Title: Critical Role for Protein Phosphatase 2A Heterotrimers in Mammalian Cell Survival
Abstract: The predominant forms of protein phosphatase 2A (PP2A), one of the major Ser/Thr phosphatases, are dimers of catalytic (C) and scaffolding (A) subunits and trimers with an additional variable regulatory subunit. In mammals, catalytic and scaffolding subunits are encoded by two genes each (α/β), whereas three gene families (B, B′, and B″) with a total of 12 genes contribute PP2A regulatory subunits. We generated stable PC12 cell lines in which the major scaffolding Aα subunit can be knocked down by inducible RNA interference (RNAi) to study its role in cell viability. Aα RNAi decreased total PP2A activity as well as protein levels of C, B, and B′ but not B″ subunits. Inhibitor experiments indicate that monomeric C and B subunits are degraded by the proteosome. Knock-down of Aα triggered cell death by redundant apoptotic and non-apoptotic mechanisms because the inhibition of RNAi-associated caspase activation failed to stall cell death. PP2A holoenzymes positively regulate survival kinase signaling, because RNAi reduced basal and epidermal growth factor-stimulated Akt phosphorylation. RNAi-resistant Aα cDNAs rescued RNAi-induced loss of the C subunit, and Aα point mutants prevented regulatory subunit degradation as predicted from each mutant's binding specificity. In transient, stable, and stable-inducible rescue experiments, both wild-type Aβ and Aα mutants capable of binding to at least one family of regulatory subunits were able to delay Aα RNAi-induced death of PC12 cells. However, only the expression of wild-type Aα restored viability completely. Thus, heterotrimeric PP2A holoenzymes containing the Aα subunit and members of all three regulatory subunit families are necessary for mammalian cell viability. The predominant forms of protein phosphatase 2A (PP2A), one of the major Ser/Thr phosphatases, are dimers of catalytic (C) and scaffolding (A) subunits and trimers with an additional variable regulatory subunit. In mammals, catalytic and scaffolding subunits are encoded by two genes each (α/β), whereas three gene families (B, B′, and B″) with a total of 12 genes contribute PP2A regulatory subunits. We generated stable PC12 cell lines in which the major scaffolding Aα subunit can be knocked down by inducible RNA interference (RNAi) to study its role in cell viability. Aα RNAi decreased total PP2A activity as well as protein levels of C, B, and B′ but not B″ subunits. Inhibitor experiments indicate that monomeric C and B subunits are degraded by the proteosome. Knock-down of Aα triggered cell death by redundant apoptotic and non-apoptotic mechanisms because the inhibition of RNAi-associated caspase activation failed to stall cell death. PP2A holoenzymes positively regulate survival kinase signaling, because RNAi reduced basal and epidermal growth factor-stimulated Akt phosphorylation. RNAi-resistant Aα cDNAs rescued RNAi-induced loss of the C subunit, and Aα point mutants prevented regulatory subunit degradation as predicted from each mutant's binding specificity. In transient, stable, and stable-inducible rescue experiments, both wild-type Aβ and Aα mutants capable of binding to at least one family of regulatory subunits were able to delay Aα RNAi-induced death of PC12 cells. However, only the expression of wild-type Aα restored viability completely. Thus, heterotrimeric PP2A holoenzymes containing the Aα subunit and members of all three regulatory subunit families are necessary for mammalian cell viability. The dynamic balance of protein kinase and phosphatase activities toward key substrates is crucial for cellular homeostasis. It is becoming increasingly apparent that