain the total reversal of NB polarity, because the now basally localized aPKC would phosphorylate PAR-1, which would cause its release from the membrane and the establishment of a new apical cortical domain at the previously basal cortex (Krahn, 2009).
PAR-1, 14-3-3 proteins, and PP2A are strongly expressed during oogenesis, and maternal contributions may account for difficulties identifying requirements during early embryogenesis. In contrast, eliminating maternal expression of these genes results in phenotypes too severe to allow the study of neurogenesis. However, overexpression of a dominant-negative form of Mts from early neurogenesis onward also caused polarity reversal only in late-stage NBs. While this experiment does not exclude the possibility that the late onset of polarity reversal in NBs is due to the perdurance of the maternal gene products, it points to a fundamental difference in the mechanism of how NB polarity is controlled immediately after delamination as opposed to subsequent asymmetric divisions. The majority of late-stage NBs showing polarity reversal were not in direct contact with the overlying epithelium and thus may rely exclusively on intrinsic polarity cues, in contrast to NBs that have just delaminated and maintain contact to the overlying epithelium. Late-stage NBs lacking contact to the overlying epithelium show a higher variability of spindle orientation as compared to early-stage NBs in close contact to the epithelium. Thus, late-stage NBs may be particularly sensitive to changes in the phosphorylation state and general activity level of Baz, because they rely on Baz as the main cue for orienting their polarity axis (Krahn, 2009).
It is interesting to note that mutations uncoupling spindle orientation from the localization of cell fate determinants commonly show fully random spindle orientation, including a variety of oblique orientations. In contrast, hyperphosphorylation of Baz at S1085 resulted very rarely in oblique orientations, and spindles were always aligned with the asymmetric crescents of cell fate determinants. Although a good explanation for why there is a strong bias for either total reversal of polarity or misorientation of the spindle by 90° is not available, the findings point to the existence of a spatial cue functioning upstream of Baz that defines a polarity axis perpendicular to the plane of the epithelium (Krahn, 2009).
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, Tws, 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).
During synaptic development, presynaptic differentiation occurs as an intrinsic property of axons to form specialized areas of plasma membrane [active zones (AZs)] that regulate exocytosis and endocytosis of synaptic vesicles. Genetic and biochemical studies in vertebrate and invertebrate model systems have identified a number of proteins involved in AZ assembly. However, elucidating the molecular events of AZ assembly in a spatiotemporal manner remains a challenge. Syd-1 (synapse defective-1 or Rho GTPase activating protein at 100F) and Liprin-α have been identified as two master organizers of AZ assembly. Genetic and imaging analyses in invertebrates show that Syd-1 works upstream of Liprin-α in synaptic assembly through undefined mechanisms. To understand molecular pathways downstream of Liprin-α, a proteomic screen was performed of Liprin-α-interacting proteins in Drosophila brains. Drosophila protein phosphatase 2A (PP2A; see MTS, the PP2A catalytic subunit) regulatory subunit B' [Wrd (Well Rounded) or PP2A-B'] was identified as a Liprin-α-interacting protein, and it was demonstrated that it mediates the interaction of Liprin-α with PP2A holoenzyme and the Liprin-α-dependent synaptic localization of PP2A. Interestingly, loss of function in syd-1, liprin-α, or wrd shares a common defect in which a portion of synaptic vesicles, dense-core vesicles, and presynaptic cytomatrix proteins ectopically accumulate at the distal, but not proximal, region of motoneuron axons. Strong genetic data show that a linear syd-1/liprin-α/wrd pathway in the motoneuron antagonizes glycogen synthase kinase-3β kinase activity to prevent the ectopic accumulation of synaptic materials. Furthermore, data is provided suggesting that the syd-1/liprin-α/wrd pathway stabilizes AZ specification at the nerve terminal and that such a novel function is independent of the roles of syd-1/liprin-α in regulating the morphology of the T-bar structural protein BRP (Bruchpilot) (Li, 2014).
During presynaptic development, small synaptic vesicle (SV) precursors, dense-core vesicles (DCVs), and synaptic cytomatrix proteins are generated in the soma, transported along the axon, and eventually incorporated into the nerve terminal. Within the nerve terminal, active zones (AZs) are specialized areas of plasma membrane containing a group of evolutionarily conserved proteins, including ELKS (glutamine, leucine, lysine, and serine-rich protein)[also called CAST (cytomatrix at the active zone-associated structural protein), Drosophila homologue is BRP (Bruchpilot)], Munc13 (mammalian uncoordinated homology 13), RIM (Rab3-interacting molecule), Syd-1 (synapse defective-1), and Liprin-α, in which the releasable pool of vesicles dock and are released on stimulation. Despite intensive studies of the proteins localized at the presynaptic density, the assembly and maintenance of AZs remains enigmatic. Studies conducted in invertebrate model organisms suggested that Syd-1, a putative RhoGAP, and Liprin-α are two master organizers of presynaptic differentiation. Genetic analyses in Caenorhabditis elegans demonstrated that Syd-1 works upstream of Liprin-α in synaptic assembly. Studies in Drosophila further confirmed this hierarchy by showing that Syd-1 regulates and retains proper localization of Liprin-α at the AZ. However, studies also found that Syd-1 regulates Liprin-α-independent processes, such as retention of Neurexin at the presynaptic side and glutamate receptor incorporation at the postsynaptic side. The morphology of the AZ is distinctly different in liprin-α and syd-1 mutants. Therefore, it is unclear how Syd-1- and Liprin-α-mediated signaling collaborate to achieve the complex regulation of presynaptic differentiation. Identifying novel Liprin-α-interacting proteins at the synapse holds the key to delineating the regulatory network mediated by these two genes (Li, 2014).
This study identified protein phosphatase 2A (PP2A) as one prominent Liprin-α-interacting protein complex through an in vivo tandem affinity purification (TAP) approach. PP2A is an abundant heterotrimeric serine/threonine phosphatase that regulates a broad range of cellular processes. PP2A is highly enriched in neurons and is implicated in Tau-mediated neurodegeneration, regulation of long-term potentiation, and presynaptic and postsynaptic apposition. The diverse functions of PP2A are attributed primarily to its many interchangeable regulatory subunits (B, B', B'', or B'''), each showing specific spatial and temporal expression patterns. The Liprin-α-interacting PP2A holoenzyme that this study identified in the fly brain contains the B' regulatory subunit [also called Wrd (Well Rounded) in fly]. Wrd is highly expressed in synapses and regulates synaptic terminal growth at the Drosophila neuromuscular junction (NMJ). Interestingly, the Liprin-α-Wrd physical interaction may be evolutionarily conserved because PP2A B56γ, the human homolog of Wrd, can bind Liprin-α1 in HEK 293 cell. However, the function of the Liprin-α-Wrd/PP2A B56γ interaction in the nervous system is unexplored (Li, 2014).
This study shows that Syd-1, Liprin-α, and Wrd work in a linear pathway to restrain the localization of vesicles and presynaptic cytomatrix proteins at the nerve terminal. Disruption of such a pathway results in ectopic accumulation of SVs and presynaptic proteins at the distal, but not proximal, end of axons (Li, 2014).
Much progress toward understanding presynaptic differentiation has been made through unbiased forward genetic screens in invertebrates. These studies have led to the identification of several key factors for AZ formation, including two evolutionarily conserved master organizer proteins of AZ assembly: syd-1 and syd-2/liprin-α. However, how Syd-1/Liprin-α organize presynaptic sites remains unclear. This study identified a new synaptic player, the PP2A B′ regulatory subunit, that is localized to the synapse by Liprin-α and mediates Syd-1/Liprin-α signaling in stabilizing AZs and their associated vesicles at the nerve terminal (Li, 2014).
Liprin-α was first identified as a protein interacting with the LAR (leukocyte antigen-related-like) family of phosphatases. Studies during the past two decades demonstrate that Liprin-α regulates presynaptic and postsynaptic development, as well as neurotransmitter release through protein–protein interactions with a range of molecules, including CAST/ELKS/BRP, RIM, CASK (calcium/calmodulin-dependent serine protein kinase), GIT (G-protein-coupled receptor kinase-interacting ArfGAP), GRIP (glutamate receptor interacting protein), LAR, CaMKII, and Liprin-β. Proteomic data confirmed the interaction between Liprin-α and BRP/RIM in Drosophila. Another important Liprin-α binding partner was identified at the presynaptic sites, the B′ regulatory subunit of PP2A (Wrd), which depends on Liprin-α for it proper synaptic localization (Li, 2014).
