Defining Components of the ?catenin Destruction Complex and Exploring Its Regulation and Mechanisms of Action during Development

Background

A subset of signaling pathways play exceptionally important roles in embryonic and post-embryonic development, and mis-regulation of these pathways occurs in most human cancers. One such pathway is the Wnt pathway. The primary mechanism keeping Wnt signaling off in the absence of ligand is regulated proteasomal destruction of the canonical Wnt effector ßcatenin (or its fly homolog Armadillo). A substantial body of evidence indicates that SCF^βTrCP mediates βcat destruction, however, an essential role for Roc1 has not been demonstrated in this process, as would be predicted. In addition, other E3 ligases have also been proposed to destroy βcat, suggesting that βcat destruction may be regulated differently in different tissues.

Methodology/Principal Findings

Here we used cultured Drosophila cells, human colon cancer cells, and Drosophila embryos and larvae to explore the machinery that targets Armadillo for destruction. Using RNAi in Drosophila S2 cells to examine which SCF components are essential for Armadillo destruction, we find that Roc1/Roc1a is essential for regulating Armadillo stability, and that in these cells the only F-box protein playing a detectable role is Slimb. Second, we find that while embryonic and larval Drosophila tissues use the same destruction complex proteins, the response of these tissues to destruction complex inactivation differs, with Armadillo levels more elevated in embryos. We provide evidence consistent with the possibility that this is due to differences in armadillo mRNA levels. Third, we find that there is no correlation between the ability of different APC2 mutant proteins to negatively regulate Armadillo levels, and their recently described function in positively-regulating Wnt signaling. Finally, we demonstrate that APC proteins lacking the N-terminal Armadillo-repeat domain cannot restore Armadillo destruction but retain residual function in negatively-regulating Wnt signaling.

Conclusions/Significance

We use these data to refine our model for how Wnt signaling is regulated during normal development.     

The SCFSlimb E3 ligase complex regulates asymmetric division to inhibit neuroblast overgrowth

Drosophila larval brain neuroblasts divide asymmetrically to balance between self‐renewal and differentiation. Here, we demonstrate that the SCF^Slimb E3 ubiquitin ligase complex, which is composed of Cul1, SkpA, Roc1a and the F‐box protein Supernumerary limbs (Slimb), inhibits ectopic neuroblast formation and regulates asymmetric division of neuroblasts. Hyperactivation of Akt leads to similar neuroblast overgrowth and defects in asymmetric division. Slimb associates with Akt in a protein complex, and SCF^Slimb acts through SAK and Akt to inhibit neuroblast overgrowth. Moreover, Beta‐transducin repeat containing, the human ortholog of Slimb, is frequently deleted in highly aggressive gliomas, suggesting a conserved tumor suppressor‐like function.     

Cleavage of the Drosophila screw prodomain is critical for a dynamic BMP morphogen gradient in embryogenesis

Dorsoventral patterning of the Drosophila embryo is regulated by graded distribution of bone morphogenetic proteins (BMPs) composed of two ligands, decapentaplegic (Dpp) a BMP2/4 ortholog and screw (Scw) a BMP5/6/7/8 family member. scw^E1 encodes an unusual allele that was isolated as a dominant enhancer of partial loss-of-function mutations in dpp. However, the molecular mechanisms that underlie this genetic interaction remain to be addressed. Here we show that scw^E1 contains a mutation at the furin cleavage site within the prodomain that is crucial for ligand production. Furthermore, our data show that Scw^E1 preferentially forms heterodimers with Dpp rather than homotypic dimers, providing a possible explanation for the dominant negative phenotype of scw^E1 alleles. The unprocessed prodomain of Scw^E1 remains in a complex with the Dpp:Scw heterodimer, and thus could interfere with interaction of the ligand with the extracellular matrix, or the kinetics of processing/secretion of the ligand in vivo. These data reveal novel mechanisms by which post-translational regulation of Scw can modulate Dpp signaling activity.     

The drosophila Bcl-2 family protein Debcl is targeted to the proteasome by the β-TrCP homologue slimb

The ubiquitin–proteasome system is one of the main proteolytic pathways. It inhibits apoptosis by degrading pro-apoptotic regulators, such as caspases or the tumor suppressor p53. However, it also stimulates cell death by degrading pro-survival regulators, including IAPs. In Drosophila, the control of apoptosis by Bcl-2 family members is poorly documented. Using a genetic modifier screen designed to identify regulators of mammalian bax-induced apoptosis in Drosophila, we identified the ubiquitin activating enzyme Uba1 as a suppressor of bax-induced cell death. We then demonstrated that Uba1 also regulates apoptosis induced by Debcl, the only counterpart of Bax in Drosophila. Furthermore, we show that these apoptotic processes involve the same multimeric E3 ligase—an SCF complex consisting of three common subunits and a substrate-recognition variable subunit identified in these processes as the Slimb F-box protein. Thus, Drosophila Slimb, the homologue of β-TrCP targets Bax and Debcl to the proteasome. These new results shed light on a new aspect of the regulation of apoptosis in fruitfly that identifies the first regulation of a Drosophila member of the Bcl-2 family.     