phosphatases are just as tightly controlled as kinases, although mechanistic details largely remain to be explored. Protein phosphatase 2A (PP2A) 1The abbreviations used are: PP2A, protein phosphatase 2A; A subunit, scaffolding subunit; C subunit, catalytic subunit; AMC, aminomethyl-coumarin; Dox, doxycycline; fmk, fluoromethyl ketone; hpRNA, hairpin RNA; RNAi, RNA interference; TO, tetracycline operator; TR, tetracycline repressor; Z, benzyloxycarbonyl. is one of four major classes of serine/threonine-specific phosphatases (for a recent review, see Ref. 1Janssens V. Goris J. Biochem. J. 2001; 353: 417-439Crossref PubMed Scopus (1542) Google Scholar) that, together with protein phosphatase 1, contributes most of the Ser/Thr phosphatase activity in cells. The PP2A catalytic subunit is a 36-kDa globular protein that associates predominantly with a 65-kDa scaffolding that associates predominantly with a 65-kD scaffolding A subunit (or PR65) to form the PP2A core dimer. In vertebrates, two genes referred to as Cα/β and Aα/β encode catalytic and scaffolding subunits, respectively. The significance of having two isoforms is unclear, although there are subtle differences in the tissue expression of the two A and C subunit genes, with α isoforms generally being more abundant (2Khew-Goodall Y. Hemmings B.A. FEBS Lett. 1988; 238: 265-268Crossref PubMed Scopus (79) Google Scholar, 3Zhou J. Pham H.T. Ruediger R. Walter G. Biochem. J. 2003; 369: 387-398Crossref PubMed Scopus (71) Google Scholar). The A subunits are hook-shaped proteins with 15 non-identical, tandem α-helical repeats (4Groves M.R. Hanlon N. Turowski P. Hemmings B.A. Barford D. Cell. 1999; 96: 99-110Abstract Full Text Full Text PDF PubMed Scopus (354) Google Scholar, 5Ruediger R. Hentz M. Fait J. Mumby M. Walter G. J. Virol. 1994; 68: 123-129Crossref PubMed Google Scholar) that link catalytic subunits to a diverse group of regulatory subunits to form PP2A heterotrimers. Regulatory subunits have been divided into three gene families, called B (or PR55), B′ (or B56 or PR61), and B″ (or PR72/130, PR59, or PR48). Each gene family contains one to two genes in lower metazoans, such as flies and worms, and three to five members in vertebrates. The three gene families have no obvious sequence or predicted structural similarities, although loose consensus sequences for A subunit interaction have been reported (6Li X. Virshup D.M. Eur. J. Biochem. 2002; 269: 546-552Crossref PubMed Scopus (69) Google Scholar). The B family of regulatory subunits consists of WD repeat-containing β-propeller proteins with variable N termini that dictate subcellular localization (7Strack S. Ruediger R. Walter G. Dagda R.K. Barwacz C.A. Cribbs J.T. J. Biol. Chem. 2002; 277: 20750-20755Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar, 8Dagda R.K. Zaucha J.A. Wadzinski B.E. Strack S. J. Biol. Chem. 2003; 278: 24976-24985Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). Proposed functions include the regulation of cytoskeletal dynamics, mitogen-activated protein kinase signaling, and apoptosis (8Dagda R.K. Zaucha J.A. Wadzinski B.E. Strack S. J. Biol. Chem. 2003; 278: 24976-24985Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar, 9Sontag E. Nunbhakdi-Craig V. Lee G. Bloom G.S. Mumby M.C. Neuron. 1996; 17: 1201-1207Abstract Full Text Full Text PDF PubMed Scopus (351) Google Scholar, 10Turowski P. Myles T. Hemmings B.A. Fernandez A. Lamb N.J. Mol. Biol. Cell. 1999; 10: 1997-2015Crossref PubMed Scopus (92) Google Scholar, 11Silverstein A.M. Barrow C.A. Davis A.J. Mumby M.C. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 4221-4226Crossref PubMed Scopus (228) Google Scholar, 12Strack S. J. Biol. Chem. 2002; 277: 41525-41532Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). B′ family regulatory subunits are phosphoproteins that have been variously implicated in the control of Wnt/β-catenin signaling, survival, and synaptic plasticity (11Silverstein A.M. Barrow C.A. Davis A.J. Mumby M.C. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 4221-4226Crossref PubMed Scopus (228) Google Scholar, 13McCright B. Rivers A.M. Audlin S. Virshup D.M. J. Biol. Chem. 1996; 271: 22081-22089Abstract Full Text Full Text PDF PubMed Scopus (327) Google Scholar, 14Seeling J.M. Miller J.R. Gil R. Moon R.T. White R. Virshup D.M. Science. 1999; 283: 2089-2091Crossref PubMed Scopus (366) Google Scholar, 15Fukunaga K. Muller D. Ohmitsu M. Bako E. DePaoli-Roach A.A. Miyamoto E. J. Neurochem. 2000; 74: 807-817Crossref PubMed Scopus (76) Google Scholar, 16Li X. Scuderi A. Letsou A. Virshup D.M. Mol. Cell. Biol. 2002; 22: 3674-3684Crossref PubMed Scopus (120) Google Scholar, 17Ruvolo P.P. Clark W. Mumby M. Gao F. May W.S. J. Biol. Chem. 2002; 277: 22847-22852Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar). Lastly, B″ regulatory subunits are nuclear, calcium-binding proteins that may regulate cell cycle progression (18Voorhoeve P.M. Hijmans E.M. Bernards R. Oncogene. 1999; 18: 515-524Crossref PubMed Scopus (89) Google Scholar, 19Yan Z. Fedorov S.A. Mumby M.C. Williams R.S. Mol. Cell. Biol. 2000; 20: 1021-1029Crossref PubMed Scopus (98) Google Scholar, 20Janssens V. Jordens J. Stevens I. Van Hoof C. Martens E. De Smedt H. Engelborghs Y. Waelkens E. Goris J. J. Biol. Chem. 2003; 278: 10697-10706Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). Although some regulatory subunits are expressed in a tissue- and developmental stage-specific pattern, any given mammalian cell is believed to express several dozen distinct PP2A heterotrimers formed by the combination of the 16 PP2A subunit genes. This complexity presents a considerable challenge, particularly because functional redundancy can complicate the interpretation of genetic ablation experiments. Representing the only PP2A subunit knocked out to date, deletion of the major catalytic subunit (Cα) leads to early embryonic lethality, because the mesoderm does not form (21Gotz J. Probst A. Ehler E. Hemmings B. Kues W. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 12370-12375Crossref PubMed Scopus (184) Google Scholar). Most PP2A catalytic subunits in the cell form heterotrimeric complexes followed by the AC core dimer and, finally, numerous lower abundance complexes (22Murata K. Wu J. Brautigan D.L. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 10624-10629Crossref PubMed Scopus (195) Google Scholar, 23Bennin D.A. Arachchige Don A.S. Brake T. McKenzie J.L. Rosenbaum H. Ortiz L. DePaoli-Roach A.A. Horne M.C. J. Biol. Chem. 2002; 277: 27449-27467Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar, 24Ogris E. Du X. Nelson K.C. Mak E.K. Yu X.X. Lane W.S. Pallas D.C. J. Biol. Chem. 1999; 274: 14382-14391Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar, 25Cayla X. Goris J. Hermann J. Hendrix P. Ozon R. Merlevede W. Biochemistry. 1990; 29: 658-667Crossref PubMed Scopus (77) Google Scholar, 26Fellner T. Lackner D.H. Hombauer H. Piribauer P. Mudrak I. Zaragoza K. Juno C. Ogris E. Genes Dev. 2003; 17: 2138-2150Crossref PubMed Scopus (87) Google Scholar, 27Moreno C.S. Park S. Nelson K. Ashby D. Hubalek F. Lane W.S. Pallas D.C. J. Biol. Chem. 2000; 275: 5257-5263Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar). Highlighting the role of PP2A in growth control, several viruses encode proteins that subvert PP2A activity by binding to either dimeric or trimeric forms of the phosphatase (1Janssens V. Goris J. Biochem. J. 2001; 353: 417-439Crossref PubMed Scopus (1542) Google Scholar). To begin to address the roles of the different forms of PP2A in mammalian cells, we report here the consequences of down-regulating the major scaffolding (Aα) subunit by inducible RNAi. We find that Aα RNAi destabilizes other PP2A subunits, impairs Akt signaling, and leads to apoptotic cell death. Rescue experiments involving Aα mutants with different regulatory subunit binding specificities demonstrate that PP2A heterotrimers containing members of all three regulatory subunit families contribute to cell survival. Plasmid Construction—Two tetracycline operator (TO) sites were inserted flanking the TATA box of the H1 promoter to create a vector for doxycycline (Dox)-inducible RNAi. The H1-TO promoter cassette was PCR-amplified from pSUPER (28Brummelkamp T.R. Bernards R. Agami R. Science. 2002; 296: 550-553Crossref PubMed Scopus (3968) Google Scholar) with the primers 5′-TCCCCCGGGCTGCAGGAATTC-3′ (forward) and 5′-GAAGATCTCTATCACTGATAGGGACTTATAAGTCTCTATCACTGATAGGGATTTCACGTTTATGGTGATTTCCC-3′ (TO sites are underlined). The PCR product was digested with EcoR1/BglII and ligated back into pSUPER. To add a neomycin resistance marker, the H1-TO cassette was re-isolated by digestion with BamHI/HindIII and ligated into pcDNA3.1 (Invitrogen) digested with BglII/HindIII. The hairpin (hp)RNA encoding sequence targeting Aα was synthesized as two complementary primers (5′-GATCCCCGGTCAAAGAGTTCTGTGAATTCAAGAGATTCACAGAACTCTTTGACCTTTTTGGAAA-3′ (forward) and 5′-AGCTTTTCCAAAAAGGTCAAAGAGTTCTGTGAATCTCTTGAATTCACAGAACTCTTTGACCGGG-3′ (reverse); Aα complementary sequence is underlined), phosphorylated, and ligated into pcDNA3.1/H1-TO. pcDNA3 plasmids expressing Aα and Aβ wild-type and Aα mutants harboring a C-terminal EE-epitope tag were described previously (3Zhou J. Pham H.T. Ruediger R. Walter G. Biochem. J. 2003; 369: 387-398Crossref PubMed Scopus (71) Google Scholar, 29Ruediger R. Fields K. Walter G. J. Virol. 1999; 73: 839-842Crossref PubMed Google Scholar) and kindly provided by Gernot Walter (University of California at San Diego). The RNAi-resistant Aα cDNAs were generated by double-stranded DNA mutagenesis according to the QuikChange protocol using Pfu Ultra polymerase (Stratagene) (sense mutagenic primer was 5′-GGGCCGCAGCCTCCCACAAAGTGAAGGAATTCTGTGAAAACCTCTCAGC-3′; base changes are underlined). For Dox-inducible expression, Aα/Aβ cDNAs were excised with HindIII/XbaI and subcloned into pcDNA5/TO (Invitrogen). All PCR products were subjected to automated sequencing. Stable Cell Line Generation—PC6-3 cells stably expressing the tetracycline repressor (12Strack S. J. Biol. Chem. 2002; 277: 41525-41532Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar) were transfected with the linearized vector expressing Aα-directed hpRNA under the control of the H1-TO promoter and selected in 500 μg/ml G418 and 2 μg/ml blasticidin essentially as described (12Strack S. J. Biol. Chem. 2002; 277: 41525-41532Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). 36 clones were expanded and screened for Dox-dependent down-regulation of Aα by immunoblotting. A-exchange cells with concomitant induction of Aα mutants were established by transfecting an inducible Aα RNAi clone with linearized, RNAi-resistant Aα cDNAs in pcDNA5/TO, followed by selection in 500 μg/ml hygromycin, 200 μg/ml G418, and 2 μg/ml blasticidin. Protein Phosphatase Assays—Aα RNAi PC6-3 cells growing in 6-well plates were lysed in buffer containing 1% Triton X-100 (v/v), 150 mm NaCl, 20 mm Tris, pH 7.5, 1 mm EDTA, 1 mm EGTA, 1 mm phenylmethylsulfonyl fluoride, 1 μg/ml leupeptin, and 1 mm benzamidine, and insoluble material was removed by centrifugation at 22,000 × g for 15 min. Cleared lysates were diluted 1:100 to 1:200 in 