A Functional Genomics Analysis of the B56 Isoforms of Drosophila Protein Phosphatase 2A*S

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.

[1]  B. Dörken,et al.  Inhibition of CPP32 blocks surface IgM‐mediated apoptosis and D4‐GDI cleavage in human BL60 Burkitt lymphoma cells , 1998, European journal of immunology.

[2]  Hervé Tricoire,et al.  BMC Genomics BioMed Central , 2004 .

[3]  Seamus J. Martin Destabilizing Influences in Apoptosis Sowing the Seeds of IAP Destruction , 2002, Cell.

[4]  M. Peifer,et al.  Puckered, a Drosophila MAPK phosphatase, ensures cell viability by antagonizing JNK-induced apoptosis , 2005, Development.

[5]  A. Davis,et al.  Actions of PP2A on the MAP kinase pathway and apoptosis are mediated by distinct regulatory subunits , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[6]  H. Jasper,et al.  JNK signaling confers tolerance to oxidative stress and extends lifespan in Drosophila. , 2003, Developmental cell.

[7]  Colin Adrain,et al.  Executioner Caspase-3, -6, and -7 Perform Distinct, Non-redundant Roles during the Demolition Phase of Apoptosis* , 2001, The Journal of Biological Chemistry.

[8]  L. Johnson,et al.  Protein Phosphatase 2A Regulates Apoptosis in Intestinal Epithelial Cells* , 2005, Journal of Biological Chemistry.

[9]  M. Miura,et al.  Molecular genetic control of caspases and JNK-mediated neural cell death. , 2002, Archives of histology and cytology.

[10]  H. Steller,et al.  Induction of apoptosis by Drosophila reaper, hid and grim through inhibition of IAP function , 2000, The EMBO journal.

[11]  H. Usui,et al.  Inhibition of the Wnt Signaling Pathway by the PR61 Subunit of Protein Phosphatase 2A* , 2001, The Journal of Biological Chemistry.

[12]  I. Kiss,et al.  Down-Regulation of RpS21, a Putative Translation Initiation Factor Interacting with P40, Produces Viable Minute Imagos and Larval Lethality with Overgrown Hematopoietic Organs and Imaginal Discs , 1999, Molecular and Cellular Biology.

[13]  C. van Hoof,et al.  PP2A: the expected tumor suppressor. , 2005, Current opinion in genetics & development.

[14]  Fengqin Gao,et al.  A Functional Role for the B56 α-Subunit of Protein Phosphatase 2A in Ceramide-mediated Regulation of Bcl2 Phosphorylation Status and Function* , 2002, The Journal of Biological Chemistry.

[15]  V. Beneš,et al.  The genomic response of the Drosophila embryo to JNK signaling. , 2001, Developmental cell.

[16]  H. Okano,et al.  Reaper-mediated inhibition of DIAP1-induced DTRAF1 degradation results in activation of JNK in Drosophila , 2002, Nature Cell Biology.

[17]  Perrimon,et al.  The Drosophila stubaristu Phenotype Is Associated With a Dosage Effect of the Putative Ribosome-Associated Protein Dp 40 on spineless , 2002 .

[18]  Suzanne Eaton,et al.  Planar cell polarization requires Widerborst, a B' regulatory subunit of protein phosphatase 2A. , 2002, Development.

[19]  D. Danley,et al.  D4-GDI, a Substrate of CPP32, Is Proteolyzed during Fas-induced Apoptosis (*) , 1996, Journal of Biological Chemistry.

[20]  Cindy L. Kanies,et al.  Protein Phosphatase 2A Dephosphorylation of Phosphoserine 112 Plays the Gatekeeper Role for BAD-Mediated Apoptosis , 2003, Molecular and Cellular Biology.

[21]  Y. Ip,et al.  A Drosophila p38 orthologue is required for environmental stress responses , 2004, EMBO reports.

[22]  L. Schwartz,et al.  Drosophila sickle Is a Novel grim-reaper Cell Death Activator , 2002, Current Biology.

[23]  Xinghai Li,et al.  B56-Associated Protein Phosphatase 2A Is Required For Survival and Protects from Apoptosis in Drosophila melanogaster , 2002, Molecular and Cellular Biology.

[24]  Raymond L. White,et al.  Regulation of β-Catenin Signaling by the B56 Subunit of Protein Phosphatase 2A , 1999 .

[25]  M. Iordanov,et al.  D-MEKK1, the Drosophila orthologue of mammalian MEKK4/MTK1, and Hemipterous/D-MKK7 mediate the activation of D-JNK by cadmium and arsenite in Schneider cells , 2006, BMC Cell Biology.

[26]  T. Ito,et al.  Ceramide Induces Bcl2 Dephosphorylation via a Mechanism Involving Mitochondrial PP2A* , 1999, The Journal of Biological Chemistry.

[27]  N. Sogame,et al.  Drosophila p53 preserves genomic stability by regulating cell death , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[28]  Simon Tavaré,et al.  Similar gene expression patterns characterize aging and oxidative stress in Drosophila melanogaster. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[29]  Sung Bae Lee,et al.  Nef induces apoptosis by activating JNK signaling pathway and inhibits NF-κB-dependent immune responses in Drosophila , 2005, Journal of Cell Science.