Phenotypic analysis of syd-1, liprin-α, and wrd mutants demonstrate that they share a unique trafficking defect, in which SVs, DCVs, presynaptic scaffolding proteins, and voltage-gated Ca2+ channels ectopically accumulate at the distal, but not the proximal, region of the axon. Genetic rescue experiments define a linear pathway, from syd-1 to liprin-α to wrd, that works cell autonomously in the presynaptic neuron to ensure proper localization of presynaptic materials to the nerve terminal and prevents ectopic accumulation. Together, these biochemical and genetic data suggest that Wrd mediates a novel Syd-1/Liprin-α function at the presynaptic site. Such a Syd-1/Liprin-α function is likely independent of their well established roles in regulating the T-bar structure protein BRP/ELKS (Li, 2014).
Two lines of evidence suggest that a Wrd-containing PP2A mediates the function of Syd-1/Liprin-α in regulating AZ stability. First, two rounds of in vivo biochemical purification using either Liprin-α or Wrd as the bait copurified Liprin-α with Wrd and the other two core subunits of PP2A, indicating the presence of a Liprin-α/Wrd/PP2A protein complex in neurons. Second, loss of GSK-3β kinase [sgg (shaggy)] function suppresses the syd-1, liprin-α, and wrd mutant distal axon phenotype, suggesting that a Wrd/PP2A-mediated phosphatase activity normally functions to antagonize a GSK-3β kinase activity in neurons to stabilize AZ and clustering of SVs at the nerve terminal (Li, 2014).
What is the primary cause for the unique distal axon phenotype in syd-1/liprin-α/wrd mutant larvae? Liprin-α was shown to interact with KIF1A (kinesin family member 1A)/Unc-104, a neuron-specific kinesin motor known to transport SV precursors containing synaptophysin, Syt, and Rab1A. It was reported that Drosophila Liprin-α regulates the trafficking of SVs through its interaction with Kinesin-1 and that liprin-α mutant peripheral nerves show accumulation of clear-core vesicles similar to kinesin heavy chain (khc) mutants. However, when this study focused on the location of the phenotypes relative to the entire axonal length, liprin-α mutant accumulation of clear-core vesicles was found to be present exclusively in the distal end (the ventrolateral peripheral nerve bundles, as well as axonal regions proximal to NMJs), whereas khc mutant larvae show massive aggregation of SV-associated proteins in the proximal end (segmental nerve bundles), and very few SV precursors reach the distal of axon. The distribution pattern of the vesicle accumulation in syd-1 and wrd mutants is the same as liprin-α mutants. Such a pattern is distinct from that of typical trafficking defects induced by mutations in vesicle-transporting motors or cargos (Li, 2014).
Although a unique vesicle trafficking defect as the primary cause for the syd-1/liprin-α/wrd mutant axonal phenotype cannot be completely excluded, a number of lines of evidence suggest a plausible explanation: AZ materials at the nerve terminal become destabilized when the syd-1/liprin-α/wrd pathway is impaired, and the floating AZ materials diffuse back to the adjacent axonal regions as ectopic docking sites for vesicles. First, Syd-1, Liprin-α, and Wrd show clear synaptic localization, with little or no axonal localization detected, consistent with a collaborative function of the three at the AZs. Second, EM analysis detected floating AZ materials in the synaptic boutons and the connected axonal regions in syd-1 mutants. Some of the floating materials are very close to or touching the bouton plasma membrane, indicating a possible defect in AZ stabilization and subsequent back-diffusion of detached AZ materials to axonal regions. Third, AZ components such as BRP, RIM, and voltage-gated Ca2+ channels are identified in the mutant distal axons along with vesicles, including SVs and DCVs, but not vesicles that transport AZ scaffolding proteins, or other synaptically localized organelles, or transport machineries. This is consistent with an ectopic accumulation of vesicles attracted by ectopic floating AZ components. Fourth, live imaging analysis found that anterogradely transported DCVs accumulate at preferred spots at the mutant distal axons, consistent with the existence of static docking sites at these axonal regions. Fifth, ectopically accumulated vesicles do not participate in release or recycling, consistent with the notion that the vesicles do not dock on the axonal plasma membrane (Li, 2014).
The fact that knockdown of a kinase (GSK-3β) rescues the distal axonal defects of syd-1/liprin-α/wrd mutants indirectly suggests that a Wrd-dependent dephosphorylation event is antagonized by a phosphorylation event (mediated by GSK-3β) to regulate AZ stability. However, these data cannot exclude the possibility that PP2A-independent functions of Wrd are involved. One way to seek direct evidence that Wrd-containing PP2A is involved in regulating AZ stability is to study the loss of function of PP2A; however, this approach has its own set of complications. As a ubiquitous heterotrimetric enzyme, the substrate specificity and subcellular localization of PP2A are greatly dependent on its regulatory subunit (such as Wrd). Mutating the catalytic or structural domain blocks overall PP2A action mediated by all regulatory subunits, which precludes analysis of Wrd-specific PP2A action. For example, mutations in MTS (the PP2A catalytic subunit) cause early lethality. Overexpression of a dominant MTS protein causes massive axonal transport defects in the entire axon, as well as defects in AZ development. Therefore, identifying common substrates shared by Wrd/PP2A and GSK-3β and studying how their phosphorylation status regulates AZ stability and/or vesicle trafficking will ultimately unravel the mechanism by which a PP2A-dependent pathway regulates presynaptic development. In this context, this study set up a model to study how synapse scaffolding proteins can regulate localized phosphorylation/dephosphorylation through recruitment of specific phosphatases or kinases (Li, 2014).
A mammalian homolog of Syd-1 was identified recently as an important regulator of presynaptic differentiation at central synapses, at least partially through its interaction with mammalian Liprin-α2. Given that Liprin-α1 interacts with PP2A B56γ (mammalian homolog of Wrd) in HEK 293 cells, it will be of interest to investigate whether the function of Drosophila Liprin-α in mediating the signaling from Syd-1 to the PP2A B′ subunit is also evolutionarily conserved during vertebrate synapse development (Li, 2014).
Establishment and maintenance of apical basal cell polarity are essential for epithelial morphogenesis and have been studied extensively using the Drosophila eye as a model system. Bazooka (Baz), a component of the Par-6 complex, plays important roles in cell polarity in diverse cell types including the photoreceptor cells. In ovarian follicle cells, localization of Baz at the apical region is regulated by Par-1 protein kinase. In contrast, Baz in photoreceptor cells is targeted to adherens junctions (AJs). To examine the regulatory pathways responsible for Baz localization in photoreceptor cells, the effects of Par-1 on Baz localization were studied in the pupal retina. Loss of Par-1 impairs the maintenance of AJ markers including Baz and apical polarity proteins of photoreceptor cells but not the establishment of cell polarity. In contrast, overexpression of Par-1 or Baz causes severe mislocalization of junctional and apical markers, resulting in abnormal cell polarity. However, flies with similar overexpression of kinase-inactive mutant Par-1 or unphosphorylatable mutant Baz protein show relatively normal photoreceptor development. These results suggest that dephosphorylation of Baz at the Par-1 phosphorylation sites is essential for proper Baz localization. This study also shows that the inhibition of protein phosphatase 2A (PP2A) mimics the polarity defects caused by Par-1 overexpression. Furthermore, Par-1 gain-of-function phenotypes are strongly enhanced by reduced PP2A function. Thus, it is proposed that antagonism between PP2A and Par-1 plays a key role in Baz localization at AJ in photoreceptor morphogenesis (Nam, 2007).
Clonal analysis suggests that Baz is crucial for targeting or maintenance of Par-6 and aPKC. In contrast, Baz protein expression was not significantly reduced in par-6 or apkc null clones, although Baz protein distribution was mislocalized to the basolateral region. This implies that Baz plays a nodal role among the Par-6 complex proteins. A caveat in this analysis is that par-6 and apkc mutant clones are very small (1-2 ommatidia) compared to the relatively large baz mutant clones, raising the possibility that par-6 and apkc mutant clones analyzed may represent rare escaper cells that survive with weaker phenotypes. However, this may not be the case because nearly identical phenotypes were seen in more than 50 par-6 or apkc mutant clones. The data suggesting the central role for Baz in Par-6 and PKC localization are also consistent with studies on embryonic epithelia, in which Baz localization to the membrane precedes localization of Par-6, implying Par-6-independent membrane localization of Baz. The requirement of Baz for the localization of Par-6/aPKC but not vice versa suggests that initial Baz localization may be independent of Par-6 and aPKC, as reported in embryonic epithelia (Nam, 2007).
