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Overexpression of the Protein Phosphatase 2A Regulatory Subunit Bγ Promotes Neuronal Differentiation by Activating the MAP Kinase (MAPK) Cascade

2002; Elsevier BV; Volume: 277; Issue: 44 Linguagem: Inglês

10.1074/jbc.m203767200

1083-351X

Stefan Strack,

Nuclear Receptors and Signaling

Protein serine/threonine phosphatase 2A (PP2A) is a multifunctional regulator of cellular signaling. Variable regulatory subunits associate with a core dimer of scaffolding and catalytic subunits and are postulated to dictate substrate specificity and subcellular location of the heterotrimeric PP2A holoenzyme. The role of brain-specific regulatory subunits in neuronal differentiation and signaling was investigated in the PC6-3 subline of PC12 cells. Endogenous Bβ, Bγ, and B′β protein expression was induced during nerve growth factor (NGF)-mediated neuronal differentiation. Transient expression of Bγ, but not other PP2A regulatory subunits, facilitated neurite outgrowth in the absence and presence of NGF. Tetracycline-inducible expression of Bγ caused growth arrest and neurofilament expression, further evidence that PP2A/Bγ can promote differentiation. In PC6-3 cells, but not non-neuronal cell lines, Bγ specifically promoted long lasting activation of the mitogen-activated protein (MAP) kinase cascade, a key mediator of neuronal differentiation. Pharmacological and dominant-negative inhibition and kinase assays indicate that Bγ promotes neuritogenesis by stimulating the MAP kinase cascade downstream of the TrkA NGF receptor but upstream or at the level of the B-Raf kinase. Mutational analyses demonstrate that the divergent N terminus is critical for Bγ activity. These studies implicate PP2A/Bγ as a positive regulator of MAP kinase signaling in neurons. Protein serine/threonine phosphatase 2A (PP2A) is a multifunctional regulator of cellular signaling. Variable regulatory subunits associate with a core dimer of scaffolding and catalytic subunits and are postulated to dictate substrate specificity and subcellular location of the heterotrimeric PP2A holoenzyme. The role of brain-specific regulatory subunits in neuronal differentiation and signaling was investigated in the PC6-3 subline of PC12 cells. Endogenous Bβ, Bγ, and B′β protein expression was induced during nerve growth factor (NGF)-mediated neuronal differentiation. Transient expression of Bγ, but not other PP2A regulatory subunits, facilitated neurite outgrowth in the absence and presence of NGF. Tetracycline-inducible expression of Bγ caused growth arrest and neurofilament expression, further evidence that PP2A/Bγ can promote differentiation. In PC6-3 cells, but not non-neuronal cell lines, Bγ specifically promoted long lasting activation of the mitogen-activated protein (MAP) kinase cascade, a key mediator of neuronal differentiation. Pharmacological and dominant-negative inhibition and kinase assays indicate that Bγ promotes neuritogenesis by stimulating the MAP kinase cascade downstream of the TrkA NGF receptor but upstream or at the level of the B-Raf kinase. Mutational analyses demonstrate that the divergent N terminus is critical for Bγ activity. These studies implicate PP2A/Bγ as a positive regulator of MAP kinase signaling in neurons. protein serine/threonine phosphatase 2A nerve growth factor mitogen-activated protein kinase extracellular signal-regulated kinase MAP/ERK kinase cytomegalovirus glutathioneS-transferase hemagglutinin green fluorescent protein tetracycline repressor The phosphorylation state of key proteins is crucial in most cellular processes and depends on the precisely orchestrated balance of protein kinases and phosphatase activities. Compared with kinases, very little is known about the regulation of protein phosphatases. PP2A,1 one of the four major classes of protein serine/threonine phosphatases in cells, is a family of abundant and ubiquitous enzymes with pleiotropic functions ranging from cell cycle regulation to synaptic plasticity (1Janssens V. Goris