Design and Analysis of Temperature Preference Behavior and its Circadian Rhythm in Drosophila

The circadian clock regulates many aspects of life, including sleep, locomotor activity, and body temperature (BTR) rhythms^1,2. We recently identified a novel Drosophila circadian output, called the temperature preference rhythm (TPR), in which the preferred temperature in flies rises during the day and falls during the night ³. Surprisingly, the TPR and locomotor activity are controlled through distinct circadian neurons³. Drosophila locomotor activity is a well known circadian behavioral output and has provided strong contributions to the discovery of many conserved mammalian circadian clock genes and mechanisms⁴. Therefore, understanding TPR will lead to the identification of hitherto unknown molecular and cellular circadian mechanisms. Here, we describe how to perform and analyze the TPR assay. This technique not only allows for dissecting the molecular and neural mechanisms of TPR, but also provides new insights into the fundamental mechanisms of the brain functions that integrate different environmental signals and regulate animal behaviors. Furthermore, our recently published data suggest that the fly TPR shares features with the mammalian BTR³. Drosophila are ectotherms, in which the body temperature is typically behaviorally regulated. Therefore, TPR is a strategy used to generate a rhythmic body temperature in these flies^5-8. We believe that further exploration of Drosophila TPR will facilitate the characterization of the mechanisms underlying body temperature control in animals.     

Expression of a heat-inducible gene in transfected mosquito cells

The evolutionary conservation of the heat shock response suggests that plasmids containing promoters from Drosophila heat shock protein (hsp) genes will be useful in the development of gene transfer procedures for cell lines representing a variety of insect species. Conditions for induction of endogenous hsp genes and for expression of the chloramphenicol acetyltransferase (CAT) gene regulated by the Drosophila hsp 70 promoter were examined in Aedes albopictus (mosquito) cells. Five hsps, ranging in size from 27,000 to 90,000 D, were induced in A. albopictus cells during incubation at 41°C in medium containing [³⁵S]methionine. Relative synthesis of these proteins at 37 and 41°C indicated that Aedes hsp 66 is homologous to Drosophila hsp 70. Detection of CAT activity in transfected mosquito cells was enhanced 10-fold under heat shock conditions (6 h, 41°C) based on maximal expression of hsp 66, relative to conditions defined for expression of hsp 70 in Drosophila cells. Analysis of the endogenous heat shock response may be essential to the optimal use of plasmids containing the Drosophila hsp 70 promoter with other insect cell types.     

Dual DNA-binding domains shape the interaction of Brh2 with DNA

Brh2, the BRCA2 ortholog in the fungus Ustilago maydis, harbors two different DNA-binding domains, one located in the N-terminal region and the other located in the C-terminal region. Here we were interested in comparing the biochemical properties of Brh2 fragments, Brh2^NT and Brh2^CT, respectively, harboring the two different DNA-binding regions to understand the mechanistic purpose of dual DNA-interaction domains. With oligonucleotide substrates to model different DNA conformations, it was found that the substrate specificity of Brh2^NT and Brh2^CT was almost indistinguishable although avidity was different depending on salt concentration. DNA annealing activity inherent in Brh2 was found to be attributable to Brh2^NT. Likewise, activity responsible for a second-end capture reaction modeling a later step in repair of DNA double-strand breaks was found attributable to Brh2^NT. Efficient annealing of DNA strands coated with RPA required full length Brh2 rather than Brh2^NT suggesting Brh2^CT contributes to the activity when RPA is present. Brh2^NT and Brh2^CT were both found capable of physically interacting with RPA. The results suggest that while the two DNA-binding regions of Brh2 appear functionally redundant in certain aspects of DNA repair, they differ in fundamental properties, and likely contribute in different ways to repair processes involving or arising from stalled DNA replication forks.     

