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• W2112520234 abstract "Members of the B56 family of protein phosphatase 2A (PP2A) regulatory subunits play crucial roles in Drosophila cell survival. Distinct functions of two B56 subunits were investigated using a combination of RNA interference, DNA microarrays, and proteomics. RNA interference-mediated knockdown of the B56-1 subunit (PP2A-B′) but not the catalytic (mts) or B56-2 subunit (wdb) of PP2A resulted in increased expression of the apoptotic inducers reaper and sickle. Co-knockdown of B56-1 with reaper, but not with sickle, reduced the apoptosis caused by depletion of the B56 subunits. Two-dimensional gel electrophoresis and mass spectrometry identified proteins modified in cells depleted of PP2A subunits. These included generation of caspase-dependent cleavage products, increases in protein abundance, and covalent modifications. Results suggested that up-regulation of the ribosome-associated protein stubarista can serve as a sensitive marker of apoptosis. Up-regulation of transcripts for multiple glutathione transferases and other proteins suggested that loss of PP2A affected pathways involved in the response to oxidative stress. Knockdown of PP2A elevated basal JNK activity and substantially decreased activation of ERK in response to oxidative stress. The results reveal that the B56-containing isoform of PP2A functions within multiple signaling pathways, including those that regulate expression of reaper and the response to oxidative stress, thus promoting cell survival in Drosophila. Members of the B56 family of protein phosphatase 2A (PP2A) regulatory subunits play crucial roles in Drosophila cell survival. Distinct functions of two B56 subunits were investigated using a combination of RNA interference, DNA microarrays, and proteomics. RNA interference-mediated knockdown of the B56-1 subunit (PP2A-B′) but not the catalytic (mts) or B56-2 subunit (wdb) of PP2A resulted in increased expression of the apoptotic inducers reaper and sickle. Co-knockdown of B56-1 with reaper, but not with sickle, reduced the apoptosis caused by depletion of the B56 subunits. Two-dimensional gel electrophoresis and mass spectrometry identified proteins modified in cells depleted of PP2A subunits. These included generation of caspase-dependent cleavage products, increases in protein abundance, and covalent modifications. Results suggested that up-regulation of the ribosome-associated protein stubarista can serve as a sensitive marker of apoptosis. Up-regulation of transcripts for multiple glutathione transferases and other proteins suggested that loss of PP2A affected pathways involved in the response to oxidative stress. Knockdown of PP2A elevated basal JNK activity and substantially decreased activation of ERK in response to oxidative stress. The results reveal that the B56-containing isoform of PP2A functions within multiple signaling pathways, including those that regulate expression of reaper and the response to oxidative stress, thus promoting cell survival in Drosophila. Protein phosphatase 2A (PP2A) 1The abbreviations used are: PP2A, protein phosphatase 2A; RNAi, RNA interference; dsRNA, double-stranded RNA; qRT-PCR, real time quantitative RT-PCR; EGFP, enhanced green fluorescent protein; 2-D, two-dimensional; 2-D electrophoresis, two-dimensional polyacrylamide gel electrophoresis; Z-VAD-fmk, benzyloxycarbonyl-Val-Ala-Asp-(OMe)-fluoromethyl ketone; ERK, extracellular signal-regulated kinase; JNK, c-jun N-terminal kinase; MAP, mitogen-activated protein; SAPK, stress-activated protein kinase; RhoGDI, Rho GDP dissociation inhibitor. is a major class of serine/threonine phosphatase that plays a myriad of roles in cellular signaling. The term PP2A refers to a group of enzymes composed of a common catalytic subunit, which forms oligomeric complexes with a large and diverse group of proteins (1Janssens V. Goris J. Protein phosphatase 2A: a highly regulated family of serine/threonine phosphatases implicated in cell growth and signalling..Biochem. J. 2001; 353: 417-439Crossref PubMed Scopus (0) Google Scholar, 2Mumby M.C. Protein phosphatase 2A: a multifunctional regulator of cell signaling.in: Arino J. Alexander D.R. Topics in Current Genetics. Springer-Verlag