J. Biochem. J. 2001; 353: 417-439Crossref PubMed Scopus (1549) Google Scholar). The predominant form of PP2A in cells has a heterotrimeric subunit structure, consisting of a core dimer of ∼36 kDa catalytic and ∼65 kDa scaffold subunits (subunits C and A, respectively) complexed to a third variable subunit. Variable subunits are encoded by three multigene families (B, B′, B") and are believed to dictate substrate specificity, subcellular localization, and regulation of PP2A by phosphorylation. There is growing evidence that incorporation of different variable subunits imparts specific cellular functions to PP2A. PP2A, containing B′ (B56, PR61) family subunits, participates in Wnt/β-catenin signaling, a signal transduction pathway necessary for vertebrate axis formation in early embryogenesis (2Hsu W. Zeng L. Costantini F. J. Biol. Chem. 1999; 274: 3439-3445Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar, 3Seeling J.M. Miller J.R. Gil R. Moon R.T. White R. Virshup D.M. Science. 1999; 283: 2089-2091Crossref PubMed Scopus (367) Google Scholar). B′ subunits bind to cyclin G1 and G2, suggesting that PP2A holoenzymes containing these subunits are involved in cell cycle regulation (4Okamoto K. Kamibayashi C. Serrano M. Prives C. Mumby M.C. Beach D. Mol. Cell. Biol. 1996; 16: 6593-6602Crossref PubMed Scopus (88) Google Scholar, 5Bennin 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, 6Okamoto 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). The B" subunit PR48 was first identified in a screen for proteins that interact with cdc6, a component of DNA prereplication complexes (7Yan Z. Fedorov S.A. Mumby M.C. Williams R.S. Mol. Cell. Biol. 2000; 20: 1021-1029Crossref PubMed Scopus (99) Google Scholar), and may mediate the obligatory role of PP2A in the initiation of chromosomal DNA replication (8Lin X.H. Walter J. Scheidtmann K. Ohst K. Newport J. Walter G. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 14693-14698Crossref PubMed Scopus (74) Google Scholar). Another B" subunit, PR59, recently identified as an interaction partner for the retinoblastoma-related p107 protein (9Voorhoeve P.M. Hijmans E.M. Bernards R. Oncogene. 1999; 18: 515-524Crossref PubMed Scopus (89) Google Scholar), could be important in the rapid dephosphorylation of p107 following DNA damage (10Voorhoeve P.M. Watson R.J. Farlie P.G. Bernards R. Lam E.W. Oncogene. 1999; 18: 679-688Crossref PubMed Scopus (41) Google Scholar). PP2A holoenzymes containing B" family subunits may thus be specialized regulators of the G1/S cell cycle transition. Despite being the first PP2A regulatory subunits to be identified, roles of members of the B-type subunit family (PR55) are still largely enigmatic. In mammals, four genes (Bα–δ) code for 54–57-kDa proteins, which are additionally diversified by alternative splicing or promoter use. Bα–δ are more than 80% identical at the amino acid level, with the greatest clustering of divergent residues at the N terminus. B-family PP2A subunits are predicted to adopt a seven-blade β-propeller fold similar to the β-subunits of heterotrimeric G proteins (11Strack 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). Bα mediates dephosphorylation of vimentin intermediate filaments in fibroblasts (12Turowski P. Myles T. Hemmings B.A. Fernandez A. Lamb N.J. Mol. Biol. Cell. 1999; 10: 1997-2015Crossref PubMed Scopus (92) Google Scholar) and has been shown to interact preferentially with microtubules and tau protein, possibly contributing to the regulation of microtubule stability by PP2A (13Sontag E. Nunbhakdi-Craig V. Bloom G.S. Mumby M.C. J. Cell Biol. 1995; 128: 1131-1144Crossref PubMed Scopus (296) Google Scholar, 14Sontag E. Nunbhakdi-Craig V. Lee G. Bloom G.S. Mumby M.C. Neuron. 1996; 17: 1201-1207Abstract Full Text Full Text PDF PubMed Scopus (352) Google Scholar). Whereas Bα and Bδ are widely expressed in different tissues, Bβ and Bγ proteins are detectable only in brain (15Mayer R.E. Hendrix P. Cron P. Matthies R. Stone S.R. Goris J. Merlevede W. Hofsteenge J. Hemmings B.A. Biochemistry. 