Regulation of the Skeletal Muscle Ryanodine Receptor/Ca2+-release Channel RyR1 by S-Palmitoylation

The ryanodine receptor/Ca²⁺-release channels (RyRs) of skeletal and cardiac muscle are essential for Ca²⁺ release from the sarcoplasmic reticulum that mediates excitation-contraction coupling. It has been shown that RyR activity is regulated by dynamic post-translational modifications of Cys residues, in particular S-nitrosylation and S-oxidation. Here we show that the predominant form of RyR in skeletal muscle, RyR1, is subject to Cys-directed modification by S-palmitoylation. S-Palmitoylation targets 18 Cys within the N-terminal, cytoplasmic region of RyR1, which are clustered in multiple functional domains including those implicated in the activity-governing protein-protein interactions of RyR1 with the L-type Ca²⁺ channel Ca_V1.1, calmodulin, and the FK506-binding protein FKBP12, as well as in “hot spot” regions containing sites of mutations implicated in malignant hyperthermia and central core disease. Eight of these Cys have been identified previously as subject to physiological S-nitrosylation or S-oxidation. Diminishing S-palmitoylation directly suppresses RyR1 activity as well as stimulus-coupled Ca²⁺ release through RyR1. These findings demonstrate functional regulation of RyR1 by a previously unreported post-translational modification and indicate the potential for extensive Cys-based signaling cross-talk. In addition, we identify the sarco/endoplasmic reticular Ca²⁺-ATPase 1A and the α_1S subunit of the L-type Ca²⁺ channel Ca_V1.1 as S-palmitoylated proteins, indicating that S-palmitoylation may regulate all principal governors of Ca²⁺ flux in skeletal muscle that mediates excitation-contraction coupling.     

The ubiquitous mitochondrial protein unfoldase CLPX regulates erythroid heme synthesis by control of iron utilization and heme synthesis enzyme activation and turnover

Heme plays a critical role in catalyzing life-essential redox reactions in all cells, and its synthesis must be tightly balanced with cellular requirements. Heme synthesis in eukaryotes is tightly regulated by the mitochondrial AAA+ unfoldase CLPX (caseinolytic mitochondrial matrix peptidase chaperone subunit X), which promotes heme synthesis by activation of δ-aminolevulinate synthase (ALAS/Hem1) in yeast and regulates turnover of ALAS1 in human cells. However, the specific mechanisms by which CLPX regulates heme synthesis are unclear. In this study, we interrogated the mechanisms by which CLPX regulates heme synthesis in erythroid cells. Quantitation of enzyme activity and protein degradation showed that ALAS2 stability and activity were both increased in the absence of CLPX, suggesting that CLPX primarily regulates ALAS2 by control of its turnover, rather than its activation. However, we also showed that CLPX is required for PPOX (protoporphyrinogen IX oxidase) activity and maintenance of FECH (ferrochelatase) levels, which are the terminal enzymes in heme synthesis, likely accounting for the heme deficiency and porphyrin accumulation observed in Clpx^−/− cells. Lastly, CLPX is required for iron utilization for hemoglobin synthesis during erythroid differentiation. Collectively, our data show that the role of CLPX in yeast ALAS/Hem1 activation is not conserved in vertebrates as vertebrates rely on CLPX to regulate ALAS turnover as well as PPOX and FECH activity. Our studies reveal that CLPX mutations may cause anemia and porphyria via dysregulation of ALAS, FECH, and PPOX activities, as well as of iron metabolism.     

A genetic screen in Drosophila for regulators of human prostate cancer progression

To uncover the mechanism by which human prostate cancer progresses, we performed a genetic screen for regulators of human prostate cancer progression using the Drosophila accessory gland, a functional homolog of the mammalian prostate. Cell growth and migration of secondary cells in the adult male accessory gland were found to be regulated by paired, N-cadherin, and E-cadherin, which are Drosophila homologues of regulators of human prostate cancer progression. Using this screening system, we also identified three genes that promoted growth and migration of secondary cells in the accessory gland. The human homologues of these candidate genes – MRGBP, CNPY2, and MEP1A – were found to be expressed in human prostate cancer model cells and to promote replication and invasiveness in these cells. These findings suggest that the development of the Drosophila accessory gland and human prostate cancer cell growth and invasion are partly regulated through a common mechanism. The screening system using the Drosophila accessory gland can be a useful tool for uncovering the mechanisms of human prostate cancer progression.     