Similar relationships among Par-6 complex proteins have also been observed during asymmetric cell division in C. elegans. PAR-3, the homolog of Baz, is properly localized in the absence of either PKC-3 (aPKC) or PAR-6, whereas it is indispensable for localization of both PKC-3 and PAR-6. These studies suggest that Baz/Par-3 is a major component in the control of cell polarity in diverse systems, including the photoreceptors in Drosophila. However, the Par-6 complex may exist in different compositions with unique functions, depending on various developmental contexts. For instance, Baz/Par-3 and Par-6 colocalize to the apical cortex of dividing neuroblasts in the Drosophila CNS and in dividing cells in C. elegans embryos, whereas Baz is distinctly localized to the AJ basal to the Par-6 domain in the apical membrane of photoreceptors. It is also striking that whereas Baz is not critically required for photoreceptor differentiation in the larval eye imaginal disc, it becomes crucial during pupal eye development. These data suggest that Baz is required for specific developmental events such as junctional reorganization and rhabdomere formation, although it is expressed in photoreceptors from the time of neuronal fate specification as well as in undifferentiated cells prior to retinal development (Nam, 2007).
Par-1 is a key regulator of Baz localization in ovarian follicle cells. In this system, Par-1 is localized to the basolateral membrane and is essential for exclusion of Baz expression from the basolateral membrane. During early embryogenesis, Par-1 is transiently restricted to the lateral membrane, but at mid-gastrulation it is localized near the apical domain immediately below the region of spot adherens junction (SAJ). Par-1 is required for the restriction of SAJ, preventing the expansion of the E-Cad SAJ marker into the lateral membrane, but not affecting Crb-Dpatj apical markers (Nam, 2007).
In contrast to follicle and embryonic epithelia, in eye imaginal discs, par-1 LOF clones show no significant apical basal polarity defects, suggesting that Par-1 is not required for cell polarity in eye imaginal disc epithelia. This raises a question of whether Par-1 function is dispensable for photoreceptor morphogenesis. In this study, analysis was focused on pupal eye development, since some cell polarity genes such as crb are not required in larval imaginal discs, although they become essential later during the pupal stage when the retina undergoes dramatic reorganization of cell junctions and the apical basal pattern in the photoreceptor cells. Analysis of pupal eyes suggests that Par-1 is required for the distal-proximal growth or maintenance of apical and AJ domains of photoreceptor cells, as Baz and apical markers often fail to form continuous AJ and rhabdomeres along the distal-proximal axis of the retina in par-1 null mutant clones. These phenotypes are similar to the defects shown previously in the eyes of Crb complex mutants. Like par-1 mutations, loss of these gene functions also affects the extension/maintenance of AJ and rhabdomeres but not apical basal cell polarity. Since Baz is essential for proper targeting of Par-6 and Crb complex proteins, loss of Par-1 function may result in mislocalization of Crb complex through affecting Baz localization, although it is possible that Par-1 may also be directly involved in localization of Crb complex proteins independent of Baz. In the eye imaginal disc, it has been reported that Par-1 is localized to the apical-marginal zone and AJ. In the pupal eye, it was also found that Par-1 is enriched in the apical region of photoreceptor clusters, although a low level of Par-1 is also detected broadly along the basolateral membrane. Thus, Par-1 localization is not restricted to the basolateral membrane but appears to be regulated in a complex pattern in different cell types (Nam, 2007).
In ovarian follicle cells, phosphorylation of Baz by Par-1 is required for proper localization of Baz to the apical region of the cells, and BazSA mutated proteins are abnormally localized to the basolateral membrane. In contrast, the current data show that under conditions of overexpression, Baz protein mutated at Par-1 phosphorylation sites is targeted to the AJ whereas wild-type Baz is ectopically localized. Overexpressed wild-type Baz may be abnormally targeted to non-AJ sites, but it is also possible that ectopic Baz may recruit AJ proteins to form ectopic AJs. Nonetheless, the data suggest that, in photoreceptor cells, Par-1-dependent phosphorylation is not essential for initial localization of Baz to AJ. Instead, dephosphorylation of Baz may be a key for the localization of Baz to AJ. This explanation is consistent with the data that the pattern of AJ and apical markers is severely disrupted by overexpression of Par-1 but not by loss of Par-1, suggesting that Baz phosphorylation by Par-1 must be suppressed to maintain photoreceptor cell polarity. The data provide genetic evidence to support that Mts is a major enzyme responsible for antagonizing the effects of Par-1-dependent Baz phosphorylation (Nam, 2007).
PP2A has been implicated in the regulation of tight junction formation in MDCK epithelial cells by interacting with aPKC (Nunbhakdi-Craig, 2002). However, it is unlikely that the Mts role in Baz localization is mediated through aPKC. First, wild-type and mutant BazSA are localized to completely different sites even though both have an intact phosphorylation site for aPKC. Second, the phenotype of Par-1 overexpression is mimicked by inhibition of Mts but not by loss of aPKC. The idea of specific antagonism between Par-1 and Mts is also supported by the enhancement of the Par-1 overexpression phenotype by reduction of mts gene dosage but not apkc. Thus, a model is proposed in which the localization of Baz to AJ in photoreceptors cells during early pupal eye development depends on the removal of Par-1-mediated phosphorylation by Mts PP2A activity. In this model, Mts plays a pivotal role in regulation of Baz localization and consequent maintenance of photoreceptor cell polarity. On the contrary, Par-1 does not play an essential role for the initial targeting of Baz to AJ, but it is required for growth or stability of AJs and rhabdomeres during photoreceptor morphogenesis. It will be interesting to see whether the antagonistic interaction of Par-1 and PP2A plays an important role in regulation of Baz localization and function in various developmental contexts in Drosophila and other animal species (Nam, 2007).
Repression by E(spl)M8 during inhibitory Notch signaling (lateral inhibition) is regulated, in part, by protein kinase CK2, but the involvement of a phosphatase has been unclear. Timekeeper (Tik), a unique dominant-negative (DN) mutation in the catalytic subunit of CK2, was used in a Gal4-UAS based assay for impaired lateral inhibition. Specifically, overexpression of Tik elicits ectopic bristles in N1 flies and suppresses the retinal defects of the gain-of-function allele Nspl. Functional dissection of the two substitutions in Tik (M161K and E165D), suggests that both mutations contribute to its DN effects. While the former replacement compromises CK2 activity by impairing ATP-binding, the latter affects a conserved motif implicated in binding the phosphatase PP2A. Accordingly, overexpression of microtubule star (mts), the PP2A catalytic subunit closely mimics the phenotypic effects of loss of CK2 functions in N1 or Nspl flies, and elicits notched wings, a characteristic of N mutations. These findings suggest antagonistic roles for CK2 and PP2A during inhibitory N signaling (Kunttas-Tatli, 2009).
Inhibitory N signaling is vital for stereotyped patterning of sense organs such as the eye and the bristles. This signaling pathway is required for proper SOP/R8 selection and involves cell-cell communications. Specifically, the future SOP/R8 cell expresses the highest levels of the N ligand, Delta, which activates N in all cells of the PNC, but the future SOP/R8. This, in turn, elicits expression of the E(spl) repressors, a family of homologous basic-helix-loop-helix (bHLH) proteins. These bHLH proteins, along with the corepressor Groucho, then antagonize ASC/Ato. As a result, cells that receive N signaling are redirected from adopting the default (SOP/ R8) neural fate. This model reflects the findings that loss of inhibitory N signaling leads to excess SOP and R8 specification, which manifest as ectopic bristles and rough eyes, respectively. It is, therefore, important to fully define the mechanisms that regulate this critical step in neural patterning (Kunttas-Tatli, 2009).