1991; 30: 3589-3597Crossref PubMed Scopus (171) Google Scholar, 16Zolnierowicz S. Csortos C. Bondor J. Verin A. Mumby M.C. DePaoli-Roach A.A. Biochemistry. 1994; 33: 11858-11867Crossref PubMed Scopus (95) Google Scholar, 17Strack S. Zaucha J.A. Ebner F.F. Colbran R.J. Wadzinski B.E. J. Comp. Neurol. 1998; 392: 515-527Crossref PubMed Scopus (150) Google Scholar, 18Strack S. Chang D. Zaucha J.A. Colbran R.J. Wadzinski B.E. FEBS Lett. 1999; 460: 462-466Crossref PubMed Scopus (67) Google Scholar), which suggests that Bβ and Bγ mediate specifically neuronal functions of PP2A. Bβ and Bγ expression is differentially regulated during development and maturation of the rat brain; Bβ protein and mRNA levels are high in late embryonic brain and decrease modestly after birth. In contrast, Bγ expression increases sharply postnatally to plateau at 2 weeks of age (17Strack S. Zaucha J.A. Ebner F.F. Colbran R.J. Wadzinski B.E. J. Comp. Neurol. 1998; 392: 515-527Crossref PubMed Scopus (150) Google Scholar). This report begins to analyze the functions of neuronal PP2A regulatory subunits in the pheochromocytoma PC12 cell line, an experimentally tractable model system of neuronal differentiation and neurite outgrowth (19Segal R.A. Greenberg M.E. Annu. Rev. Neurosci. 1996; 19: 463-489Crossref PubMed Scopus (908) Google Scholar, 20Green S.H. Russo A.F. Green S.H. Neuronal Cell Lines. 7. Academic Press, San Diego1995: 222-237Google Scholar, 21Greene L.A. Aletta J.M. Rukenstein A. Green S.H. Methods Enzymol. 1987; 147: 207-216Crossref PubMed Scopus (247) Google Scholar, 22Huang E.J. Reichardt L.F. Annu. Rev. Neurosci. 2001; 24: 677-736Crossref PubMed Scopus (3398) Google Scholar). Upon NGF treatment, PC12 cells develop into sympathetic neuron-like cells with elaborate neurites capable of generating action potentials (23Toledo-Aral J.J. Brehm P. Halegoua S. Mandel G. Neuron. 1995; 14: 607-611Abstract Full Text PDF PubMed Scopus (149) Google Scholar). Although NGF binding to the TrkA receptor tyrosine kinase activates several signaling pathways, sustained activation of the MAP kinase cascade is both obligatory and sufficient for neurite outgrowth and differentiation (24Qui M.S. Green S.H. Neuron. 1992; 9: 705-717Abstract Full Text PDF PubMed Scopus (360) Google Scholar, 25Traverse S. Gomez N. Paterson H. Marshall C. Cohen P. Biochem. J. 1992; 288: 351-355Crossref PubMed Scopus (807) Google Scholar, 26Cowley S. Paterson H. Kemp P. Marshall C.J. Cell. 1994; 77: 841-852Abstract Full Text PDF PubMed Scopus (1854) Google Scholar). Evidence from several laboratories supports a model in which NGF promotes persistent activation of the small GTPase Rap1. Rap1 then recruits the serine/threonine kinase B-Raf to the membrane and maintains its activity by poorly understood mechanisms ((Refs. 27York R.D. Yao H. Dillon T. Ellig C.L. Eckert S.P. McCleskey E.W. Stork P.J. Nature. 1998; 392: 622-626Crossref PubMed Scopus (762) Google Scholar, 28Kao S. Jaiswal R.K. Kolch W. Landreth G.E. J. Biol. Chem. 2001; 276: 18169-18177Abstract Full Text Full Text PDF PubMed Scopus (318) Google Scholar, 29Wu C. Lai C.F. Mobley W.C. J. Neurosci. 2001; 21: 5406-5416Crossref PubMed Google Scholar, but also see Ref. 30Zwartkruis F.J. Wolthuis R.M. Nabben N.M. Franke B. Bos J.L. EMBO J. 1998; 17: 5905-5912Crossref PubMed Scopus (191) Google Scholar). Raf family kinases phosphorylate the dual-specificity kinase MEK1, which in turn activates MAP kinases of the ERK (extracellular signal-regulated kinase) family. ERKs are broad specificity serine/threonine kinases that target cytosolic as well as nuclear substrates, including the transcription factors Myc and Elk1 (31Pearson G. Robinson F. Beers Gibson T., Xu, B.E. Karandikar M. Berman K. Cobb M.H. Endocr. Rev. 2001; 22: 153-183Crossref PubMed Scopus (3564) Google Scholar). Here, I show that NGF-mediated differentiation of a PC12 subline leads to expression of neuronal PP2A regulatory subunits. One of these subunits, Bγ, can promote neuronal differentiation through the MAP kinase pathway by activating B-Raf. The PC6-3 cell line (32Pittman R.N. Wang S. DiBenedetto A.J. Mills J.C. J. Neurosci. 