GmbH, Heidelberg, Germany2003: 45-72Google Scholar, 3Gallego M. Virshup D.M. Protein serine/threonine phosphatases: life, death, and sleeping..Curr. Opin. Cell Biol. 2005; 17: 197-202Crossref PubMed Scopus (134) Google Scholar). The most common forms of PP2A contain a core dimer, composed of the catalytic subunit and a scaffold protein termed the A subunit. The scaffold subunit mediates formation of heterotrimeric holoenzymes by binding to additional regulatory subunits. There are four characterized families of PP2A regulatory subunits that target PP2A to specific substrates and signaling complexes. An important class of regulatory subunit is encoded by the PPP2R5 gene family, which has also been referred to as B56, B′, and PR61. The mammalian B56 gene family contains five members, each of which is processed into multiple splice variants. Members of the B56 family target PP2A to a variety of cellular processes including transformation (4Chen W. Possemato R. Campbell K.T. Plattner C.A. Pallas D.C. Hahn W.C. Identification of specific PP2A complexes involved in human cell transformation..Cancer Cell. 2004; 5: 127-136Abstract Full Text Full Text PDF PubMed Scopus (281) Google Scholar, 5Janssens V. Goris J. Van Hoof C. PP2A: the expected tumor suppressor..Curr. Opin. Genet. Dev. 2005; 15: 34-41Crossref PubMed Scopus (365) Google Scholar), Wnt signaling (6Seeling J.M. Miller J.R. Gil R. Moon R.T. White R. Virshup D.M. Regulation of β-catenin signaling by the B56 subunit of protein phosphatase 2A..Science. 1999; 283: 2089-2091Crossref PubMed Scopus (366) Google Scholar, 7Yamamoto H. Hinoi T. Michiue T. Fukui A. Usui H. Janssens V. Van Hoof C. Goris J. Asashima M. Kikuchi A. Inhibition of the Wnt signaling pathway by the PR61 subunit of protein phosphatase 2A..J. Biol. Chem. 2001; 276: 26875-26882Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar, 8Yang J. Wu J. Tan C. Klein P.S. PP2A:B56[cepsilon] is required for Wnt/β-catenin signaling during embryonic development..Development. 2003; 130: 5569-5578Crossref PubMed Scopus (76) Google Scholar), cell polarization (9Hannus M. Feiguin F. Heisenberg C.P. Eaton S. Planar cell polarization requires Widerborst, a B′ regulatory subunit of protein phosphatase 2A..Development. 2002; 129: 3493-3503Crossref PubMed Google Scholar), and circadian rhythm (10Sathyanarayanan S. Zheng X. Xiao R. Sehgal A. Posttranslational regulation of Drosophila PERIOD protein by protein phosphatase 2A..Cell. 2004; 116: 603-615Abstract Full Text Full Text PDF PubMed Scopus (213) Google Scholar). PP2A plays both positive and negative roles in pathways that regulate cell survival and apoptosis. Induction of apoptosis by the second messenger ceramide, a membrane sphingolipid, correlates with stimulation of a fraction of PP2A associated with mitochondria in HL60 cells and the murine interleukin-3-dependent cell line NSF/N1.H7 (11Ruvolo P.P. Deng X. Ito T. Carr B.K. May W.S. Ceramide induces bcl2 dephosphorylation via a mechanism involving mitochondrial PP2A..J. Biol. Chem. 1999; 274: 20296-20300Abstract Full Text Full Text PDF PubMed Scopus (306) Google Scholar). The function of PP2A in ceramide-induced apoptosis involves inhibition of the antiapoptotic activity of Bcl2 by dephosphorylation of serine 70. The ceramide-enhanced dephosphorylation of this site is associated with recruitment of PP2A to Bcl2 via interaction with the B56α regulatory subunit (12Ruvolo P.P. Clark W. Mumby M. Gao F. May W.S. A Functional role for the B56 α-subunit of protein phosphatase 2A in ceramide-mediated regulation of Bcl2 phosphorylation status and function..J. Biol. Chem. 2002; 277: 22847-22852Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar). PP2A also plays a positive role in the apoptosis caused by withdrawal of survival factors from interleukin-3-dependent FL5.12 cells (a murine pro-B-cell lymphoid cell line). In this case, PP2A associates with the Bad proapoptotic protein and dephosphorylates serine 112. Dephosphorylation of this site leads to subsequent dephosphorylation of additional sites, dissociation of 14-3-3 proteins, and unmasking of the apoptotic activity of Bad (13Chiang C.W. Harris G. Ellig C. Masters S.C. Subramanian R. Shenolikar S. Wadzinski B.E. Yang E. Protein phosphatase 2A activates the proapoptotic function of BAD in interleukin-3-dependent lymphoid cells by a mechanism requiring 14-3-3 dissociation..Blood. 