2 mg/ml bovine serum albumin, 50 mm Tris, pH 7.5, 2 mm EDTA, 2 mm EGTA, 2 mm dithiothreitol, 1 mm benzamidine, and 1 mg/ml leupeptin. Myelin basic protein was phosphorylated with [γ-33P]ATP by protein kinase A as described (8Dagda R.K. Zaucha J.A. Wadzinski B.E. Strack S. J. Biol. Chem. 2003; 278: 24976-24985Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar) and diluted to ∼10,000 cpm/μl in the same buffer. Phosphatase reactions were started by the addition of 5 μl of diluted lysate to 20 μl of diluted substrate, incubated for 30 min at 30 °C with intermittent agitation on an Eppendorf shaking incubator, and terminated by the addition of trichloroacetic acid to a final concentration of 20% (w/v). Following centrifugation at 22,000 × g, acid-soluble 33P-phosphate was quantified by liquid scintillation counting. PP2A activity was determined by subtracting the activity in the presence of 2.5 nm okadaic acid from the total activity (30Cohen P. Methods Enzymol. 1991; 201: 389-398Crossref PubMed Scopus (205) Google Scholar). Less than 20% substrate dephosphorylation occurred under all assay conditions. Survival and Apoptosis Assays—Aα RNAi PC6-3 cells were cultured (37 °C with 5% CO2) in PC12 growth medium (10% horse serum and 5% fetal bovine serum in RPMI 1640) containing 200 μg/ml G418 and 2 μg/ml blasticidin. A-exchange PC6-3 cell medium additionally contained 200 μg/ml hygromycin. For survival/apoptosis assays, cells were seeded in 24- or 96-well plates at 50,000 or 2,000 cells/well, respectively, followed by the addition of 1 μg/ml Dox or ethanol vehicle (final 0.1%) at different time points. Cell density was assayed by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfenyl)-2H-tetrazolium (MTS tetrazolium) reduction to formazan according to the manufacturer's instructions (CellTiter 96® AQueous nonradioactive cell proliferation assay, Promega). Formazan production was quantified after 2–4 h by an absorbance measurement at 490 nm using a 96-well plate reader. Previous apoptosis studies with PC6-3 cells have documented excellent correlation between cell counts and metabolic activity as assayed by tetrazolium salt reduction (31Pittman R.N. Wang S. DiBenedetto A.J. Mills J.C. J. Neurosci. 1993; 13: 3669-3680Crossref PubMed Google Scholar). For apoptosis assays based on nuclear morphology, fixed cells in 24-well plates were stained with 1 μg/ml Hoechst 33342 and analyzed by epifluorescence microscopy as described (8Dagda R.K. Zaucha J.A. Wadzinski B.E. Strack S. J. Biol. Chem. 2003; 278: 24976-24985Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). For trypan blue exclusion and annexin V assays, cells were trypsinized, stained with either a 0.2% trypan blue dye solution or Cy3-conjugated annexin V according to the manufacturer's protocol (United States Biological, San Antonio, TX) and counted in a hemocytometer under transmission or epifluorescence illumination. Caspase-3/7 activity was determined by cleavage of the fluorescent substrate acetyl-Asp-Glu-Val-Asp-aminomethyl-coumarin (Ac-DEVD-AMC; Biomol, Hamburg, Germany). Cells cultured in the presence or absence of 50 μm of the pan-caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp (OMe) fluoromethylketone (Z-VAD-fmk; Calbiochem Novabiochem) were treated with 20 μm Ac-DEVD-AMC for 24 to 48 h prior to fluorescence measurement (excitation at 360 nm, emission at 460 nm) using a 96-well plate reader. Transfection Experiments—COS-M6 or PC6-3 cells were transfected using LipofectAMINE 2000 (BD Biosciences), and EE epitope-tagged A subunits were immunoprecipitated as described previously (8Dagda R.K. Zaucha J.A. Wadzinski B.E. Strack S. J. Biol. Chem. 2003; 278: 24976-24985Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). β-galactosidase