Voltar
Artigo Acesso aberto Revisado por pares
BIBTEX RIS

Distinct Protein Phosphatase 2A Heterotrimers Modulate Growth Factor Signaling to Extracellular Signal-regulated Kinases and Akt

2005; Elsevier BV; Volume: 280; Issue: 43 Linguagem: Inglês

10.1074/jbc.m506986200

ISSN

1083-351X

Autores

Michael J. Van Kanegan, Deanna G. Adams, Brian E. Wadzinski, Stefan Strack,

Tópico(s)

PI3K/AKT/mTOR signaling in cancer

Resumo

A key regulator of many kinase cascades, heterotrimeric protein serine/threonine phosphatase 2A (PP2A), is composed of catalytic (C), scaffold (A), and variable regulatory subunits (B, B′, B″ gene families). In neuronal PC12 cells, PP2A acts predominantly as a gatekeeper of extracellular signal-regulated kinase (ERK) activity, as shown by inducible RNA interference of the Aα scaffolding subunit and PP2A inhibition by okadaic acid. Although okadaic acid potentiates Akt/protein kinase B and ERK phosphorylation in response to epidermal, basic fibroblast, or nerve growth factor, silencing of Aα paradoxically has the opposite effect. Epidermal growth factor receptor Tyr phosphorylation was unchanged following Aα knockdown, suggesting that chronic Akt and ERK hyperphosphorylation leads to compensatory down-regulation of signaling molecules upstream of Ras and blunted growth factor responses. Inducible exchange of wild-type Aα with a mutant with selective B′ subunit binding deficiency implicated PP2A/B′ heterotrimers as Akt modulators. Conversely, silencing of the B-family regulatory subunits Bα and Bδ led to hyperactivation of ERK stimulated by constitutively active MEK1. In vitro dephosphorylation assays further support a role for Bα and Bδ in targeting the PP2A heterotrimer to dephosphorylate and inactivate ERKs. Thus, receptor tyrosine kinase signaling cascades leading to Akt and ERK activation are modulated by PP2A holoenzymes with distinct regulatory properties. A key regulator of many kinase cascades, heterotrimeric protein serine/threonine phosphatase 2A (PP2A), is composed of catalytic (C), scaffold (A), and variable regulatory subunits (B, B′, B″ gene families). In neuronal PC12 cells, PP2A acts predominantly as a gatekeeper of extracellular signal-regulated kinase (ERK) activity, as shown by inducible RNA interference of the Aα scaffolding subunit and PP2A inhibition by okadaic acid. Although okadaic acid potentiates Akt/protein kinase B and ERK phosphorylation in response to epidermal, basic fibroblast, or nerve growth factor, silencing of Aα paradoxically has the opposite effect. Epidermal growth factor receptor Tyr phosphorylation was unchanged following Aα knockdown, suggesting that chronic Akt and ERK hyperphosphorylation leads to compensatory down-regulation of signaling molecules upstream of Ras and blunted growth factor responses. Inducible exchange of wild-type Aα with a mutant with selective B′ subunit binding deficiency implicated PP2A/B′ heterotrimers as Akt modulators. Conversely, silencing of the B-family regulatory subunits Bα and Bδ led to hyperactivation of ERK stimulated by constitutively active MEK1. In vitro dephosphorylation assays further support a role for Bα and Bδ in targeting the PP2A heterotrimer to dephosphorylate and inactivate ERKs. Thus, receptor tyrosine kinase signaling cascades leading to Akt and ERK activation are modulated by PP2A holoenzymes with distinct regulatory properties. At least 99% of protein phosphorylation in eukaryotic cells occurs on Ser and Thr residues. The greater than 300 protein Ser/Thr kinases in the human genome are opposed by less than 30 protein Ser/Thr phosphatases, of which protein