2001; 97: 1289-1297Crossref PubMed Scopus (131) Google Scholar, 14Chiang C.W. Kanies C. Kim K.W. Fang W.B. Parkhurst C. Xie M. Henry T. Yang E. Protein phosphatase 2A dephosphorylation of phosphoserine 112 plays the gatekeeper role for BAD-mediated apoptosis..Mol. Cell. Biol. 2003; 23: 6350-6362Crossref PubMed Scopus (128) Google Scholar). PP2A also plays a positive role in tumor necrosis factor α-induced apoptosis of rat IEC-6 cells through a process that involves both Bcl-2 and Bad (15Ray R.M. Bhattacharya S. Johnson L.R. Protein phosphatase 2A regulates apoptosis in intestinal epithelial cells..J. Biol. Chem. 2005; 280: 31091-31100Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). In addition to roles in promoting apoptosis in response to specific stimuli, PP2A plays a more fundamental role in promoting cell survival. Silencing of PP2A by RNA interference results in apoptosis of multiple cell types. Knockdown of PP2A in the Schneider 2 Drosophila cell line results in an apoptotic response mediated by the combined loss of the two Drosophila B56 subunits (16Silverstein A.M. Barrow C.A. Davis A.J. Mumby M.C. Actions of PP2A on the MAP kinase pathway and apoptosis are mediated by distinct regulatory subunits..Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 4221-4226Crossref PubMed Scopus (228) Google Scholar, 17Li X. Scuderi A. Letsou A. Virshup D.M. B56-associated protein phosphatase 2A is required for survival and protects from apoptosis in Drosophila melanogaster..Mol. Cell. Biol. 2002; 22: 3674-3684Crossref PubMed Scopus (120) Google Scholar). RNAi-mediated knockdown of PP2A also causes apoptosis in the rat PC12 neuronal cell line (18Strack S. Cribbs J.T. Gomez L. Critical role for protein phosphatase 2A heterotrimers in mammalian cell survival..J. Biol. Chem. 2004; 279: 47732-47739Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar). The ability of PP2A to prevent apoptosis in PC12 cells requires multiple classes of regulatory subunits including the B56 family. The catalytic and scaffold subunits as well as members of the PPP2R2 (B), PPP2R3 (PR72), and PPP2R5 (B56) regulatory subunits of human PP2A have all been identified as prosurvival proteins in an RNAi screen (19Mackeigan J.P. Murphy L.O. Blenis J. Sensitized RNAi screen of human kinases and phosphatases identifies new regulators of apoptosis and chemoresistance..Nat. Cell Biol. 2005; 7: 591-600Crossref PubMed Scopus (472) Google Scholar). The ability of PP2A to play either positive or negative roles in apoptosis is likely to depend on individual holoenzymes and on the organism, cell type, and apoptotic stimulus. A deficiency in understanding the functions of PP2A in cellular regulatory processes, including those that regulate cell survival and apoptosis, is a lack of information about the substrates and signaling pathways that are targeted by different forms of the enzyme. In mammalian cells, the problem is complicated by a large number of regulatory subunit isoforms and splice variants. The simplified repertoire of PP2A regulatory subunits in Drosophila melanogaster provides a simpler system in which individual isoforms can be investigated. Previous studies showed that the Drosophila B56 subunits (B56-1/PP2A-B′ and B56-2/widerborst) are required for cell survival and prevent programmed cell death (16Silverstein A.M. Barrow C.A. Davis A.J. Mumby M.C. Actions of PP2A on the MAP kinase pathway and apoptosis are mediated by distinct regulatory subunits..Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 4221-4226Crossref PubMed Scopus (228) Google Scholar, 17Li X. Scuderi A. Letsou A. Virshup D.M. B56-associated protein phosphatase 2A is required for survival and protects from apoptosis in Drosophila melanogaster..Mol. Cell. Biol. 2002; 22: 3674-3684Crossref PubMed Scopus (120) Google Scholar). To gain insights into the molecular functions of these subunits, we used a combination of RNA interference, DNA microarrays, and proteomics to identify proteins and signaling pathways targeted by PP2A and the B56 family of regulatory subunits. Schneider S2 cells were maintained in Drosophila serum-free medium (Invitrogen) and seeded at 1 × 106 cells/well in 6-well culture plates for RNAi experiments as described previously (16Silverstein A.M. Barrow C.A. Davis A.J. Mumby M.C. Actions of PP2A on the MAP kinase pathway and apoptosis are mediated by distinct regulatory subunits..Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 4221-4226Crossref PubMed Scopus (228) Google Scholar). Twenty-four hours later, 15 μg of dsRNA was added to the culture medium. Double-stranded RNA corresponding to enhanced green fluorescent protein (EGFP) and the catalytic, B56-1, B56-2, and PR72 subunits of Drosophila PP2A were prepared as described previously (16Silverstein A.M. Barrow C.A. Davis A.J. Mumby M.C. Actions of PP2A on the MAP kinase pathway and apoptosis are mediated by distinct regulatory subunits..Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 4221-4226Crossref PubMed Scopus (228) Google Scholar). For knockdown of reaper and sickle mRNA, regions of 500–800 bp were amplified by PCR using gene-specific primers from cDNA prepared from S2 cells using similar methods. Cells were harvested 3–5 days after addition of dsRNA. In experiments where the B56-1 and -2 subunits were knocked down simultaneously, twice the normal amount of EGFP dsRNA was used in the control treatments. In some experiments the cells were incubated with Z-VAD-fmk (30 μm final concentration) during the dsRNA treatment to block apoptosis. In some two-dimensional gel electrophoresis experiments, cells that had not been treated with dsRNA were incubated with actinomycin D (20 nm) for 24 h to induce apoptosis. Total protein and RNA were isolated from dsRNA-treated S2 cells using the TriPure reagent (Roche Diagnostics) following the manufacturer’s instructions. Total protein extracts for analysis by two-dimensional gel electrophoresis were prepared from three wells of S2 cells treated with the same dsRNA. The protein pellet was dried in a vacuum concentrator and dissolved in 100 μl of 2-D rehydration buffer (7 m urea, 2 m thiourea, 4% (w/v) CHAPS, 50 mm DTT, 3% (v/v) Ampholines, 1 mm sodium orthovanadate, 1 μm okadaic acid, and 1× Complete Mini protease mixture (Roche Applied Science)). The concentration of the solubilized protein was determined by Amido Black protein assay (20Schaffner W. Weissmann C. A rapid, sensitive, and specific method for the determination of protein in dilute solution..Anal. Biochem. 1973; 56: 502-514Crossref PubMed Scopus (1953) Google Scholar). Total RNA was isolated from the same samples by precipitation of the aqueous phase from the phenol-chloroform extraction with isopropanol. The RNA precipitate was washed once in 75% ethanol, dried at room temperature, and resuspended in RNase-free water. Contaminating DNA was removed by treating with DNase I (Ambion). The RNA quality was examined by analysis on a 1% agarose gel to determine the presence of intact ribosomal RNA. The 260/280 ratio was then measured, and only samples with ratios between 1.8 and 2.1 were used for subsequent analyses. Preparation of cRNA, hybridization to Drosophila Genome Chip arrays (DrosGenome 1), and scanning were performed according to the manufacturer’s protocol (Affymetrix, Santa Clara, CA). Briefly 5 μg of RNA was converted into cDNA by reverse transcription using the SuperScript Choice cDNA Synthesis kit (Invitrogen). Biotin-labeled cRNA was generated from the double-stranded cDNA using the BioArray High Yield RNA Transcript Labeling kit (Enzo Life Sciences, Farmingdale, NY). After purification with Rneasy spin columns (Qiagen, Valencia, CA), the labeled cRNA was fragmented and hybridized to the arrays in the University of Texas Southwestern Medical Center Microarray Core Facility. The hybridized chips were stained and scanned using a GeneArray scanner (Affymetrix). The probe-level data from 16 arrays were analyzed using GeneSpring® software, version 7.2 (Silicon Genetics). Following import of the Affymetrix CEL files into GeneSpring, background correction, normalization, and summarization were carried out using the Robust Multichip Average with GC content background correction (GC-RMA) algorithm. The expression of each gene was then normalized to its median across all chips. For those knockdown experiments that were replicated (triplicates for EGFP, catalytic subunit, B56-1, and B56-1 and -2 and duplicates for B56-2 alone), the mean signal was used to compare transcript levels in the PP2A dsRNA-treated samples with those from the control EGFP dsRNA-treated samples. Transcripts whose levels were reproducibly changed were identified using the following criteria. 