[30]  N. Sogame,et al.  The Damage-Responsive Drosophila Gene sickle Encodes a Novel IAP Binding Protein Similar to but Distinct from reaper, grim, and hid , 2002, Current Biology.

[31]  W. Earnshaw,et al.  Caspase‐6 gene disruption reveals a requirement for lamin A cleavage in apoptotic chromatin condensation , 2002, The EMBO journal.

[32]  Henry C. Chang,et al.  Protein phosphatase 2A positively and negatively regulates Ras1-mediated photoreceptor development in Drosophila. , 1996, Genes & development.

[33]  Xiangzhong Zheng,et al.  Posttranslational Regulation of Drosophila PERIOD Protein by Protein Phosphatase 2A , 2004, Cell.

[34]  W. Hahn,et al.  Identification of specific PP2A complexes involved in human cell transformation. , 2004, Cancer cell.

[35]  N. Holbrook,et al.  Cellular response to oxidative stress: Signaling for suicide and survival * , 2002, Journal of cellular physiology.

[36]  Jeffrey P. MacKeigan,et al.  Sensitized RNAi screen of human kinases and phosphatases identifies new regulators of apoptosis and chemoresistance , 2005, Nature Cell Biology.

[37]  Sharda P. Singh,et al.  Cloning, expression and biochemical characterization of one Epsilon-class (GST-3) and ten Delta-class (GST-1) glutathione S-transferases from Drosophila melanogaster, and identification of additional nine members of the Epsilon class. , 2003, The Biochemical journal.

[38]  V. Janssens,et al.  Protein phosphatase 2A: a highly regulated family of serine/threonine phosphatases implicated in cell growth and signalling. , 2001, The Biochemical journal.

[39]  W. Schaffner,et al.  A rapid, sensitive, and specific method for the determination of protein in dilute solution. , 1973, Analytical biochemistry.

[40]  S. Shenolikar,et al.  Protein phosphatase 2A activates the proapoptotic function of BAD in interleukin- 3-dependent lymphoid cells by a mechanism requiring 14-3-3 dissociation. , 2001, Blood.

[41]  H. Müller,et al.  The Drosophila Caspase Inhibitor DIAP1 Is Essential for Cell Survival and Is Negatively Regulated by HID , 1999, Cell.

[42]  D. Virshup,et al.  Protein serine/threonine phosphatases: life, death, and sleeping. , 2005, Current opinion in cell biology.

[43]  H. Koh,et al.  JNK pathway mediates apoptotic cell death induced by tumor suppressor LKB1 in Drosophila , 2006, Cell Death and Differentiation.

[44]  G. Rubin,et al.  Drosophila p53 Binds a Damage Response Element at the reaper Locus , 2000, Cell.

[45]  Gerald M. Rubin,et al.  Drosophila melanogaster MNK/Chk2 and p53 Regulate Multiple DNA Repair and Apoptotic Pathways following DNA Damage , 2004, Molecular and Cellular Biology.

[46]  N. Perrimon,et al.  The Drosophila stubarista phenotype is associated with a dosage effect of the putative ribosome-associated protein D-p40 on spineless. , 1993, Genetics.

[47]  W. Gehring,et al.  Phosphorylation status of the SCR homeodomain determines its functional activity: essential role for protein phosphatase 2A,B′ , 2000, The EMBO journal.

[48]  G. Rubin,et al.  The Toll and Imd pathways are the major regulators of the immune response in Drosophila , 2002, The EMBO journal.

[49]  B. Fanburg,et al.  Reactive oxygen species in cell signaling. , 2000, American journal of physiology. Lung cellular and molecular physiology.

[50]  Lisa L Gomez,et al.  Critical Role for Protein Phosphatase 2A Heterotrimers in Mammalian Cell Survival* , 2004, Journal of Biological Chemistry.

[51]  M. Cheetham,et al.  The Chaperone Environment at the Cytoplasmic Face of the Endoplasmic Reticulum Can Modulate Rhodopsin Processing and Inclusion Formation* , 2003, Journal of Biological Chemistry.

[52]  A. Bergmann,et al.  Drosophila mixed lineage kinase/slipper, a missing biochemical link in Drosophila JNK signaling. , 2003, Biochimica et biophysica acta.

[53]  J. Hemingway,et al.  Characterization of the promoters of Epsilon glutathione transferases in the mosquito Anopheles gambiae and their response to oxidative stress. , 2005, The Biochemical journal.

[54]  S. Srinivasula,et al.  sickle, a Novel Drosophila Death Gene in the reaper/hid/grim Region, Encodes an IAP-Inhibitory Protein , 2002, Current Biology.

[55]  M. Mumby Protein phosphatase 2A: A multifunctional regulator of cell signaling , 2004 .

[56]  Sharda P. Singh,et al.  Catalytic function of Drosophila melanogaster glutathione S-transferase DmGSTS1-1 (GST-2) in conjugation of lipid peroxidation end products. , 2001, European journal of biochemistry.

[57]  N. Perrimon,et al.  Sequential activation of signaling pathways during innate immune responses in Drosophila. , 2002, Developmental cell.

[58]  M. Mumby,et al.  Identification of Phosphoproteins and Their Phosphorylation Sites in the WEHI-231 B Lymphoma Cell Line* , 2004, Molecular & Cellular Proteomics.