Calcineurin and its regulation by Sra/RCAN is required for completion of meiosis in Drosophila

Ca²⁺ signaling pathways play important roles to complete meiosis from metaphase II arrest in vertebrate oocytes. However, less is known about the molecular mechanism of completion of meiosis in Drosophila females. Here, we provide direct evidence that calcineurin, a Ca²⁺/calmodulin (CaM)-dependent phosphatase, is essential for meiotic progression beyond metaphase I in Drosophila oocytes. Oocytes from germline clones lacking CanB2, a calcineurin regulatory subunit B, failed to complete meiosis after egg activation, and laid eggs exhibited a meiotic arrested anaphase I chromosome configuration. Genetic analyses suggest that calcineurin activity is regulated by Sarah (Sra), a family member of regulators of calcineurin (RCANs), through a Sra phosphorylation-dependent mechanism. Our results support a view in which the phosphorylation of Sra not only acts to relieve the inhibitory effects of Sra, but also acts to activate calcineurin, thus explaining the role of RCAN proteins as positive regulators of calcineurin.     

Post-translational control of nitrate reductase activity responding to light and photosynthesis evolved already in the early vascular plants

Regulation of nitrate reductase (NR) by reversible phosphorylation at a conserved motif is well established in higher plants, and enables regulation of NR in response to rapid fluctuations in light intensity. This regulation is not conserved in algae NR, and we wished to test the evolutionary origin of the regulatory mechanism by physiological examination of ancient land plants. Especially a member of the lycophytes is of interest since their NR is candidate for regulation by reversible phosphorylation based on sequence analysis. We compared Selaginella kraussiana, a member of the lycophytes and earliest vascular plants, with the angiosperm Arabidopsis thaliana, and also tested the moss Physcomitrella patens. Interestingly, optimization of assay conditions revealed that S. kraussiana NR used NADH as an electron donor like A. thaliana, whereas P. patens NR activity depended on NADPH. Examination of light/darkness effects showed that S. kraussiana NR was rapidly regulated similar to A. thaliana NR when a differential (Mg²⁺ contra EDTA) assay was used to reveal activity state of NR. This implies that already existing NR enzyme was post-translationally activated by light in both species. Light had a positive effect also on de novo synthesis of NR in S. kraussiana, which could be shown after the plants had been exposed to a prolonged dark period (7 days). Daily variations in NR activity were mainly caused by post-translational modifications. As for angiosperms, the post-translational light activation of NR in S. kraussiana was inhibited by 3-(3,4-dichlorophenyl)-1*1-dimethylurea (DCMU), an inhibitor of photosynthesis and stomata opening. Evolutionary, a post-translational control mechanism for NR have occurred before or in parallel with development of vascular tissue in land plants, and appears to be part of a complex mechanisms for coordination of CO₂ and nitrogen metabolism in these plants.     

Calcium-regulated GTPase activity in the calcium-binding protein calexcitin

Calexcitin (CE) is a calcium-binding protein, closely related to sarcoplasmic calcium-binding proteins, that is involved in invertebrate learning and memory. Early reports indicated that both Hermissenda and squid CE also could bind GTP; however, the biochemical significance of GTP-binding and its relationship to calcium binding have remained unclear. Here, we report that the GTPase activity of CE is strongly regulated by calcium. CE possessed a P-loop-like structure near the C-terminal similar to the phosphate-binding regions in other GTP-binding proteins. Site-directed mutagenesis of this region showed that Gly¹⁸², Phe¹⁸⁶ and Gly¹⁸⁷ are required for maximum affinity, suggesting that the GTP-binding motif is G-N-x-x-[FM]-G. CE cloned from Drosophila CNS possessed a similar C-terminal sequence and also bound and hydrolyzed GTP. GTPase activity in Drosophila CE was also strongly regulated by Ca²⁺, exhibiting over 23-fold higher activity in the presence of 0.3 μM calcium. Analysis of the conserved protein motifs defines a new family of Ca²⁺-binding proteins representing the first example of proteins endowed with both EF-hand calcium binding domains and a C-terminal, P-loop-like GTP-binding motif. These results establish that, in the absence of calcium, both squid and Drosophila CE bind GTP at near-physiological concentrations and hydrolyze GTP at rates comparable to unactivated ras. Calcium functions to increase GTP-binding and GTPase activity in CE, similar to the effect of GTPase activating proteins in other low-MW GTP-binding proteins. CE may, therefore, act as a molecular interface between Ca²⁺ cytosolic oscillations and the G protein-coupled signal transduction.     