phosphatase 1 and protein phosphatase 2A (PP2A) 2The abbreviations used are:PP2Aprotein phosphatase 2AEGFepidermal growth factorFGF2fibroblast growth factor-2NGFnerve growth factorERKextracellular signal-regulated kinaseMAPKmitogen-activated protein kinaseMEK(1)MAPK/ERK kinase(1)OAokadaic acidRNAiRNA interferenceshRNAshort hairpin RNADoxdoxycycline 2The abbreviations used are:PP2Aprotein phosphatase 2AEGFepidermal growth factorFGF2fibroblast growth factor-2NGFnerve growth factorERKextracellular signal-regulated kinaseMAPKmitogen-activated protein kinaseMEK(1)MAPK/ERK kinase(1)OAokadaic acidRNAiRNA interferenceshRNAshort hairpin RNADoxdoxycycline contribute the bulk of activity in most cell types. The notion of Ser/Thr phosphatases as promiscuous and constitutively active enzymes that simply provide the substrates for regulated kinase signaling has been challenged by the discovery of batteries of catalytic subunit-interacting proteins, which impart substrate specificity, subcellular localization, and responsiveness to phosphorylation (1Janssens V. Goris J. Biochem. J. 2001; 353: 417-439Crossref PubMed Scopus (1532) Google Scholar, 2Ceulemans H. Bollen M. Physiol. Rev. 2004; 84: 1-39Crossref PubMed Scopus (526) Google Scholar). In the case of PP2A, the predominant holoenzyme is formed by association of a core dimer of catalytic and scaffold subunits with one of a least 12 regulatory subunits in vertebrates. Since vertebrate A and C subunits are each encoded by two genes and since many regulatory subunits are diversified by alternative splicing, several dozen different PP2A heterotrimers are likely to exist in any given cell type. The three unrelated regulatory subunit gene families (B, B′, B″) are almost certain to utilize different mechanisms to control enzymatic activity and cellular localization of PP2A. The B-family (PR55) of regulatory subunits consists of predicted β-propellers with divergent N-terminal tails that act as subcellular targeting signals (3Dagda R.K. Barwacz C.A. Cribbs J.T. Strack S. J. Biol. Chem. 2005; 280: 27375-27382Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar, 4Dagda 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, 5Strack 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). In addition to interacting with phosphatase substrates (6Seeling J.M. Miller J.R. Gil R. Moon R.T. White R. Virshup D.M. Science. 1999; 283: 2089-2091Crossref PubMed Scopus (365) Google Scholar, 7Okamoto K. Li H. Jensen M.R. Zhang T. Taya Y. Thorgeirsson S.S. Prives C. Mol. Cell. 2002; 9: 761-771Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar, 8Bennin 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, 9Ruvolo 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 (136) Google Scholar, 10Ito A. Kataoka T.R. Watanabe M. Nishiyama K. Mazaki Y. Sabe H. Kitamura Y. Nojima H. EMBO J. 2000; 19: 562-571Crossref PubMed Scopus (144) Google Scholar, 11Berry M. Gehring W. EMBO J. 2000; 19: 2946-2957Crossref PubMed Scopus (56) Google Scholar, 12Firulli B.A. Howard M.J. McDaid J.R. McIlreavey L. Dionne K.M. Centonze V.E. Cserjesi P. Virshup D.M. Firulli A.B. Mol. Cell. 2003; 12: 1225-1237Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar), B′-family (also referred to as B56, PR61) subunits are heavily phosphorylated, which may confer regulation by second messengers (13McCright B. Rivers A.M. Audlin S. Virshup D.M. J. Biol. Chem. 1996; 271: 22081-22089Abstract Full Text Full Text PDF PubMed Scopus (326) Google Scholar, 14Xu Z. Williams B.R.G. Mol. Cell. Biol. 2000; 20: 5285-5299Crossref PubMed Scopus (98) Google Scholar, 15Usui H. Inoue R. Tanabe O. Nishito Y. Shimizu M. Hayashi H. Kagamiyama H. Takeda M. FEBS Lett. 1998; 430: 312-316Crossref PubMed Scopus (78) Google Scholar, 16Fukunaga K. Muller D. Ohmitsu M. Bako E. DePaoli-Roach A.A. Miyamoto E. J. Neurochem. 