1993; 13: 3669-3680Crossref PubMed Google Scholar) was obtained from Henry Paulson (University of Iowa). This PC12 subline was found to display less propensity to form cell aggregates and could be transfected with higher efficiency than its parental cell line. PC6-3 cells were grown on uncoated plastic in RPMI 1640 medium containing 10% horse serum and 5% fetal bovine serum in a 5% CO2 incubator. HEK293 and COS-M6 cells were cultured in Dulbecco's modified Eagle's medium/high glucose containing 10% fetal bovine serum. cDNAs of B subunits were isolated by reverse transcriptase-polymerase chain reaction from rat brain total RNA (Access RT-PCR kit, Promega, Madison, WI), subcloned into a pcDNA3.1 mammalian expression vector under control of the cytomegalovirus (CMV) promoter and FLAG epitope-tagged at the N terminus by PCR. The addition of the FLAG epitope did not affect the activity of Bγ in neurite outgrowth and MAP kinase assays (not shown). The Bγ 1-35α chimera was constructed by PCR-amplifying Bα 5′ and Bγ 3′ sequences and ligating the fragments utilizing an introduced silent NheI site. Plasmids encoding FLAG-ERK2 and Myc-B-Raf were obtained from Philip Stork (Vollum Institute) and Richard Marais (Royal Cancer Hospital, London), respectively. Hemagglutinin (HA)-MEK1 wild-type and dominant-negative MEK1 K97R plasmids were provided by Jeffrey Pessin (University of Iowa). HA-tagged B′ (B56) α-subunit plasmid and B′ antibodies were obtained from David Virshup (University of Utah). PP2A/A, Bα/δ, Bβ, and Bγ antibodies were from Brian Wadzinski (Vanderbilt University) (17Strack S. Zaucha J.A. Ebner F.F. Colbran R.J. Wadzinski B.E. J. Comp. Neurol. 1998; 392: 515-527Crossref PubMed Scopus (150) Google Scholar). The following reagents were obtained commercially: antibodies to the PP2A C subunit, ERK1/2, B-Raf, protein G-agarose (Santa Cruz Biotechnology, Santa Cruz, CA); FLAG-tag antibody (M2) and its agarose conjugate (Sigma); 2.5S NGF, glutathioneS-transferase (GST)-ERK2, GST-MEK1 (Upstate Biotechnology Inc., Lake Placid, NY); GST-Elk1, phospho-Ser-383 Elk1 antibody, phospho-ERK1/2 antibody (Cell Signaling Technologies, Beverly, MA); Myc- and HA-tag-directed antibodies (Covance, Princeton, NJ); U0126, K252A (Calbiochem). PC6-3 cells were transfected at 20–30% confluency with 1 μg of DNA (0.8 μg of PP2A subunit plasmid, 0.2 μg of pEGFP-C1, a green fluorescent protein (GFP) expression vector), and 2 μl of LipofectAMINE 2000 (Invitrogen) per well of a 12-well plate according to the manufacturer's instructions. After 36–48 h, neurite outgrowth of living cells was analyzed by capturing digital images from 4–5 randomly selected fields on an inverted epifluorescence microscope. Transmitted and mercury light sources were adjusted to superimpose phase contrast and fluorescence signals on the same image (Kodak MDS290 documentation system). Images were analyzed using NIH Image software by a second, naive experimenter, who either counted cells with neurites longer than 2-cell body diameters or measured the area of a rectangle bounding the cell body and neurites. Tetracycline-inducible, stably Bγ-expressing PC6-3 cell lines were generated by two rounds of antibiotic selection essentially as described by the vendor of the T-Rex system (Invitrogen). Briefly, PC6-3 cells were transfected with the linearized vector pcDNA6/TR encoding the tetracycline repressor (TR) protein. After selection in blasticidine (5 μg/ml), 48 clones were expanded and tested for TR function by transfection with a pcDNA5/TO/LacZ reporter plasmid and chemiluminescent β-galactosidase assay (Galacto-Star, Tropix, Bedford, MA). One clone (PC6-3/TR156) displayed 25-fold induction of β-galactosidase activity upon treatment with doxycycline and was transfected with linearized pcDNA5/TO/FLAG-Bγ, a plasmid encoding FLAG-tagged Bγ under control of a chimeric CMV promoter/tetracycline operator. Cells were selected