Earlier studies suggested that transcription of E(spl) and the ensuing rise in protein levels was, perhaps, sufficient for restriction of the R8/SOP fate. Accumulating evidence, however, suggests that phosphorylation of E(spl) proteins is important for repression. Evidence has so far been obtained for M8 and its structurally related repressor Hairy, and in either case phosphorylation by CK2 augments repression in the eye and/or the bristle. It has, however, remained unclear whether protein phosphatases act to oppose CK2 functions. The characterization of such a regulation would open the possibility that phosphorylation and repression by E(spl) (inhibitory N signaling) is dynamically controlled in vivo. A role for PP2A has been implicated in studies showing ectopic bristle defects upon increased dosage of the regulatory subunits widerborst (wdb) or twins (tws) and in screens for modifiers of N. However, interactions between PP2A and alleles of N, such as Nspl have not yet been described. These studies provide new insights into the genetic behaviors of Tik and its revertant allele TikR, and implicate a tripartite regulatory nexus, involving CK2, PP2A and inhibitory N signaling (Kunttas-Tatli, 2009).
Both Tik and TikR lack CK2 kinase activity (in vitro). The severe clock defect of Tik/1 flies is, however, not observed in TikR/1 animals (Lin, 2002), and in this sense TikR meets the criteria of a revertant allele. These studies suggest that the TikR protein is not only devoid of kinase activity, but more importantly is deficient for binding CK2b, a prerequisite for CK2-holoenzyme formation and for proper functions in vivo. The most parsimonious interpretation is that misfolding of TikR prevents its incorporation into the holoenzyme. It seems reasonable to, therefore, suggest that the ability of Tik to incorporate into and 'poison' the endogenous holoenzyme (by binding CK2b) underlies its strong DN effects in vivo. However, it has been generally thought that these effects of Tik primarily reflect the M161K, but not the E165D, substitution. These studies on site-specific variants, suggest that these substitutions have additive effects on activity and N signaling, and Tik is likely to therefore be a 'double hit (Kunttas-Tatli, 2009).
The studies in N1 and Nspl backgrounds provide evidence that both substitutions in Tik affect proper CK2 functions. How might one interpret the effects on Nspl? Unlike the bristle, where N signaling occurs only after the specification of the bristle PNCs, the development of patterned founding R8 photoreceptors requires N signaling in a biphasic manner in the MF of the developing third instar eye disc. At the anterior margin of the MF, N elicits ato expression (for R8 specification), whereas in the MF it drives expression of E(spl) enabling refinement of a single R8 cell from the PNCs. Nspl only perturbs the latter. Specifically, Nspl renders R8 precursors hypersensitive to inhibitory N signaling, and consequently impairs R8 differentiation. These impaired R8s are defective in the presentation of signals such as Hedgehog and Decapentaplegic, whose activities are necessary for ato expression at the anterior margin of the MF. As a result, the reduced ato expression in the MF of Nspl perpetuates throughout retinal histogenesis, and elicits the rough and reduced eye of Nspl. Consistent with the notion that this allele renders R8s sensitive to inhibitory N signaling, the retinal defect of Nspl are strongly suppressed by conditions that attenuate E(spl) activity, such as halved dosage of Delta or E(spl), or by reduced CK2 activity (Kunttas-Tatli, 2009).
The dominant-negative effects of CK2a-M161K and CK2a-E165D in N1 and in Nspl animals are likely to involve the ability of either variant to robustly interact with CK2b and efficiently incorporate into the endogenous holoenzyme, in a manner akin to wild type CK2alpha. It is suggested that incorporation of the former variant attenuates endogenous CK2 activity. In contrast, the dominant-negative effects of the E165D substitution might not involve impaired CK2 kinase activity, but instead reflect its ability to perturb the interaction of endogenous CK2 with PP2A, an interaction that is increasingly suspect in the regulation of this protein phosphatase. These possibilities are addressed below (Kunttas-Tatli, 2009).
The effects of CK2alpha-M161K in N1 or in Nspl are easier to reconcile given its position in the ATP-binding site. This substitution substantially impairs kinase activity, and consequently ectopic CK2alpha-M161K mimics the neural defects of knockdown of this enzyme by RNAi. It would therefore seem to be the case that ectopic CK2alpha-M161K binds CK2beta, efficiently incorporates into the endogenous CK2-holoenzyme and attenuates activity, and this lowered activity impairs phosphorylation of, and repression by, endogenous E(spl). If so, this will reduce the 'strength' of inhibitory N signaling and elicit ectopic bristles in N1, and suppress the eye/R8 defects of Nspl. The effects of CK2alpha-M161K in these three developmental contexts are consistent with this model (Kunttas-Tatli, 2009).
However, the behavior of the E165D substitution was unexpected. The suggestion that this substitution exerts a negative impact on CK2 functions is supported by multiple findings, in addition to the extraordinary conservation of Glu165 in metazoan CK2alpha subunits. First, CK2alpha-E165D elicits ectopic bristles in N1 and suppresses the retinal defects of Nspl, and these effects are observed with multiple independent insertions and with multiple drivers. Second, CK2alpha-E165D restores eye size and the hexagonal phasing of the facets in Nspl, akin to Tik or CK2alpha-M161K. Third, CK2alpha-E165D appears to restore Ato expression anterior to the MF and increases the number of Sens-positive R8 cells at its posterior margin. Therefore, its effects closely correlate, in time and space, to R8 cell specification, which is defective in Nspl. Together, these results suggest that the E165D substitution impairs CK2 functions. These functions, however, might not involve perturbed kinase activity per se, but may instead be related to the interaction of this enzyme with PP2A (Kunttas-Tatli, 2009).
Studies with mts overexpression are of interest, because this is the first demonstration that increased dosage of the PP2A catalytic subunit elicits developmental defects that are hallmarks of loss of N functions. Specifically, mts overexpression elicits ectopic bristles and notched wings in N1 flies, and suppresses the retinal defects of Nspl. Furthermore, its effects on restored ommatidial phasing and eye size (facet numbers) are comparable to those seen with Tik, CK2alpha-M161K or CK2alpha-E165D. These studies lead to the suggestion that interaction of PP2A with CK2 down-regulates phosphatase activity, perhaps by competing with the regulatory subunit such as Wdb, which is essential for target recognition and dephosphorylation. Such a mechanism would reflect the mutually exclusive binding of the catalytic (Mts) subunit of PP2A with Wdb or SV40 t-antigen. If so, ectopic Mts would override the binding capacity of endogenous CK2, and upon recruiting Wdb attenuate repression by E(spl) through dephosphorylation (Kunttas-Tatli, 2009).
This model could account for the dominant-negative effects of CK2alpha-E165D. In this case, ectopic CK2alpha-E165D would bind CK2beta, incorporate into the endogenous CK2-holoenzyme, and impair PP2A binding and downregulation. Its effects should therefore mimic Mts overexpression, a proposal that is consistent with the findings. If so, overexpression of CK2-E165D probably leads to enhanced PP2A activity. In contrast, the effects of ectopic CK2alpha-M161K more likely reflect a negative influence on CK2 activity itself, and suggest that this variant may represent a more precise dominant-negative construct of CK2 (Kunttas-Tatli, 2009).
The possibility arises that a precise regulation of repression by E(spl) proteins involves a balance between the opposing activities of CK2 and PP2A, perhaps involving direct interactions. Indeed, direct interactions between CK2 and PP2A have been identified by proteomic analysis in the mouse model and in cultured cells. While consensus sequences for kinases are easier to identify computationally and biochemically, similar analysis with phosphatases has been less forthcoming. For example, in the case of Period (Per), the central clock protein, coordinated activities of CK2, CK1, and PP2A are required for proper function. While Per is phosphorylated by CK2 and CK1 in vitro and in vivo, evidence for its dephosphorylation by PP2A is lacking especially as it relates to its site preference(s). In the future it will be important to determine whether E(spl) proteins are direct targets of PP2A, and if so how a balance between PP2A and CK2 activities regulates repression. PP2A may play a similar role in the regulation of mammalian Hes6 (the homolog of fly M8), given its phosphorylation by CK2. A reversible switch could be important in neural patterning to confer a rapid and precise temporal control over the onset of repression, or prevent a protracted block to the neural fate once resolution of the PNC has occurred and the SOPs and R8s have been selected (Kunttas-Tatli, 2009).