2000; 74: 807-817Crossref PubMed Scopus (76) Google Scholar). Members of the B″-family of PP2A subunits (PR72/130, PR59, PR48) feature two calcium binding EF hands with presumed structural rather than regulatory functions (17Janssens 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). Striatin and SG2NA have been referred to as B″ regulatory subunits (18Moreno 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). It may be more appropriate to refer to them as PP2A dimer-associated proteins since their stability does not depend on association with the PP2A core enzyme (17Janssens 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, 19Strack S. Cribbs J.T. Gomez L. J. Biol. Chem. 2004; 279: 47732-47739Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar) and since they lack the A subunit binding consensus motif common to B, B′, and B″ subunits (20Li X. Virshup D.M. Eur. J. Biochem. 2002; 269: 546-552Crossref PubMed Scopus (69) Google Scholar). protein phosphatase 2A epidermal growth factor fibroblast growth factor-2 nerve growth factor extracellular signal-regulated kinase mitogen-activated protein kinase MAPK/ERK kinase(1) okadaic acid RNA interference short hairpin RNA doxycycline protein phosphatase 2A epidermal growth factor fibroblast growth factor-2 nerve growth factor extracellular signal-regulated kinase mitogen-activated protein kinase MAPK/ERK kinase(1) okadaic acid RNA interference short hairpin RNA doxycycline A growing body of evidence indicates that PP2A has complex inhibitory and stimulatory effects on hormone and growth factor signaling, in particular the extracellular-signal regulated (ERK)/mitogen-activated protein kinase (MAPK) cascade (21Millward T.A. Zolnierowicz S. Hemmings B.A. Trends Biochem. Sci. 1999; 24: 186-191Abstract Full Text Full Text PDF PubMed Scopus (706) Google Scholar). PP2A substrates include G-protein-coupled receptors and receptor tyrosine kinases (22Spurney R.F. J. Pharmacol. Exp. Ther. 2001; 296: 592-599PubMed Google Scholar, 23Fan G.H. Yang W. Sai J. Richmond A. J. Biol. Chem. 2001; 276: 16960-16968Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar, 24Hashigasako A. Machide M. Nakamura T. Matsumoto K. J. Biol. Chem. 2004; 279: 26445-26452Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar), receptor-associated proteins (25El-Shemerly M.Y. Besser D. Nagasawa M. Nagamine Y. J. Biol. Chem. 1997; 272: 30599-30602Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar, 26Gual P. Giordano S. Anguissola S. Parker P.J. Comoglio P.M. Oncogene. 2001; 20: 156-166Crossref PubMed Scopus (36) Google Scholar, 27Hartley D. Cooper G.M. J. Cell. Biochem. 2002; 85: 304-314Crossref PubMed Scopus (89) Google Scholar, 28Hupfeld C.J. Resnik J.L. Ugi S. Olefsky J.M. J. Biol. Chem. 2005; 280: 1016-1023Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar), and all three kinases of the ERK/MAPK cascade core module (Raf (29Abraham D. Podar K. Pacher M. Kubicek M. Welzel N. Hemmings B.A. Dilworth S.M. Mischak H. Kolch W. Baccarini M. J. Biol. Chem. 2000; 275: 22300-22304Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar, 30Ory S. Zhou M. Conrads T.P. Veenstra T.D. Morrison D.K. Curr. Biol. 2003; 13: 1356-1364Abstract Full Text Full Text PDF PubMed Scopus (243) Google Scholar, 31Dhillon A.S. Meikle S. Yazici Z. Eulitz M. Kolch W. EMBO J. 2002; 21: 64-71Crossref PubMed Scopus (228) Google Scholar, 32Kubicek M. Pacher M. Abraham D. Podar K. Eulitz M. Baccarini M. J. Biol. Chem. 2002; 277: 7913-7919Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar), MAPK/ERK kinase (MEK) (33Yu L.G. Packman L.C. Weldon M. Hamlett J. Rhodes J.M. J. Biol. Chem. 2004; 279: 41377-41383Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar, 34Westermarck J. Li S.P. Kallunki T. Han J. Kahari V.M. Mol. Cell Biol. 2001; 21: 2373-2383Crossref PubMed Scopus (177) Google Scholar, 35Heriche J.K. Lebrin F. Rabilloud T. Leroy D. Chambaz E.M. Goldberg Y. Science. 