1) The change in normalized mean signal between the EGFP dsRNA-treated and PP2A dsRNA-treated samples was at least 2-fold. 2) The difference between EGFP and PP2A was statistically significant using one-way parametric analysis of variance with a p value cutoff of 0.05 (false positive rate of 5%). The changes in transcript levels are expressed as the -fold change in signal between PP2A dsRNA samples and the EGFP dsRNA samples. RT-PCR to detect knockdown of PP2A subunit mRNA was performed as described previously (16Silverstein A.M. Barrow C.A. Davis A.J. Mumby M.C. Actions of PP2A on the MAP kinase pathway and apoptosis are mediated by distinct regulatory subunits..Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 4221-4226Crossref PubMed Scopus (228) Google Scholar) using the superscript one-step RT-PCR system (Invitrogen). For real time quantitative reverse transcription PCR, 2 μg of total RNA, isolated as described for the microarray experiments, was used in reverse transcription reactions using TaqMan reverse transcription reagents (Applied Biosystems). The PCRs were performed in triplicate using the SYBR Green PCR kit (Applied Biosystems) according to the manufacturer’s instructions. Fluorescence changes during real time PCR were measured with an ABI PRISM 7900 sequence detection system or an ABI 7500 qRT-PCR instrument (Applied Biosystems). All gene-specific primers were designed using Primer Expression software (Applied Biosystems). A primer pair against Drosophila actin was used as a control for all experiments. The following primer pairs were used for qRT-PCR: reaper forward, 5′-CACAGTGGAGATTCCTGGCC; reaper reverse, 5′-TGTACTGGCGCAGGGTTTC; sickle forward, 5′-GTGCAAGGTCCTGAAGCAAT; sickle reverse, 5′-GTGGCCTTTAGTTTGCTGGA; GstD2 forward, 5′-GCCGCACGGTCATCATG; GstD2 reverse, 5′-TGGTGTTCAGTAGCTTCTTGTTCAG; CG5224 forward, 5′-CCCCTGTCGTGCTGTTCTG; CG5224 reverse, 5′-TTACGTTGACCAGTCGCAAGTC; GstE6 forward, 5′-TTGCCTATTTGGTCTCGAAATATG; GstE6 reverse, 5′-ACAGCCCGCTTGAGAGGAT; GstE7 forward, 5′-ACCAGTTCGTGCCGTCAAAT; GstE7 reverse, 5′-CCGAGTGTTTACCTCCACGAAT; actin forward, 5-CGCGAAAAGATGACTCAGATTATG; actin reverse, 5′-CCGCTTGGATGGCAACAT. The amount of each target mRNA relative to actin mRNA was calculated using the comparative CT (ΔCT) method. The difference in the amounts of target mRNA between the EGFP dsRNA-treated and PP2A subunit dsRNA-treated cells was calculated as described in Applied Biosystems User Bulletin 2. Extracted S2 cell protein (400 μg) in 225 μl of 2-D rehydration buffer was used to rehydrate 11-cm immobilized pH 4–7 gradient gels (pH 4–7 Immobiline DryStrips, Amersham Biosciences) overnight following the manufacturer’s protocol. After rehydration, the strips were placed in a Multiphor II system (Amersham Biosciences) and focused according to the following program: 10 V-h at 500 V, 500 V-h at 300 V, 17,500 V-h at 3500 V, 25,000 V-h at 3500 V, and 17,000 V-h at 300 V. The focused strips were equilibrated for 10 min in DTT equilibration solution (50 mm Tris-HCl, pH 8.8, 38% glycerol, 6 m urea, 3% SDS, 5 mg/ml dithiothreitol) followed by 10 min in iodoacetamide equilibration solution (same as above but with 45 mg/ml iodoacetamide replacing the DTT). The equilibrated strips were placed on top of 12.5% IPG+1 Criterion gels (Bio-Rad), and the focused proteins were resolved by SDS-polyacrylamide gel electrophoresis. For some experiments, high resolution 2-D gel electrophoresis was carried out using the same methods but with Immobiline IPG strips that were 18 cm long and second dimension SDS gels that were 20 cm long. Following electrophoresis the gels were stained with SYPRO Ruby dye (Molecular Probes), and images were acquired with a Typhoon imaging system (Amersham Biosciences). The 2-D gel images were analyzed with Ettan Progenesis software (Amersham Biosciences). After spot detection, background subtraction, and normalization to total spot intensity, gel images from EGFP control and PP2A dsRNA-treated samples were warped to align matched protein spots