Drosophila visual signaling, a G-protein-coupled phospholipase Cbeta (PLCbeta)-mediated mechanism, is regulated by eye-protein kinase C (PKC) that promotes light adaptation and fast deactivation, most likely via phosphorylation of the scaffolding protein inactivation no afterpotential D (INAD) and TRP (transient receptor potential). To reveal the critical phosphatases that dephosphorylate INAD, several biochemical analyses were used, and protein phosphatase 2A (PP2A) was identified as a candidate. Importantly, the catalytic subunit of PP2A, Microtubule star (MTS), is copurified with INAD, and an elevated phosphorylation of INAD by eye-PKC was observed in three mts heterozygotes. To explore whether PP2A (MTS) regulates dephosphorylation of INAD by counteracting eye-PKC [INAC (inactivation no afterpotential C] in vivo, ERG recordings were performed. inaCP209 was semidominant, because inaCP209 heterozygotes displayed abnormal light adaptation and slow deactivation. Interestingly, the deactivation defect of inaCP209 heterozygotes was rescued by the mtsXE2258 heterozygous background. In contrast, mtsXE2258 failed to modify the severe deactivation of norpAP16, indicating that MTS does not modulate NORPA (no receptor potential A) (PLCbeta). Together, these results strongly indicate that dephosphorylation of INAD is catalyzed by PP2A, and a reduction of PP2A can compensate for a partial loss of function in eye-PKC, restoring the fast deactivation kinetics in vivo. It is thus proposed that the fast deactivation of the visual response is modulated in part by the phosphorylation of INAD (Wang, 2008).
This study presents the results from several distinct biochemical and electrophysiological assays, each shedding light on the key role that PP2A plays in Drosophila visual transduction. In vitro assays show that PP2A dephosphorylates the scaffolding protein INAD, opposing the activity of eye-PKC to phosphorylate INAD. An increased level of INAD phosphorylation was shown in in three distinct mts heterozygotes, wherein the catalytic C subunit of PP2A has been rendered ineffective. Utilizing ERG recordings, in partial loss-of-function mutants of mtsXE2258, inaCP209, and the combined double mutant, it was found that PP2A and eye-PKC have opposing physiological functions, and that a balance between the activities of eye-PKC and PP2A is central for the proper deactivation of the visual response. These results strongly indicate that PP2A appears to impact signaling proteins operating downstream of NORPA in the visual cascade. Integrating in vivo and in vitro findings into the current model of eye-PKC-mediated regulation of INAD, it is concluded that reversible phosphorylation of INAD is dependent on the opposing enzymatic actions of eye-PKC and PP2A and that phosphorylation of INAD is critical for fast deactivation of the visual signaling process (Wang, 2008).
The multiple biochemical assays that were conducted strongly support PP2A as a key phosphatase responsible for the dephosphorylation of INAD. Based on both its inhibition profile with okadaic acid, and its copurification alongside the FPLC fraction with positive phosphatase activity, PP2A was identified as the prime candidate for mediating INAD dephosphorylation. After finding that a purified A/C dimer of PP2A dephosphorylates INAD in vitro, immunocomplex kinase assays were performed. As expected, a reduction in PP2A catalytic efficiency causes a significant increase in the measurable fraction of phosphorylated INAD. By examining three distinct mts heterozygotes, each carrying a C subunit mutation resulting in a partial loss of PP2A function, a significant increase was demonstrated in INAD phosphorylation levels with mtsXE2258 exhibiting the most dramatic increase. The enhanced INAD phosphorylation observed in the mts extracts strongly suggests that PP2A is closely positioned in the INAD complex to promote timely dephosphorylation of INAD. Consistently, it was demonstrated that PP2A can be coisolated with INAD, thus representing a newly identified component of the INAD macromolecular complex (Wang, 2008).
For insights into the role PP2A plays in Drosophila vision, mtsXE2258 heterozygotes were studied and a surprisingly normal ERG waveform was found. Although a reduction in the level of active PP2A in vivo has no effect on visual function, it was found that inaCP209 heterozygotes exhibit abnormal light adaptation, as well as delayed deactivation of visual signaling. inaCP209 and mtsXE2258 both encode for enzymes; why would missing one functional copy of the PP2A gene not affect normal visual electrophysiology, whereas missing a functional copy of the eye-PKC gene drastically slows deactivation? inaCP209 must be a semidominant mutation; in other words, inaC is haploinsufficient. An explanation for the haploinsufficiency of the eye-PKC gene is that the substrate repertoire of eye-PKC is defined by substrate colocalization to the INAD macromolecular complex to which eye-PKC is tethered. This hypothesis is in good agreement with the observation that the interaction with INAD is essential for the in vivo function of eye-PKC to modulate the visual response (Wang, 2008).
Although MTS is also tethered to the INAD signaling complex, unlike eye-PKC, the abundance of MTS relative to INAD in photoreceptors is likely to make it less sensitive to a reduction of its gene dosage to effect visual electrophysiology. Alternatively, it is possible that anchoring to the INAD complex by PP2A may be regulated by the interaction via its B subunit instead of the C subunit. Therefore, a reduction of MTS may not significantly modify its presence in the INAD complex (Wang, 2008).
To elucidate the functional interplay, in vivo, between eye-PKC and MTS, mtsXE2258 and inaCP209 double heterozygotes were characterized. A significant discovery was made that only the slow deactivation defect was rescued in the mtsXE2258 heterozygous background. The selective rescue of deactivation defects by mtsXE2258, with no rescue of the abnormal light adaptation found in inaCP209 heterozygotes, suggests that PP2A regulates proteins that lie downstream of eye-PKC, a conclusion that is also supported by the inability of mtsXE2258 to restore the slow deactivation defect in a hypomorphic allele of norpP16 (Wang, 2008).
A concomitant reduction of the PP2A activity would lead to increased phosphorylation of multiple substrates contributing to the observed normal, fast deactivation kinetics found in the double mutant. Potential PP2A substrates may include INAD, TRP, and eye-PKC. Like other conventional PKCs, eye-PKC is most likely a phosphoprotein and hence its catalytic activity is sensitive to PP2A. It is expected that a reduction of PP2A should increase the autophosphorylation of eye-PKC, bringing about an enhanced catalytic capability. However, the presumably increased eye-PKC activity fails to restore the light adaptation abnormality, suggesting that the modulation of eye-PKC represents a lesser role of PP2A in vivo (Wang, 2008).
It is possible that PP2A dephosphorylates TRP, thus regulating deactivation kinetics. Consistently, a lack of phosphorylation at Ser982 of TRP leads to slowed deactivation of the visual response. However, trpP343, a null allele affecting the trp gene, displays a less severe defect in deactivation than that of inaCP209, suggesting eye-PKC phosphorylation of TRP does not play a major role in the normal deactivation. The eye-PKC-dependent phosphorylation of INAD is being undertaken, and preliminary results suggest that a loss of phosphorylation in INAD also results in slowed deactivation kinetics. This finding together with the biochemical results supports the hypothesis that the phosphatase PP2A directly regulates phosphorylation states of INAD to impact fast deactivation of the visual signaling. A light-dependent conformation change has been reported in the fifth postsynaptic density-95/Discs large/zona occludens-1 (PDZ) domain of INAD and it has been proposed that eye-PKC might orchestrate this event. These findings are in agreement with the current studies supporting a critical role of INAD phosphorylation to promote fast deactivation. It will be of great interest to elucidate how phosphorylation of INAD leads to the conformation switch in its fifth PDZ domain (Wang, 2008).
In addition to INAD, PP2A may dephosphorylate other, yet-to-be identified substrates. The observations that mtsXE2258 modified the severe deactivation defect of inaCP209 homozygotes, suggests that a reduction of MTS increases phosphorylation of proteins, which are regulated by non-eye-PKC serine/threonine protein kinases. Several protein kinases including CaMKII, and NinaC (neither inactivation nor afterpotential C) have been shown to modulate deactivation, and may play a role in regulating deactivation in the absence of eye-PKC (Wang, 2008).
In summary, this study demonstrates the roles of PP2A and eye-PKC in orchestrating reversible phosphorylation of INAD, and that phosphorylation of INAD is most likely involved in fast deactivation kinetics of the visual signaling in Drosophila. The multiple biochemical findings support the critical role of PP2A to dephosphorylate INAD. Electrophysiological characterization strongly indicates that a reduction of PP2A compensate for a partial loss of function in eye-PKC leading to rescuing the slow deactivation defect (Wang, 2008).