1997; 276: 952-955Crossref PubMed Scopus (249) Google Scholar), and ERK (36Zhou B. Wang Z.X. Zhao Y. Brautigan D.L. Zhang Z.Y. J. Biol. Chem. 2002; 277: 31818-31825Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar, 37Alessi D.R. Gomez N. Moorhead G. Lewis T. Keyse S.M. Cohen P. Curr. Biol. 1995; 5: 283-295Abstract Full Text Full Text PDF PubMed Scopus (321) Google Scholar)). Here, we have begun to dissect the contribution of different PP2A holoenzymes to growth factor signaling in PC12 cells. The net effect of total PP2A silencing or inhibition was ERK and Akt hyperphosphorylation, most likely as a consequence of direct dephosphorylation of these kinases by PP2A. Cautioning against exclusive reliance on silencing approaches, protracted inhibition of PP2A by RNA interference (RNAi) resulted in a compensatory uncoupling of growth factor receptors from their kinase effectors. A combination of mutant Aα subunit exchange, regulatory subunit RNAi, and in vitro dephosphorylation assays indicated that PP2A/B′ heterotrimers regulate Akt, whereas PP2A/Bα and Bδ directly dephosphorylate ERKs. Cell Culture—Parental PC6-3 cells were cultured (37 °C, 5% CO2) in RPMI 1640 containing 10% horse and 5% fetal bovine serum (both heat-inactivated). Aα-RNAi cell medium was supplemented with 2 μg/ml blasticidin and 200 μg/ml G418 to maintain tetracycline repressor and inducible short hairpin (sh)RNA constructs, respectively. Medium of Aα-exchange cells additionally included 200 μg/ml hygromycin for selection of the inducible Aα DTP177AAA construct (19Strack S. Cribbs J.T. Gomez L. J. Biol. Chem. 2004; 279: 47732-47739Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar). Antibiotics were omitted from cultures seeded for experiments. COS-M6 cells were cultured in Dulbecco's modified Eagle's medium, 10% fetal bovine serum. Antibodies—the pan-A (rat monoclonal 6G3) antibody was kindly provided by Gernot Walter (University of California, San Diego). Polyclonal antibodies against B′α and B′β were a gift from David Virshup (University of Utah), and the polyclonal PR59 antibody was from Egon Ogris (Vienna Biocenter). Commercial sources of antibodies were as follows: FLAG epitope (M2 and its agarose conjugate, Sigma); PP2A catalytic subunit (Pharmingen); total ERK (Santa Cruz Biotechnology, Santa Cruz, CA); phospho-Ser-473 Akt, total Akt, phospho-ERK1/2, phospho-Smad1/5/8, phospho-Tyr-1045 epidermal growth factor (EGF) receptor (Cell Signaling, Beverly, MA); phospho-Tyr (4G10) and total EGF receptor (Upstate Biotechnology, Lake Placid, NY). Other Reagents—Rat nerve growth factor (NGF, 2.5 S), mouse EGF, and recombinant human fibroblast growth factor-2 (FGF2) were purchased from Upstate Biotechnology, Sigma, and Alomone Labs (Jerusalem, Israel), respectively. Growth factors were stored at –20 °C in lyophilized aliquots and dissolved to 100× in medium prior to use. Okadaic acid and microcystin-LR were purchased from Alexis (Lausanne, Switzerland). Plasmids encoding FLAG-ERK2, Ha-Ras V12, the EGF receptor, and FLAG-B″/PR72 were donated by Phillip Stork (Vollum Institute), Jeffrey Pessin (State University of New York (SUNY) Stony Brook), Nancy Lill (University of Iowa), and Eric Cohen (University of Montreal), respectively. pFC-MEK1, a plasmid encoding constitutively active MEK1 (S218,222D), was supplied as part of the PathDetect Elk1 trans-reporting system (Stratagene, La Jolla, CA). The coding sequence for rat B′β was isolated from rat brain RNA by the reverse-transcriptase-PCR (sequence deposited in GenBank™ as AY251278). FLAG epitope tags were added via PCR to the N terminus of B′β and to the C terminus of Bα and Bδ followed by ligation of PCR products into the pcDNA5/TO expression vector (Invitrogen). MAPK Reporter Assays—The PathDetect Elk1 trans-reporting system was modified for the dual-luciferase assay (Promega, Madison, WI) to quantify ERK activation according to the manufacturers' instructions. PC6-3 cells were plated at 100,000–150,000 cells/well in 24-well plates and transfected 24 h later in triplicate using Lipofectamine 2000 (BD Biosciences). Aα-RNAi and Aα-exchange cells were transfected with 0.5 μg/well reporter plasmid mix (by mass: 92.5% pFR-Luc, 5% pFA2-Elk1, 2.5% pRL-SV40) and 0.5–2 ng/well activator plasmid (Ha-Ras V12, MEK1 S218,222D) or pcDNA3.1 empty vector. Doxycycline (1 μg/ml) or 0.1% ethanol vehicle was added at the same time. PC6-3 cells subjected to transient RNAi were transfected with 0.25 μg/well pSUPER-based plasmids (38Brummelkamp T.R. Bernards R. Agami R. Science. 2002; 296: 550-553Crossref PubMed Scopus (3963) Google Scholar) expressing shRNAs, 0.25 μg/well reporter plasmid mix, and 0.5 ng/well MEK1 S218,222D. After 3 days, cells were preincubated for 1 h in medium with one-tenth original serum concentration ±300 nm okadaic acid (OA) followed by 5–6 h of stimulation with growth factors as indicated. Cultures were lysed and subjected to dual-luciferase assays using a Berthold Sirius tube luminometer. Photinus and Renilla luciferase activity ratios were expressed relative to basal conditions without ERK activator plasmids or growth factor stimulation. Quantitative Immunoblotting with Phospho-specific Antibodies—Aα-RNAi or Aα-exchange cells were seeded at 150,000 cell/well in 24-well plates, in some experiments transfected with EGF receptor plasmids, and treated ±doxycycline (Dox) for 3 days. Cells were serum-starved ±300 nm OA for at least 2 h prior to adding growth factors at staggered times. After washing with phosphate-buffered saline, cells were harvested in SDS sample buffer supplemented with 1 mm EDTA and 1 μm microcystin-LR, and lysates were probe tip-sonicated to shear the DNA. Protein concentrations were determined by a dot blot assay and normalized prior to SDS-polyacrylamide electrophoresis and electrophoretic transfer of samples to nitrocellulose membranes. Enhanced chemiluminescence (SuperSignal, Pierce) images were captured using a Kodak Imager 440, and band intensities were quantified with the ImageJ software gel analyzer plug-in. Phospho-specific antibody signals were divided by total protein antibody signals to control for loading differences. All signal intensities scaled linearly with the amount of lysate loaded. RNAi of Bα and Bδ—Double-stranded oligonucleotides encoding shRNAs were ligated into the H1 promoter-driven pSUPER plasmid as described (38Brummelkamp T.R. Bernards R. Agami R. Science. 