on different gels. The software was then used to identify and quantitate spots whose normalized intensities were different in control and treated samples as well as spots that were uniquely present (non-matched) in individual samples. The 2-D gel analysis was carried out in four to five separate experiments, and those spots whose intensities were altered by at lease 2-fold in three or more experiments were considered to be reproducibly affected by loss of PP2A subunits. Protein spots of interest were excised from SYPRO Ruby-stained 2-D gels illuminated on a UV light box. Peptides for each spot were generated by in-gel digestion with trypsin as described previously (21Shu H. Chen S. Bi Q. Mumby M. Brekken D.L. Identification of phosphoproteins and their phosphorylation sites in the WEHI-231 B lymphoma cell line..Mol. Cell. Proteomics. 2004; 3: 279-286Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). The dried peptides were desalted with 0.1% TFA using ZipTips (Millipore) and eluted with 2,5-dihydroxybenzoic acid matrix solution (Agilent) directly onto MALDI sample plates. MS and MS/MS data were acquired by MALDI-Q-TOF mass spectrometry using a QSTAR Pulsar-I quadrupole time-of-flight tandem mass spectrometer (Applied Biosystems). The MS and MS/MS data were searched against the National Center for Biotechnology Information (NCBI) non-redundant Drosophila protein database using Knexus software (Genomic Solutions, Inc.) as described previously (21Shu H. Chen S. Bi Q. Mumby M. Brekken D.L. Identification of phosphoproteins and their phosphorylation sites in the WEHI-231 B lymphoma cell line..Mol. Cell. Proteomics. 2004; 3: 279-286Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). To be considered a positive identification, the mass spectra of at least three different peptides from each spot had to match the database entry for that protein. Assay for caspase-3-like enzyme activity was performed using the ApoAlert Caspase Colorimetric Assay kit (Clontech) as described previously (16Silverstein A.M. Barrow C.A. Davis A.J. Mumby M.C. Actions of PP2A on the MAP kinase pathway and apoptosis are mediated by distinct regulatory subunits..Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 4221-4226Crossref PubMed Scopus (228) Google Scholar). Caspase activities were normalized for total protein recovered in each sample and expressed as the relative activity in PP2A dsRNA-treated cells relative to control cells treated with EGFP dsRNA. The values are reported as the mean ± S.D. from three independent experiments. Lysates from S2 cells treated with dsRNA were assayed for activation of the Drosophila homologs of the ERK (rolled), JNK (basket), and p38 MAP kinases (Mpk2 and p38b) using phosphospecific antibodies. S2 cells were treated with control or PP2A dsRNA for 4 days and then subjected to oxidative or osmotic stress. The dsRNA-treated cells were harvested and incubated for 10 min in medium containing no additions, 2 mm hydrogen peroxide, 0.3 m NaCl, or 0.3 m sorbitol. The treated cells were collected by centrifugation for 30 s at 18,000 × g, lysed in 5% SDS sample buffer (62.5 mm Tris-HCl, pH 6.7, 10% glycerol, 5% SDS, 0.1% bromphenol blue, 1% 2-mercaptoethanol), and heated at 95 °C for 10 min. Protein concentrations were determined by Amido Black assay (20Schaffner W. Weissmann C. A rapid, sensitive, and specific method for the determination of protein in dilute solution..Anal. Biochem. 1973; 56: 502-514Crossref PubMed Scopus (1953) Google Scholar), and 40 μg of protein was resolved by SDS-PAGE. Proteins were transferred to nitrocellulose membranes and immunoblotted with antibodies against MAP kinases and PP2A subunits. Antibodies against the catalytic and B56-1 subunits of PP2A were used as described previously (16Silverstein A.M. Barrow C.A. Davis A.J. Mumby M.C. Actions of PP2A on the MAP kinase pathway and apoptosis are mediated by distinct regulatory subunits..Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 4221-4226Crossref PubMed Scopus (228) Google Scholar). Antibodies that recognize Drosophila MAP kinases were obtained from the following sources: anti-phospho-p44/42 MAP kinase (Thr-202/Tyr-204) rabbit polyclonal, anti-phospho-p38 MAP kinase (Thr-180/Tyr-182) rabbit polyclonal, and anti-phospho-SAPK/JNK (Thr-183/Tyr-185) rabbit polyclonal antibodies were from Cell Signaling Technology; anti-ERK, anti-p38, and anti-JNK1 (FL) rabbit polyclonal antibodies were from Santa Cruz Biotechnology. Each of these antibodies has been shown to cross-react with the Drosophila homologs of these protein kinases (22Sathyanarayana P. Barthwal M.K. Lane M.E. Acevedo S.F. Skoulakis E.M. Bergmann A. Rana A. Drosophila mixed lineage kinase/slipper, a missing biochemical link in Drosophila JNK signaling..Biochim. Biophys. Acta. 2003; 1640: 77-84Crossref PubMed Scopus (20) Google Scholar). Immunoblotting was performed according to the manufacturer’s protocol. Genome-wide screens for changes in gene expression were carried out to identify signaling pathways altered by knockdown of the catalytic and B56 subunits of PP2A. Affymetrix Drosophila Genome Array chips representing 13,500 genes were used to identify transcripts that were differentially expressed in S2 cells in which these subunits had been knocked down by RNAi. To detect transcripts whose changes were due to loss of PP2A subunits and not simply to dsRNA, RNA from cells treated with a control dsRNA (derived from the sequence of EGFP) was used as the base line for all comparisons. Genes whose transcript levels were reproducibly altered by PP2A dsRNA treatment were identified as those whose levels changed by a least 2-fold relative to the EGFP dsRNA-treated controls and were statistically significant according to one-way analysis of variance with a p value cutoff of 0.05. Knockdown of the PP2A catalytic subunit resulted in up-regulation of 30 transcripts and down-regulation of 32 transcripts. The largest group of affected transcripts with a common biological function was composed of six up-regulated genes involved in the insect defense response. Five of these genes (CG5224, GstE3, GstE6, GstE7, and GstE8) encode Drosophila glutathione S-transferases, which play roles in insecticide metabolism and protection from oxidative stress. Another defense response gene, the CG18522 oxidoreductase, and two other oxidoreductases (Cyp6a8 and CG2064) were also up-regulated. Transcripts that were down-regulated included a number of genes involved in signal transduction including pebble (a guanyl-nucleotide exchange factor), Cdc2 (a cell cycle kinase), Toll (a receptor involved in activation of the immune response), Serrate (a ligand for the Notch receptor), and CrebA (a cAMP-dependent protein kinase-regulated transcriptional co-activator). The complete set of transcripts altered by knockdown of the PP2A catalytic subunit can be found in Supplemental Table S1. Because depletion of the PP2A catalytic subunit induces apoptosis of Drosophila S2 cells, it was important to differentiate transcripts whose expression was altered by the depletion of PP2A from those altered by the induction of apoptosis. RNA interference experiments were consequently carried out in the presence of the caspase inhibitor Z-VAD-fmk. Exposure of S2 cells to Z-VAD-fmk inhibited apoptosis caused by knockdown of the catalytic subunit as well as apoptosis induced by actinomycin D (data not shown). Treatment of Z-VAD-fmk blocked up-regulation of two transcripts (CG14291 and CG13510) and down-regulation of five transcripts (CG3739, CG10026, CG8468, sugarless, and CG6199). The results indicated that most of the changes in transcript levels observed in cells depleted of the PP2A catalytic subunit were either independent of apoptosis or caused by changes that occurred upstream of caspase activation (Supplemental Table S1). Knockdown of the B56-1 subunit resulted in up-regulation of 57 transcripts and down-regulation of 36 transcripts. Although there was limited overlap with transcripts altered by knockdown of the catalytic subunit, there were similarities in the classes of transcripts up-regulated (Supplemental Table S5). The largest group of functionally related transcripts up-regulated by knockdown of the B56-1 subunit were eight genes involved in the defense response including the glutathione S-transferases CG5224 and GstD2 (Supplemental Table S2). Knockdown of B56-1 also led to increases in several transcripts that are up-regulated during the Drosophila innate immune response including Attacin-C and Attacin-B, CG16713, reape" @default.