Signaling by Hedgehog (Hh) proteins shapes most tissues and organs in both vertebrates and invertebrates, and its misregulation has been implicated in many human diseases. Although components of the signaling pathway have been identified, key aspects of the signaling mechanism and downstream targets remain to be elucidated. An enhancer/suppressor screen was performed in Drosophila to identify novel components of the pathway and 26 autosomal regions were identified that modify a phenotypic readout of Hh signaling. Three of the regions include genes that contribute constituents to the pathway: patched, engrailed, and hh. One of the other regions includes the gene microtubule star (mts) that encodes a subunit of protein phosphatase 2A. mts is necessary for full activation of Hh signaling. A second region includes the gene second mitotic wave missing (swm). swm is recessive lethal and is predicted to encode an evolutionarily conserved protein with RNA binding and Zn+ finger domains. Characterization of newly isolated alleles indicates that swm is a negative regulator of Hh signaling and is essential for cell polarity (Casso, 2008).
This screen identified twenty-six autosomal regions that modified a smo hypomorphic phenotype in a dosage-sensitive manner. Two aspects of its design were key to its success. First, its two-generation crossing scheme eliminated background effects by homogenizing the genetic backgrounds of both experimental and control flies. It also generated reasonably large numbers of both classes of progeny so that a good estimate of an average phenotype could be obtained. These features allowed monitoring of subtle variations in wing vein morphology, despite the significant strain differences among the many lines tested. Second, its high scoring threshold rendered it relatively insensitive to changes in Hh signaling strength, thereby helping to submerge weak influences. Key to this property was the ptcGAL4 driver that was used to express smo RNAi; it functioned in part as a 'genetic buffer.' Since ptcGAL4 is itself responsive to Hh, a modifier that increased Hh signaling would also be predicted to increase the expression of ptcGAL4 and smo RNAi, while a modifier that decreased Hh signaling might be expected to decrease the expression of the ptcGAL4 and smo RNAi. ptcGAL4 therefore buffered against changes in signaling strength and decreased the effects of genetic factors that enhance or suppress signaling; as a consequence, only highly penetrant and consistent phenotypes were scored (Casso, 2008).
The screen netted many of the known core components of the Hh signaling pathway, including smo, ptc, hh, and en. mts and swm were two genes whose haplo-insufficiency phenotypes were sufficiently strong to score above the threshold set by the genetic tests. Many other known regulators of Hh signaling were not identified in this screen. There are perhaps multiple reasons, including the high scoring threshold of the smo RNAi screen, or the possibility that not all pathway regulators have haplo-insufficiency phenotypes. skinny hedgehog or suppressor of fused were not included among those identified in the screen, despite the fact that deficiencies that removed them interacted with smo RNAi. The reason is that mutant alleles of these genes that were tested did not yield similar interaction phenotypes. Since many examples were observed of interaction between null alleles of Hh pathway regulators and smo RNAi but consistent failure of hypomorphic alleles to interact, lack of interaction is not viewed as evidence against a gene being a smo RNAi enhancer/suppressor. The possibility that stronger alleles might interact cannot be discounted. It was surprising that hemizygosity of cos2 did not show an interaction with smo RNAi. This could be because it is not haplo-insufficient in the particular assay or because of the complex positive and negative roles cos2 plays in Hh signaling. Finally, there was no apparent overlap between the regions identified and the mutant lines that were identified in previous screens for modifiers of Hh phenotypes; the smo RNAi assay may be less sensitive but more specific (Casso, 2008).
mts lies within 1 of 16 regions that enhanced the smo RNAi phenotype, suggesting that its wild-type function augments the Hh response. mts encodes the catalytic (C) subunit of PP2A, a heterotrimeric phosphatase that has two regulatory subunits, B and B'. It was previously identified as a Hh pathway regulator in CL8 cells (Nybakken, 2005); the current study provides in vivo evidence for a role in Hh signaling during development. Three proteins in Hh signal transduction have been shown to be functionally phosphorylated. Phosphorylation of the Smo C terminus is induced by Hh and is required for surface accumulation of Smo and normal activation of the pathway. Thus, reduction of PP2A activity and increased phosphorylation of Smo would not be expected to decrease Hh signaling and enhance the smo RNAi phenotype. Other possible targets of Mts are Ci and Cos2. Phosphorylation of Ci by PKA, casein kinase 1α, and GSK3β is required to convert Ci from its full-length form to its transcriptional repressor form, Ci-75. Hh signaling blocks this proteolytic transformation and also promotes conversion of Ci to an activator form. A decrease in phosphatase activity might increase levels of phosphorylated Ci to effect enhanced conversion to Ci-75 and reduced levels of Ci activator. Levels of Hh signaling would be predicted to decrease. Alternatively, Mts might control phosphorylation of Cos2 by Fu. Phosphorylation of Cos2 prevents its binding to Smo and release of Smo from Cos2 increases the cell surface accumulation of Smo that is necessary for pathway activation. Therefore, a reduction of Smo on the plasma membrane due to loss of PP2A activity might attenuate Hh pathway activation (Casso, 2008).
While the catalytic subunit of PP2A carries enzymatic phosphatase activity, the substrate specificity of PP2A is directed by its regulatory subunits. The phenotypes of mutants in genes that encode the B and B' regulatory subunits of PP2A, twins and widerborst (wdb), respectively, are interesting to consider in the context of Hh signaling. Wing discs in the twinsP mutant have mirror symmetrical posterior compartment duplications that are associated with ectopic compartment borders. Symmetric wing duplications have also been observed after ectopic expression of Hh or Dpp, or after loss of en/inv induces an ectopic compartment border. Since loss of PP2A function should reduce Hh signaling, it is not obvious how loss of the B twins regulatory subunit leads to an ectopic signaling center. Understanding this interesting aspect of the twins phenotype warrants further investigation (Casso, 2008).
Misexpression of PP2A can cause cell planar polarity defects in the wing. Misexpression of mts, wdb, or mutant alleles of these genes disrupted wing hair polarity. Like mts, reducing wdb expression with RNAi reduced Hh signaling in CL8 cells (Nybakken, 2005). This evidence, as well as the wing hair polarity phenotype of swm mutants, raises the possibility that PP2A links Hh signaling with cell polarity. The PCP and Hh pathways may be parallel and independent if PP2A activity is simply common to both, but evidence that Hh is required to establish PCP in the Drosophila embryonic and adult epidermis has recently been described. The phenotype of swm mutants provides additional evidence for an association of Hh signaling with cell polarity (Casso, 2008).
swm was first identified as l(2)37Dh in a screen for recessive lethal alleles within Df(2L)E55 (37D2-38A1). It was shown to exhibit synthetic lethality as an enhancer of Minutes. Among the mutant chromosomes from the current screen that failed to complement swm, one had a Minute-like phenotype. No changes in the swm coding sequence were found in this mutant; rare escapers that eclosed as heterozygotes with the verified swm alleles had a variety of phenotypes including loss of ocelli, thin macrochetae, and deformed legs. In contrast to swm mutant escapers, however, both their eyes and wings were phenotypically normal (Casso, 2008).
swm was identified as a suppressor of the roughex eye phenotype. Alleles of ptc were also isolated in this screen. These interactions between rux, ptc, and swm were confirmed. Since Ptc is a negative regulator of the Hh pathway and ptc mutations are therefore likely to elevate Hh signaling, and since Hh plays a key role in eye morphogenesis, the rux phenotype is apparently sensitive to Hh levels. Therefore the identification of both ptc and swm mutants as rux suppressors is interpreted as a consequence of the same mechanism -- an increase in Hh signaling caused by a decrease in the level of a negative regulator (Casso, 2008).
The results provide several additional lines of evidence that swm negatively regulates Hh signaling. swm mutants dominantly suppress smo hypomorphic phenotypes (smo RNAi and smo5A, enhance a Hh gain-of-function phenotype (hhMrt), and increase targets of Hh signaling such as Ptc and Ci. These effects on Hh signaling seem to occur through swm activity in the anterior compartment since swm RNAi expressed in these cells is sufficient to suppress smo RNAi. Although these interactions implicate Swm, it has not been determined how and where Swm impacts signal transduction or what its molecular function might be. Swm protein has features suggestive of a function in nucleic acid metabolism -- it has a putative RRM RNA binding domain and a CCCH Zn+ finger, and a GFP-Swm fusion that was examined localized to nuclei in cultured cells. Presumably, Swm affects expression, production, or presentation of proteins involved in Hh signaling or signal transduction. However, swm function is not specific to Hh signaling, since many aspects of the phenotype (e.g., ectopic venation, wing hair polarity, cell size, and interaction with Minutes) are not attributable to defects in Hh signaling (Casso, 2008).
swm is expressed broadly in both embryos and larvae, and in wing discs, it appears to be required in all cells. Null alleles, which are cell lethal in a swm/+ background, share some, but not all characteristics of Minute ribosomal protein mutants. Although swm mutants do not have thin bristles as is characteristic of Minutes, they are recessive lethal and developmentally delayed, and they interact genetically with Minutes and Minute-like loci. The wings of the Minute locus RpL38 have defects which are similar to swm wings -- extra venation, expanded distance between veins 3 and 4, wing hair polarity abnormalities, and increased cell size. Although RpL3845-72, Df(2R)M41A10, and M41A4 suppressed hhMrt, they did not interact with smo RNAi or smo5A (Casso, 2008).