Cold stress causes rapid but differential changes in properties of plasma membrane H-ATPase of camelina and rapeseed

Camelina (Camelina sativa) and rapeseed (Brassica napus) are well-established oil-seed crops with great promise also for biofuels. Both are cold-tolerant, and camelina is regarded to be especially appropriate for production on marginal lands. We examined physiological and biochemical alterations in both species during cold stress treatment for 3 days and subsequent recovery at the temperature of 25 °C for 0, 0.25, 0.5, 1, 2, 6, and 24 h, with particular emphasis on the post-translational regulation of the plasma membrane (PM) H⁺-ATPase (EC3.6.3.14). The activity and translation of the PM H⁺-ATPase, as well as 14-3-3 proteins, increased after 3 days of cold stress in both species but recovery under normal conditions proceeded differently. The increase in H⁺-ATPase activity was the most dramatic in camelina roots after recovery for 2 h at 25 °C, followed by decay to background levels within 24 h. In rapeseed, the change in H⁺-ATPase activity during the recovery period was less pronounced. Furthermore, H⁺-pumping increased in both species after 15 min recovery, but to twice the level in camelina roots compared to rapeseed. Protein gel blot analysis with phospho-threonine anti-bodies showed that an increase in phosphorylation levels paralleled the increase in H⁺-transport rate. Thus our results suggest that cold stress and recovery in camelina and rapeseed are associated with PM H⁺-fluxes that may be regulated by specific translational and post-translational modifications.     

A Follicle Rupture Assay Reveals an Essential Role for Follicular Adrenergic Signaling in Drosophila Ovulation

Ovulation is essential for the propagation of the species and involves a proteolytic degradation of the follicle wall for the release of the fertilizable oocyte. However, the precise mechanisms for regulating these proteolytic events are largely unknown. Work from our lab and others have shown that there are several parallels between Drosophila and mammalian ovulation at both the cellular and molecular levels. During ovulation in Drosophila, posterior follicle cells surrounding a mature oocyte are selectively degraded and the residual follicle cells remain in the ovary to form a corpus luteum after follicle rupture. Like in mammals, this rupturing process also depends on matrix metalloproteinase 2 (Mmp2) activity localized at the posterior end of mature follicles, where oocytes exit. In the present study, we show that Mmp2 activity is regulated by the octopaminergic signaling in mature follicle cells. Exogenous octopamine (OA; equivalent to norepinephrine, NE) is sufficient to induce follicle rupture when isolated mature follicles are cultured ex vivo, in the absence of the oviduct or ovarian muscle sheath. Knocking down the alpha-like adrenergic receptor Oamb (Octoampine receptor in mushroom bodies) in mature follicle cells prevents OA-induced follicle rupture ex vivo and ovulation in vivo. We also show that follicular OA-Oamb signaling induces Mmp2 enzymatic activation but not Mmp2 protein expression, likely via intracellular Ca²⁺ as the second messenger. Our work develops a novel ex vivo follicle rupture assay and demonstrates the role for follicular adrenergic signaling in Mmp2 activation and ovulation in Drosophila, which is likely conserved in other species.     

Drosophila TRPML Forms PI(3,5)P2-activated Cation Channels in Both Endolysosomes and Plasma Membrane

Transient Receptor Potential mucolipin (TRPML) channels are implicated in endolysosomal trafficking, lysosomal Ca²⁺ and Fe²⁺ release, lysosomal biogenesis, and autophagy. Mutations in human TRPML1 cause the lysosome storage disease, mucolipidosis type IV (MLIV). Unlike vertebrates, which express three TRPML genes, TRPML1–3, the Drosophila genome encodes a single trpml gene. Although the trpml-deficient flies exhibit cellular defects similar to those in mammalian TRPML1 mutants, the biophysical properties of Drosophila TRPML channel remained uncharacterized. Here, we show that transgenic expression of human TRPML1 in the neurons of Drosophila trpml mutants partially suppressed the pupal lethality phenotype. When expressed in HEK293 cells, Drosophila TRPML was localized in both endolysosomes and plasma membrane and was activated by phosphatidylinositol 3,5-bisphosphate (PI(3,5)P₂) applied to the cytoplasmic side in whole lysosomes and inside-out patches excised from plasma membrane. The PI(3,5)P₂-evoked currents were blocked by phosphatidylinositol 4,5-bisphosphate (PI(4,5)P₂), but not other phosphoinositides. Using TRPML A487P, which mimics the varitint-waddler (Va) mutant of mouse TRPML3 with constitutive whole-cell currents, we show that TRPML is biphasically regulated by extracytosolic pH, with an optimal pH about 0.6 pH unit higher than that of human TRPML1. In addition to monovalent cations, TRPML exhibits high permeability to Ca²⁺, Mn²⁺, and Fe²⁺, but not Fe³⁺. The TRPML currents were inhibited by trivalent cations Fe³⁺, La³⁺, and Gd³⁺. These features resemble more closely to mammalian TRPML1 than TRPML2 and TRPML3, but with some obvious differences. Together, our data support the use of Drosophila for assessing functional significance of TRPML1 in cell physiology.