• W2112520234 created "2016-06-24" @default.
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• W2112520234 creator A5032467019 @default.
• W2112520234 creator A5049212235 @default.
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• W2112520234 date "2007-02-01" @default.
• W2112520234 modified "2023-09-25" @default.
• W2112520234 title "A Functional Genomics Analysis of the B56 Isoforms of Drosophila Protein Phosphatase 2A" @default.
• W2112520234 cites W1528266879 @default.
• W2112520234 cites W1754711307 @default.
• W2112520234 cites W1841140288 @default.
• W2112520234 cites W1860335819 @default.
• W2112520234 cites W1902946247 @default.
• W2112520234 cites W1903528283 @default.
• W2112520234 cites W1964269904 @default.
• W2112520234 cites W1964293578 @default.
• W2112520234 cites W1964538721 @default.
• W2112520234 cites W1964739066 @default.
• W2112520234 cites W1966343869 @default.
• W2112520234 cites W1967181393 @default.
• W2112520234 cites W1971212979 @default.
• W2112520234 cites W1979212686 @default.
• W2112520234 cites W1981076681 @default.
• W2112520234 cites W1983755623 @default.
• W2112520234 cites W1988298690 @default.
• W2112520234 cites W1988969017 @default.
• W2112520234 cites W1996952871 @default.
• W2112520234 cites W2001054344 @default.
• W2112520234 cites W2004127615 @default.
• W2112520234 cites W2011169071 @default.
• W2112520234 cites W2014082672 @default.
• W2112520234 cites W2026481478 @default.
• W2112520234 cites W2033808303 @default.
• W2112520234 cites W2034007192 @default.
• W2112520234 cites W2039039582 @default.
• W2112520234 cites W2039520182 @default.
• W2112520234 cites W2039884859 @default.
• W2112520234 cites W2043107604 @default.
• W2112520234 cites W2056561005 @default.
• W2112520234 cites W2069656813 @default.
• W2112520234 cites W2082497364 @default.
• W2112520234 cites W2088811318 @default.
• W2112520234 cites W2090504233 @default.
• W2112520234 cites W2093655143 @default.
• W2112520234 cites W2101803513 @default.
• W2112520234 cites W2102532761 @default.
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• W2112520234 cites W2109010802 @default.
• W2112520234 cites W2109023063 @default.
• W2112520234 cites W2118609604 @default.
• W2112520234 cites W2119121234 @default.
• W2112520234 cites W2125499046 @default.
• W2112520234 cites W2126167700 @default.
• W2112520234 cites W2128849220 @default.
• W2112520234 cites W2129128106 @default.
• W2112520234 cites W2133232851 @default.
• W2112520234 cites W2133650476 @default.
• W2112520234 cites W2137817844 @default.
• W2112520234 cites W2154903535 @default.
• W2112520234 cites W2163023090 @default.
• W2112520234 cites W2163668204 @default.
• W2112520234 cites W2172249877 @default.
• W2112520234 cites W4238581030 @default.
• W2112520234 cites W4240253200 @default.
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