2002; 296: 550-553Crossref PubMed Scopus (3963) Google Scholar). The following sequences were targeted for RNAi: Bα (α3), 5′-AATCCAGTCTCATAGCAGAGG-3′; Bα (α4), 5′-AAGTGGCAAGCGAAAGAAAGA-3′; Bδ (δ1), 5′-AACAGAATGCTGCTCATTTTC-3′; Bδ (δ2), 5′-AACACTCGGAGGGATGTTACA-3′. [32P]ERK2 Dephosphorylation Assays—2 × 106 COS-M6 cells were plated into a 100-mm dish and cotransfected 24 h later with 10.8 μgof FLAG-ERK2 and 1.2 μg of MEK1 S218,222D plasmid using Lipofectamine 2000. After 3 days, cells were metabolically labeled for 3 h with 0.6 mCi/ml 32PO42− (ICN, Irvine, CA) in phosphate-free Dulbecco's modified Eagle's medium containing 1% dialyzed fetal bovine serum. Cells were rinsed once with phosphate-buffered saline and lysed in 1.5 ml of immunoprecipitation buffer (1% Triton X-100, 150 mm NaCl, 20 mm Tris, pH 7.5, 1 mm EDTA, 1 mm EGTA, 2 mm dithiothreitol, 1 mm β-glycerophosphate, 1 mm Na3VO4, 1 mm Na4P2O7, 1 μm microcystin-LR, 1 mm phenylmethylsulfonyl fluoride, 1 μg/ml leupeptin, 1 mm benzamidine). Debris was pelleted (20,000 × g, 15 min), and 32P-labeled FLAG-ERK2 was immunoprecipitated from the cleared lysate using FLAG antibody (M2)-conjugated agarose (3 h at 4 °C). Beads were washed once with 2 ml of immunoprecipitation buffer and four times with 2 ml of immuno-precipitation buffer lacking phosphatase inhibitors. PP2A heterotrimers containing FLAG-tagged regulatory subunits were immunoisolated from COS-M6 as described (4Dagda 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). [32P]ERK2 and PP2A FLAG-immunoprecipitates were stored at –20 °C in buffer containing 50% (v/v) glycerol, 10% (v/v) ethylene glycol, 20 mm Tris, pH 7.5, 5 mm dithiothreitol, 0.5 mm EGTA for up to 1 week. PP2A preparations were adjusted for equal catalytic subunit content after quantitative immunoblotting. For phosphatase assays, 32P-labeled FLAG-ERK2 beads (∼10,000 cpm/reaction) were resuspended in buffer containing 50 mm Tris, pH 7.5, 50 μg/ml bovine serum albumin, 2 mm dithiothreitol, 0.5 mm EDTA, 0.5 mm EGTA, 1 mm benzamidine, 5 μg/ml leupeptin. FLAG peptide (50 μg/ml) was added to dissociate FLAG-tagged substrate and phosphatase from the antibody beads for more efficient dephosphorylation. Reactions were started by the addition of 20 μl of diluted [32P]ERK2 substrate to 5 μl of PP2A holoenzyme preparation, incubated for 30 min at 30 °C with intermittent agitation on an Eppendorf shaking incubator and then terminated by the addition of trichloroacetic acid to a final concentration of 20% (w/v). Following centrifugation at 22,000 × g acid-soluble 32PO42− was quantified by liquid scintillation counting. Percent ERK2 dephosphorylation was calculated after subtraction of counts from a "blank" reaction containing, instead of PP2A, immunoprecipitates from a mock transfection. PP2A Modulates the MAPK Cascade at the Level of ERK—Inducible knockdown of Aα, the principal PP2A scaffold subunit, promotes apoptosis in PC6-3 cells, a subline of neuronal PC12 pheochromocytoma cells. The loss of viability of Aα-RNAi cells starts 4 days after induction by Dox and is associated with attenuated Akt phosphorylation in response to EGF treatment (19Strack S. Cribbs J.T. Gomez L. J. Biol. Chem. 2004; 279: 47732-47739Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar). Viability, as well as growth factor responsiveness, are rescued by constitutive or inducible expression of an RNAi-resistant Aα cDNA (Ref. 19Strack S. Cribbs J.T. Gomez L. J. Biol. Chem. 2004; 279: 47732-47739Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar and data not shown). To further explore the effect of Aα silencing on growth factor signaling, ERK/MAPK reporter assays were carried out in Aα-RNAi cells treated ±Dox for 3 days to induce shRNA expression, which results in 20–25% inhibition of PP2A activity (19Strack S. Cribbs J.T. Gomez L. J. Biol. Chem. 2004; 279: 47732-47739Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar). Cultures were