activity-based survival assays were performed by co-transfecting β-galactosidase and A subunit expression plasmids at a 1:10 mass ratio. After 0–11 days in Dox, survival was determined with a chemiluminescent β-galactosidase assay (Galacto Star, Applied Biosystems). Antibodies—Pan-A (rat monoclonal 6G3), Aα (rat monoclonal 6F9), and Aβ (rabbit polyclonal) antibodies were kindly provided by Gernot Walter (University of California at San Diego). Polyclonal antibodies against B′α and B′δ were a gift from David Virshup (University of Utah), and the polyclonal PR59 antibody and pan-B antibody were from Egon Ogris (Vienna Biocenter) and Brian Wadzinski (Vanderbilt University), respectively. Antibodies obtained from commercial sources recognized the FLAG epitope (mouse monoclonal M2, Sigma; rabbit polyclonal, Affinity Bioreagents, Golden, CO), the EE epitope (Babco, Richmond, CA), the PP2A catalytic subunit (BD Pharmingen), striatin (Santa Cruz Biotechnology, Santa Cruz, CA), and Ser(P)-473 Akt and total Akt (Cell Signaling, Beverly, MA). Knock-down of PP2A/Aa by Inducible RNAi—Stable knock-down of gene expression is a prerequisite for carrying out long term growth and survival studies in a homogenous cell population, and stable expression of double-stranded RNA to down-regulate endogenous mRNAs is increasingly used as a less time consuming alternative to traditional gene knock-outs (32Hannon G.J. Conklin D.S. Methods Mol. Biol. 2004; 257: 255-266PubMed Google Scholar). An inducible RNAi approach avoids a possible selection bias and allows for the analysis of essential gene products. We modified the H1 promoter, which is widely used to drive small hpRNA expression for RNAi (28Brummelkamp T.R. Bernards R. Agami R. Science. 2002; 296: 550-553Crossref PubMed Scopus (3968) Google Scholar), by replacing evolutionarily non-conserved sequences before and after the TATA box with the bacterial TO sequence. A similar inducible construct with a single TO site between the TATA box and the transcription start site was reported recently (33Van De Wetering M. Oving I. Muncan V. Pon Fong M.T. Brantjes H. Van Leenen D. Holstege F.C. Brummelkamp T.R. Agami R. Clevers H. EMBO Rep. 2003; 4: 609-615Crossref PubMed Scopus (463) Google Scholar) and has since become commercially available (pSUPERIOR; OligoEngine, Seattle, WA). The induction principle is illustrated in Fig. 1A. In cells expressing the tetracycline repressor (TR) protein, TR silences the hybrid H1-TO promoter in the absence of tetracycline or its more stable analog, Dox. Upon the addition of an inducer, TR dissociates from the promoter, allowing hpRNA expression and RNAi to proceed. We found the H1-TO promoter to lack detectable leak expression and to effect gene knock-down with a potency comparable to the parental H1 promoter. Importantly, this inducible system appears to require the repressor function of the native TR protein in that TR cannot be replaced with the more widely used VP16 transactivation domain fusions of the TR DNA binding domain (34Gossen M. Freundlieb S. Bender G. Muller G. Hillen W. Bujard H. Science. 1995; 268: 1766-1769Crossref PubMed Scopus (2040) Google Scholar) (data not shown). To study the role of PP2A holoenzymes in cell survival, we chose to target the scaffolding Aα subunit because it is the best-characterized PP2A subunit in terms of structure/function (4Groves M.R. Hanlon N. Turowski P. Hemmings B.A. Barford D. Cell. 1999; 96: 99-110Abstract Full Text Full Text PDF PubMed Scopus (354) Google Scholar, 29Ruediger R. Fields K. Walter G. J. Virol. 