While the interactions between swm and Minutes, as well as the similar phenotypes of swm and the RpL38 genes, might indicate a direct role in ribosome function, both Drosophila Swm and one of its two vertebrate homologs (RBM-27) are nuclear. The presence of RRM sequences in Swm and its homologs might suggest a role in RNA binding or metabolism, and the RRM of RBM-27 binds RNA. However, RRMs can have a structural role in protein-protein interactions independent of RNA binding, so the molecular function of Swm and its homologs cannot be determined by genetic methods alone. The fact is intriguing that the other vertebrate homolog, RBM-26, was identified as se70-2, an autoantigen that is recognized by sera of cutaneous T-cell lymphoma patients and has been used as a diagnostic marker for this tumor. In addition, the mouse RBM-26/se70-2 locus was identified as one of four genes deleted in a region required for normal murine skeletal, cartilage, and craniofacial development. Perhaps the roles of Hh that extend beyond pattern formation to cell cycle regulation, growth control, and cell polarity signify that Hh signal transduction integrates inputs from all three pathways. The pleiotropy of swm and mts may reflect these multiple inputs (Casso, 2008).
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).
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).
Approximately one-third of the Drosophila kinome has been ascribed some cell-cycle function. However, little is known about which of its 117 protein phosphatases (PPs) or subunits have counteracting roles. This study investigated mitotic roles of PPs through systematic RNAi. It was found that G2-M progression requires Puckered, the JNK MAP-kinase inhibitory phosphatase and PP2C in addition to string (Cdc25). Strong mitotic arrest and chromosome congression failure occurs after Pp1-87B downregulation. Chromosome alignment and segregation defects also occurs after knockdown of PP1-Flapwing, not previously thought to have a mitotic role. Reduction of several nonreceptor tyrosine phosphatases produced spindle and chromosome behavior defects, and for corkscrew, premature chromatid separation. RNAi of the dual-specificity phosphatase, Myotubularin, or the related Sbf 'antiphosphatase' resulted in aberrant mitotic chromosome behavior. Finally, for PP2A, knockdown of the catalytic or A subunits led to bipolar monoastral spindles, knockdown of the Twins B subunit led to bridged and lagging chromosomes, and knockdown of the B' Widerborst subunit led to scattering of all mitotic chromosomes. Widerborst is associated with MEI-S332 (Shugoshin) and is required for its kinetochore localization. This study has identified cell-cycle roles for 22 of 117 Drosophila PPs. Involvement of several PPs in G2 suggests multiple points for its regulation. Major mitotic roles are played by PP1 with tyrosine PPs and Myotubularin-related PPs having significant roles in regulating chromosome behavior. Finally, depending upon its regulatory subunits, PP2A regulates spindle bipolarity, kinetochore function, and progression into anaphase. Discovery of several novel cell-cycle PPs identifies a need for further studies of protein dephosphorylation (Chen, 2007).
P2A is a heterotrimeric serine/threonine phosphatase composed of invariant catalytic (C) and structural (A) subunits together with one member of a family of B regulatory subunits thought to direct the AC core to different substrates. The Drosophila gene for the catalytic subunit of type 2A protein serine/threonine phosphatase (PP2A) is known as microtubule star (mts) because mutant embryos show multiple individual centrosomes with disorganized astral arrays of microtubules. In agreement with this mutant phenotype, it was found that S2 cells depleted for Mts (PP2A-C) displayed aberrant elongated arrays of microtubules with a high proportion (5- to 10-fold increase over the control) of bipolar monoastral spindles or monopolar spindles emanating from a single centrosomal mass. This phenotype is also consistent with the observations in Xenopus egg extracts where mitotic microtubules grow longer and bipolar spindles can not be assembled after inhibition of PP2A by low concentrations of okadaic acid (OA). It is speculated that the monopolar spindle phenotype after mts dsRNA treatment is a consequence of the spindle collapse rather than a failure in centrosome duplication or separation because most of the RNAi-treated cells showed well-separated centrosomes during prophase. In support of this view, spindle bipolarity can be rescued by restoration of microtubule dynamics in OA-treated Xenopus egg extracts (Chen, 2007).
In Drosophila, as in many other eukaryotes, mitosis-specific phosphorylation of histone H3 requires Aurora B activity, but the identity of the opposing phosphatase remains unclear. Because P-H3 (Ser 10) levels were used for monitoring the mitotic index in this analysis, it is possible that a high mitotic index observed after RNAi for PPs may also reflect a defect in dephosphorylating P-H3 in the absence of PPs upon mitotic exit. The phosphorylation state of this histone was therefore studied after RNAi for PPs that displayed a significant increase in the mitotic index in the screen. The immunostaining of control cells showed that P-H3 signals began to decrease at early telophase and then disappeared completely at late telophase. After RNAi knockdown of Mts (PP2A-C) or Pp1-87B, however, the majority of mitotic cells were arrested at prometaphase, but late telophase figures could occassionally be found showing an abnormal accumulation of P-H3 on decondensed chromosomes. To better assess the effect of depletion of these two PPs on P-H3 dephosphorylation, the spindle-assembly checkpoint was inactivated by simultaneously knocking down BubR1. It was found that this rescued the prometaphase arrest of cells simultaneously depleted for Mts or Pp1-87B; this allowed a study of telophase cells. P-H3 was present in the majority of such telophase cells compared to control cells, indicating that both PPs are required for P-H3 dephosphorylation. These results are in accordance with previous studies showing that reduction of PP1 activity can partially suppress defects in the mitotic histone H3 phosphorylation in yeast and C. elegans (Chen, 2007).
Downregulation of Pp2A-29B, the structural A subunit, revealed almost identical aberrant phenotypes to those observed after mts (PP2A-C) RNAi. Consistently, knockdown of Pp2A-29B (PP2A-A) led to a reduction of the protein level of Mts (PP2A-C) (Chen, 2007).
The Drosophila genome contains 4 B-type PP2A regulatory subunits, twins/tws/aar (B sub-type), widerborst/wdb (B' sub-type), Pp2A-B' (B' sub-type), and Pp2A-B" (B" sub-type), but mitotic defects have so far only been reported for mutants of tws. Consistent with the phenotype of tws mutants, it was observed that RNAi for this gene led to an increased proportion of anaphase figures showing lagging chromosomes and chromosome bridges (Chen, 2007).