also treated for 5 h with 300 nm okadaic acid to acutely inhibit PP2A. At this concentration, okadaic acid is a selective inhibitor of PP2A, and possibly, the related PP4, PP5, and PP6 catalytic subunits in intact cells (39Favre B. Turowski P. Hemmings B.A. J. Biol. Chem. 1997; 272: 13856-13863Abstract Full Text Full Text PDF PubMed Scopus (280) Google Scholar, 40Honkanen R.E. Golden T. Curr. Med. Chem. 2002; 9: 2055-2075Crossref PubMed Scopus (230) Google Scholar). Okadaic acid increased MAPK reporter activation dramatically (170 ± 41-fold, n = 3), whereas Aα silencing had a more modest, but still significant, effect (2.7 ± 0.3-fold, n = 3; Fig. 1A). Similar effects were observed by immunoblotting with an antibody specific for ERK1/2 dually phosphorylated on Thr and Tyr residues in the activation loop (Fig. 1A). To determine the level at which PP2A inhibits the MAPK cascade, cells were transfected with constitutively active Ras (oncogenic Ha-Ras V12) or MEK1 (S218,222D). Plasmid concentrations were titrated to achieve 100–150-fold reporter activation over empty vector transfected cells. Okadaic acid and Aα silencing stimulated ERK activity further, to about 600- and 400-fold above basal, respectively (Fig. 1B). Importantly, Ras V12 and MEK1 S218,222D-induced reporter activations were equivalently augmented by disabling the phosphatase. The most parsimonious interpretation of these results is that PP2A inhibits the ERK/MAPK cascade predominantly at the level of the terminal kinases, the ERKs. PP2A Silencing Attenuates, whereas PP2A Inhibition Promotes Growth Factor-dependent Akt and ERK Phosphorylation—To further investigate the loss of EGF responsiveness following Aα silencing (19Strack S. Cribbs J.T. Gomez L. J. Biol. Chem. 2004; 279: 47732-47739Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar), Aα-RNAi cells were pretreated with okadaic acid or Dox as before followed by stimulation with various growth factors. EGF stimulation led to transient activation of Akt (detected with a phospho-Ser-473 specific antibody) and ERK, both peaking at 5 min (Fig. 2A). Okadaic acid potentiated basal as well as EGF-stimulated Akt and ERK phosphorylation, whereas Aα silencing attenuated phosphorylation of both kinases. Opposite effects of PP2A inhibition and knockdown were also observed on FGF2- and NGF-stimulated Akt and ERK phosphorylation (Fig. 2B and not shown). On the other hand, phosphorylation of the transcription factors Smad1/5/8 following stimulation of bone morphogenetic protein-2 Ser/Thr kinase receptors was unaffected by either manipulation of PP2A, demonstrating specificity for receptor tyrosine kinase pathways (Fig. 2C). ERK/MAPK reporter assays confirmed that although okadaic acid stimulated growth factor-dependent transcriptional activity by 4–20-fold, Aα knockdown resulted in a paradoxical loss of responsiveness to EGF, FGF2, and NGF (∼50%, Fig. 2D). This blunted reporter activation was observed over a wide range of EGF and NGF concentrations (Fig. 2, E and F). A detailed time course of NGF-dependent Akt and ERK phosphorylation was examined in ±Dox-treated Aα-RNAi cells (representative immunoblot in Fig. 3A, quantification of three independent experiments in Fig. 3, B and C). Aα knockdown decreased peak ERK and Akt phosphorylation somewhat and had a dramatic effect on the sustained phase of NGF signaling. The diminished responsiveness of growth factor signaling cascades following Aα-RNAi not only contrasted with the effects of pharmacological PP2A inhibition but also with the increase in basal and constitutively active Ras/MEK1-dependent ERK activity seen after