1999; 73: 839-842Crossref PubMed Google Scholar). We used TR-expressing PC6-3 cells as a host for the inducible RNAi cassette (12Strack S. J. Biol. Chem. 2002; 277: 41525-41532Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). PC6-3 cells are a subline of rat PC12 pheochromocytoma cells that differentiate to a sympathetic neuronal-like phenotype upon the addition of nerve growth factor (31Pittman R.N. Wang S. DiBenedetto A.J. Mills J.C. J. Neurosci. 1993; 13: 3669-3680Crossref PubMed Google Scholar). Cells were selected in G418, and multiple, independently isolated clones were scored for Dox-inducible down-regulation of the endogenous A subunit by immunoblotting. Two positive clones were characterized further and exhibited identical phenotypes in survival and PP2A subunit degradation assays. Experiments with one of these clones are reported here. Proteosomal Degradation of PP2A Holoenzymes after Aα RNAi—Dox addition resulted in a time-dependent (∼70% by day 4) decrease in A subunit expression as detected with an antibody that recognizes both α and β gene products (Fig. 1, B and C). An Aα-specific antibody showed the same time course of down-regulation, indicating that Aα is the predominant scaffolding subunit isoform expressed in PC6-3 cells and that Aα down-regulation does not cause an appreciable compensatory induction of Aβ. Previous studies in Drosophila S2 cells showed that RNAi of the A or C subunit leads to the loss of the other PP2A subunits (11Silverstein A.M. Barrow C.A. Davis A.J. Mumby M.C. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 4221-4226Crossref PubMed Scopus (228) Google Scholar, 16Li X. Scuderi A. Letsou A. Virshup D.M. Mol. Cell. Biol. 2002; 22: 3674-3684Crossref PubMed Scopus (120) Google Scholar). Consistent with this result, Aα knock-down in mammalian cells was paralleled by a significant decrease in PP2A catalytic subunit immunoreactivity (Fig. 1, B and C), which was well matched by a decline in total PP2A phosphatase activity (Fig. 1D). In addition to the PP2A C subunit, levels of B family subunits (detected by a pan-B specific antibody) as well as B′ family subunits (detected with B′α- and B′δ-specific antibodies) fell dramatically following Aα knock-down (Fig. 2). In contrast, neither the B″ subunit PR59 nor the PP2A-associated protein striatin responded to Dox treatment for 3 days. These data suggest that C, B, and B′ family subunits are stable only when complexed to the A subunit, whereas B″ and striatin proteins can exist independently of PP2A in the cell. We have demonstrated previously that ectopically expressed, monomeric Bγ mutants are rapidly cleared by ubiquitination and proteosomal degradation (7Strack S. Ruediger R. Walter G. Dagda R.K. Barwacz C.A. Cribbs J.T. J. Biol. Chem. 2002; 277: 20750-20755Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). To examine the mechanism of PP2A subunit removal following Aα knock-down, cells were treated with MG132, a proteosomal inhibitor, or leupeptin, which inhibits lysosomal degradation, in the absence or presence of Dox. Proteosome inhibition restored C and B family subunits almost to control levels, whereas leupeptin had no effect. Neither inhibitor prevented the loss of B′ family subunits following Aα RNAi (Fig. 2). Aα Knock-down Kills Cells by Apoptotic and Non-apoptotic Mechanisms—RNAi-mediated knock-down of the single A and C subunit genes in Drosophila S2 cells causes apoptotic cell death (11Silverstein A.M. Barrow C.A. Davis A.J. Mumby M.C. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 4221-4226Crossref PubMed Scopus (228) Google Scholar, 16Li X. Scuderi A. Letsou A. Virshup D.M. Mol. Cell. Biol. 2002; 22: 3674-3684Crossref PubMed Scopus (120) Google Scholar). We observed a drastic loss of PC6-3 cell viability starting 4 days after Dox treatment to induce Aα-directed hpRNA expression, with almost complete cell loss by 8 days after the Dox treatment (Fig. 3A). In a second, independently isolated clonal cell line with slower kinetics of Aα down-regulation, cell death was first detectable 6 days after Dox addition (data not shown). These results indicate that PC6-3 cells can tolerate a ∼70% drop in Aα levels before viability is compromised. As a control, inducible RNAi of the Bα regulatory subunit of PP2A was associated with a modest (∼20%) decrease in cell number over the same time course, which appeared to be due to a slowing of proliferation rather than mortality (Fig. 3A and data not shown). Starting 4 days after Dox treatment, Aα RNAi was associated with cell rounding and the appearance of membrane blebs (Fig. 3B, inset). Additionally, we observed increases in membrane permeability, annexin V staining, and condensed/fragmented nuclear morphology (Fig. 3B), which occurred over the same time course as the loss of viability (Fig. 3A). As further evidence of apoptotic cell death, Aα RNAi was found to result in a time-dependent increase in caspase-3/7 activity, which could be abolished by the general caspase inhibitor Z-VAD-fmk (50 μm; Fig. 3C). In the same experiments, however, caspase inhibition was unable to prevent the cell death caused by PP2A down-regulation (Fig. 3D). Importantly, the same dose of Z-VAD-fmk completely blocked apoptosis induced by 100 nm stauroporine (data not shown), as was reported previously for PC12 cells (35Ivins K.J. Ivins J.K. Sharp J.P. Cotman C.W. J. Biol. Chem. 1999; 274: 2107-2112Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar). The inability of Z-VAD-fmk to restore viability in PP2A-depleted cells stands in marked contrast to Drosophila studies, which showed that caspase knock-down overcomes PP2A RNAi-mediated cell death (16Li X. Scuderi A. Letsou A. Virshup D.M. Mol. Cell. Biol. 2002; 22: 3674-3684Crossref PubMed Scopus (120) Google Scholar). It would appear that in mammalian cells the loss of PP2A activity initiates redundant caspase-dependent (apoptotic) and caspase-independent cell death programs. Aα Knock-down Impairs Akt Phosphorylation—The Ser/Thr kinase Akt/PKB transduces survival signals from a variety of growth factor receptors (36Nicholson K.M. Anderson N.G. Cell. Signal. 2002; 14: 381-395Crossref PubMed Scopus (1388) Google Scholar). We analyzed Akt signaling by immunoblotting with an antibody that recognizes Ser(P)-473, which is tightly correlated with activity. At the time point analyzed, 3 days with or without Dox, Aα subunit level and cellular PP2A activity are diminished by ∼60 and 20%, respectively (Fig. 1), whereas cells are still completely viable (Fig. 3). We found that Dox treatment decreased basal Akt phosphorylation by 50%. In cells stimulated for 5 and 15 min with epidermal growth factor (10 ng/ml), Aα RNAi caused a similar reduction in phospho-Akt without changing total Akt protein levels (Fig. 4). These results indicate that PP2A holoenzymes are necessary for proper activation of the survival kinase Akt. RNAi Resistant Aα Mutants Prevent Holoenzyme Degradation—Gernot Walter's group has identified several mutations in the N-terminal half of Aα that modulate its ability to interact with select regulatory subunit families without effecting C subunit association (29Ruediger R. Fields K. Walter G. J. Virol. 1999; 73: 839-842Crossref PubMed Google Scholar). For instance, Glu-100 and Glu-101 of Aα form critical salt bridges with basic residues on regulatory subunits (7Strack S. Ruediger R. Walter G. Dagda R.K. Barwacz C.A. Cribbs J.T. J. Biol. Chem. 2002; 277: 20750-20755Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar), and the Aα EE100RR mutant can hence only form PP2A dimers in the cell. The Aα DWF139HA