In metazoans, the B' regulatory subunits of PP2A have evolved into two related subclasses with conserved central regions and diverged amino and carboxy termini. The protein encoded by widerborst (wdb) is more closely related to the human α and ɛ subunits (79%-80% identity) than to the β, γ, or δ subunits (69%-75% identity). Whereas RNAi for tws led to lagging chromosomes, wdb RNAi led to dramatic scattering of chromosomes throughout the spindle. Whether this dramatic effect of wdb RNAi on chromosome segregation reflected any particular subcellular localization of this regulatory subunit was examined. To this end, a GFP-tagged Wdb was expressed in S2 cells. During interphase and prophase, Wdb::GFP partially colocalized with the centromeric marker CID (CENP-A). After spindle formation, Wdb::GFP was found adjacent and external to the centromeres. Although less pronounced, this distribution remained during chromosome segregation at anaphase. Because MEI-S332 (Drosophila Shugoshin) is a dynamic centromeric marker, its distribution was examined in wdb RNAi cells. In control cells, MEI-S332 localized in a band between each pair of the centromeres at metaphase. After downregulation of wdb, however, greatly reduced MEI-S332 staining was found on the metaphase chromosome. In contrast, depletion of MEI-S332 by RNAi did not affect the normal localization of the Wdb B' PP2A subunit. Thus, it is concluded that the Wdb B' subunit is required for correct localization of MEI-S332 but not vice versa. Whether the two proteins existed in the same complex was examined. To address this, a Protein A (PrA)-tagged form of MEI-S332 was expressed in S2 cells to purify potential protein complexes and identify its components by mass spectrometry. The catalytic C (Mts), the structural A (PP2A-29B), and the regulatory B' (Wdb) and B (Tws) subunits of PP2A were identified associated with MEI-S332. Three recent studies also identified PP2A complexed to the B' subunit bound to Shugoshin (Sgo) in human and yeast cells, where they are thought to protect centromeric cohesin subunits from phosphorylation that would promote premature sister-chromatid separation. As with the archetypal family member, Drosophila MEI-S332, the Shugoshins function primarily to protect sister chromatids from separation in the first meiotic division but are also present in mitotic divisions. Consistent with these observations in Drosophila S2 cells, it has been found that depletion of PP2A in human cells led to premature dissociation of Shugoshin 1 (Sgo1) from the kinetochore and loss of mitotic centromere cohesion. The finding of Shugoshin complexed to PP2A/B' in yeast and human, and now in Drosophila, points to a highly evolutionally conserved role for this particular PP2A heterotrimer in regulating sister-chromatid cohesion. Interestingly, Tws B regulatory subunit was also recovered associated with MEI-S332. How this subunit of PP2A might function with MEI-S332 should be the subject of future investigations (Chen, 2007).
Only a moderatedly elevated mitotic index (by approximately 10%) was observed after downregulation of the second Drosophila B' regulatory subunit (Pp2A-B'/B56-1). However, when this second B' subunit was simultaneously knocked down with Wdb, this led to similar phenotypes seen in Mts (PP2A-C) or Pp2A-29B (PP2A-A)-depleted cells. Western-blot analysis showed that the Mts (PP2A-C) level decreased after simultaneous knockdown of both B' subunits, suggesting that this phenotype could be partially due to the loss of PP2A catalytic subunit, although the possibility that the two B' subunits share partially redundant mitotic functions cannot be excluded (Chen, 2007).
Cell-cycle kinases represent a large family of enzymes governing the cell division cycle. It is therefore not surprising that a considerable number of counteracting cell-cycle phosphatases (19% of the genes for tested) were identified in the current study. In addition to finding all the well-known PPs required for cell-cycle progression in Drosophila (Mts, Tws, String, Pp4-19C, and Pp1-87B), the Drosophila counterparts of some eight PPs implicated in cell-cycle functions were identified from studies on other organisms together with six PPs for which novel cell-cycle roles were ascribe. These results were validated by confirming the observed phenotypes with a second nonoverlapping dsRNA. In two cases (flw and csw), their mitotic roles were confirmed through the analysis of phenotypes in mutant larval neuroblasts. The RNAi phenotypes of catalytic subunits were evaluated by observing similar phenotypes after downregulation of the corresponding regulatory subunits (e.g., Pp4-19C and PPP4R2r, Mts/PP2A-C and Pp2A-29B/PP2A-A, and simultaneous RNAi of the two PP2A-B' regulatory subunits). Although a recent large-scale RNAi screen based solely on flow cytometry in Drosophila S2 cells identified many regulators of the cell cycle, cell size, and cell death, this study showed a very low degree of overlap with the current analysis (only six), reflecting the need for more sensitive small-scale screens that can examine the functional requirements of assayed proteins in greater detail. These results have provided novel insights into the cell-cycle functions of the Drosophila PPs, and it is likely that, in many cases, these functions have been conserved in other metazoans including humans. This study should guide future work aimed at elucidating the significance and mechanisms of the balanced activities of PKs and PPs in regulating the cell division cycle. The challenge ahead will be to match up the functions of the PPs that were identified with their corresponding counteracting PKs and to identify their common key substrates (Chen, 2007).
Multiple regulatory mechanisms control the activity of the protein serine/threonine phosphatase 2A catalytic subunit (PP2Ac), including post-translational modifications and its association with regulatory subunits and interacting proteins. Alpha4 is a PP2Ac-interacting protein that is hypothesized to play a role in PP2Ac ubiquitination via its interaction with the E3 ubiquitin ligase Mid1, which targets phosphatase 2A for degradation. This report shows that murine alpha4 serves as a necessary adaptor protein that provides a binding platform for both PP2Ac and Mid1. Aa novel ubiquitin-interacting motif (UIM) was identified within alpha4 (amino acid residues 46-60), and the interaction between alpha4 and ubiquitin was analyze using NMR. Consistent with other UIM-containing proteins, alpha4 is monoubiquitinated. Interestingly, deletion of the UIM within alpha4 enhances its association with polyubiquitinated proteins. Lastly, it was demonstrated that addition of wild-type alpha4 but not an alpha4 UIM deletion mutant suppresses PP2Ac polyubiquitination. Thus, the polyubiquitination of PP2Ac is inhibited by the UIM within alpha4. These findings reveal direct regulation of PP2Ac polyubiquitination by a novel UIM within the adaptor protein alpha4 (McConnell, 2010).
Search PubMed for articles about Drosophila Microtubule star
Bielinski, V. A. and Mumby, M. C. (2007). Functional analysis of the PP2A subfamily of protein phosphatases in regulating Drosophila S6 kinase. Exp Cell Res 313: 3117-3126. PubMed ID: 17570358
Casso, D. J., (2008). A screen for modifiers of Hedgehog signaling in Drosophila melanogaster identifies swm and mts. Genetics 178: 1399-1413. PubMed ID: 18245841
Chabu, C. and Doe, C. Q. (2008). Dap160/intersectin binds and activates aPKC to regulate cell polarity and cell cycle progression. Development 135(16): 2739-46. PubMed ID: 18614576
Chabu, C. and Doe, C. Q. (2009). Twins/PP2A regulates aPKC to control neuroblast cell polarity and self-renewal. Dev. Biol. 330(2): 399-405. PubMed ID: 19374896
Chen, F., et al. (2007). Multiple protein phosphatases are required for mitosis in Drosophila. Curr. Biol. 17: 293-303. PubMed ID: 17306545
Cygnar, K. D., Gao, X., Pan, D. and Neufeld, T. P. (2005). The phosphatase subunit tap42 functions independently of target of rapamycin to regulate cell division and survival in Drosophila. Genetics 170: 733-740. PubMed ID: 15802506
Eichhorn, P. J., Creyghton, M. P. and Bernards, R. (2009). Protein phosphatase 2A regulatory subunits and cancer. Biochim. Biophys. Acta 1795: 1-15. PubMed ID: 18588945
Hannus, M., Feiguin, F., Heisenberg, C. P. and Eaton, S. (2002). Planar cell polarization requires Widerborst, a B' regulatory subunit of protein phosphatase 2A. Development 129: 3493-3503. PubMed ID: 12091318
Jia, H., Liu, Y., Yan, W. and Jia, J. (2009). PP4 and PP2A regulate Hedgehog signaling by controlling Smo and Ci phosphorylation. Development 136: 307-316. PubMed ID: 19088085
Junttila, M. R., Puustinen, P., Niemela, M., Ahola, R., Arnold, H., Bottzauw, T., Ala-aho, R., Nielsen, C., Ivaska, J., Taya, Y. et al. (2007). CIP2A inhibits PP2A in human malignancies. Cell 130: 51-62. PubMed ID: 17632056
Kong, M., Fox, C. J., Mu, J., Solt, L., Xu, A., Cinalli, R. M., Birnbaum, M. J., Lindsten, T. and Thompson, C. B. (2004). The PP2A-associated protein alpha4 is an essential inhibitor of apoptosis. Science 306: 695-698. PubMed ID: 15499020
Krahn, M. P., Egger-Adam, D. and Wodarz, A. (2009). PP2A antagonizes phosphorylation of Bazooka by PAR-1 to control apical-basal polarity in dividing embryonic neuroblasts. Dev. Cell 16(6): 901-8. PubMed ID: 19531360
Kunttas-Tatli, E., Bose, A., Kahali, B., Bishop, C. P. and Bidwai, A. P. (2009). Functional dissection of Timekeeper (Tik) implicates opposite roles for CK2 and PP2A during Drosophila neurogenesis. Genesis [Epub ahead of print]. PubMed ID: 19536808
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date revised: 21 November 2016
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