Aα knockdown (Fig. 1). It was therefore most likely that the blunted growth factor response reflects a cellular adaptation to a chronic decrease in PP2A activity, presumably mediated by feedback of the disinhibited kinases. Aα-RNAi Uncouples EGF Receptor Activation from Downstream Signaling—To pinpoint the site of this compensatory response to Aα silencing, the activation state of the EGF receptor was assessed. Low expression levels (<30,000 receptors/cell in parental PC12 cells (41Chandler L.P. Chandler C.E. Hosang M. Shooter E.M. J. Biol. Chem. 1985; 260: 3360-3367Abstract Full Text PDF PubMed Google Scholar)) precluded an analysis of the endogenous EGF receptor. Instead, Aα-RNAi cells were transfected with EGF receptor plasmids at a concentration that preserved the ligand dependence of Tyr phosphorylation. Although Akt and ERK stimulation by EGF was blunted as before, Aα knockdown had no effect on expression levels of the EGF receptor, total Tyr phosphorylation, or phosphorylation of Tyr-1045, an autophosphorylation site that recruits the ubiquitin-protein isopeptide ligase c-Cbl (42Waterman H. Katz M. Rubin C. Shtiegman K. Lavi S. Elson A. Jovin T. Yarden Y. EMBO J. 2002; 21: 303-313Crossref PubMed Scopus (226) Google Scholar) (Fig. 4). Thus, Aα silencing attenuated growth factor-dependent ERK and Akt phosphorylation downstream of the EGF receptor. Since Aα silencing stimulated oncogenic Ras-dependent ERK activation (Fig. 1B), compensatory uncoupling may occur at the level of receptor tyrosine kinase adaptor proteins or guanylate exchange factors. B′-family Regulatory Subunits Mediate Akt Modulation by PP2A—The observation that PP2A keeps both Akt and ERK signaling in check raised the question whether different PP2A regulatory subunits might be involved. If so, cells would have the ability to throttle the output of both kinase cascades independently. Aα-exchange PC6-3 cell lines were previously generated in which Dox treatment silences the endogenous Aα subunit, concomitant with expression of an RNAi-resistant Aα subunit mutant defective in binding to select regulatory subunit families (19Strack S. Cribbs J.T. Gomez L. J. Biol. Chem. 2004; 279: 47732-47739Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar). In Aα(+-+)-exchange cells, wild-type Aα is replaced with the Aα DTP177AAA mutant, which binds to B and B″ but not B′ subunits (19Strack S. Cribbs J.T. Gomez L. J. Biol. Chem. 2004; 279: 47732-47739Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar, 43Ruediger R. Fields K. Walter G. J. Virol. 1999; 73: 839-842Crossref PubMed Google Scholar) (Fig. 5A). Quantitative Aα subunit exchange is evident not only as the disappearance of wild-type Aα and appearance of the epitope-tagged, slightly larger mutant but also as the selective loss of B′α and B′β protein after 3 days in Dox (Fig. 5B) because monomeric B′ (and B) family subunits are unstable in cells (5Strack 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, 19Strack S. Cribbs J.T. Gomez L. J. Biol. Chem. 2004; 279: 47732-47739Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar, 44Li X. Scuderi A. Letsou A. Virshup D.M. Mol. Cell Biol. 2002; 22: 3674-3684Crossref PubMed Scopus (120) Google Scholar, 45Silverstein 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). The loss of B′-family subunits was not accompanied by detectable changes in the levels of catalytic or other regulatory subunits (Fig. 5B), and cells did not show signs of apoptotic cell death until 7 days in the presence of Dox (19Strack S. Cribbs J.T. Gomez L. J. Biol. Chem. 2004; 279: 47732-47739Abstract Full Text Ful

Referência(s)