for plasmid integration in 500 μg/ml hygromycin, 2 μg/ml blasticidine. Eighty-four clones were expanded and tested for inducible Bγ expression by MAP kinase reporter assays (below) and immunoblotting. Eight clones showed more than 3-fold doxycycline-inducible MAP kinase activity and a high level of Bγ expression. Cells were cultured in 24-well plates for up to 4 days in the presence of 1 μg/ml doxycycline, 20 ng/ml NGF, or vehicle. [3H]Thymidine (2 μCi/ml) was added to the medium for 6 h followed by a wash with phosphate buffered saline and incubation with 0.5 ml/well 5% (w/v) trichloroacetic acid for 20 min at 4 °C to remove unincorporated label. DNA was solubilized with 0.5 ml/well 0.1 n NaOH and scintillation-counted. Cells cultured in 24-well plates were transfected at 40–70% confluency with 1 μl/well LipofectAMINE 2000 (Invitrogen) and 500 ng/well DNA comprising 250 ng of PP2A regulatory subunit plasmid (or empty vector), 125 ng of pFR-Luc (GAL4 promoter-driven luciferase), 12.5 ng of pFA-Elk1 (CMV promoter-driven GAL4 DNA binding domain-Elk1 fusion protein, Elk1 PathDetect Trans-reporter assay, Stratagene, La Jolla, CA), and 112.5 ng of pSV40-βGal (SV40 promoter-driven β-galactosidase). After 16–18 h of serum starvation (1/10 normal serum concentration) and 36–48 h following transfection, cells were washed once in phosphate-buffered saline and lysed in 100 μl/well lysis buffer (luciferase assay kit, Promega). Luciferase activity was measured from cleared lysates (20,000 × g, 15 min) with a tube luminometer according to the manufacturer's instructions and normalized to β-galactosidase activity determined from the same lysates by a chemiluminescent assay (Galacto-Star, Tropix). PC6-3 cells were cultured in 12-well plates to 40–70% confluency and transfected with 2 μl/well LipofectAMINE 2000 and 1 μg/well DNA (750 ng of Bγ plasmid or empty vector plus 250 ng of epitope-tagged kinase (B-Raf, MEK1, or ERK2) plasmid). 36–48 h after transfection and following an overnight incubation with low serum medium (1% horse serum, 0.5% fetal calf serum), cells were lysed in 150 μl/well immunoprecipitation/kinase lysis buffer (1% Triton X-100, 150 mm NaCl, 20 mm Tris pH 7.5, 1 mmEDTA, 1 mm EGTA, 1 mm β-glycerolphosphate, 1 mm Na3V04, 1 mmNa4P2O7, 1 μmmicrocystin-LR, 1 mm phenylmethylsulfonyl fluoride, 1 μg/ml leupeptin, 1 mm benzamidine) and sonicated for 2 s at low intensity with a probe tip sonicator. Debris was pelleted (20,000 × g, 15 min), and kinases were immunoprecipitated from the cleared lysate with 0.5–2 μg of epitope-tagged antibodies and 6 μl of protein G-agarose for 4 h at 4 °C. Immunoprecipitates were washed with 6 ml of immunoprecipitation/kinase lysis buffer and 1.5 ml of kinase assay buffer (25 mm Tris pH 7.5, 10 mmMgCl2, 2 mm dithiothreitol, 5 mmβ-glycerolphosphate, 0.1 mmNa3V04, 1 μm microcystin-LR). Kinase assays were started by adding 25 μl of assay mix to the immunoprecipitates; the mixture was incubated for 30 min at 30 °C with intermittent agitation, and assays were stopped by the addition of 10 μl of 4× SDS-sample buffer containing 100 mm EDTA. The assay mixes contained 40 μg/ml GST-Elk1 substrate and 200 μm ATP in kinase assay buffer. MEK1 assays additionally included 20 μg/ml GST-ERK2, and B-Raf assays included GST-ERK2 in addition to 2 μg/ml GST-MEK1. Kinase assays were analyzed by SDS-PAGE and immunoblotting with phospho-Ser-383 Elk1 antibodies and chemiluminescent detection (SuperSignal, Pierce) on a Kodak Imager 440. Serially diluted samples were analyzed to ensure quantification in the linear range of detection. Phospho-Ser-383 Elk1 immunoreactivity was quantified from digital images using NIH Image software, subtracting negligible background phosphorylation from immunoprecipitates of mock-transfected cells. PC6-3 cells are a subline of PC12 cells. Upon withdrawal of NGF, differentiated PC6-3 cells undergo rapid apoptosis even in the presence of serum, which may make them a better model of sympathetic neurons than PC12 cells (32Pittman R.N. Wang S. DiBenedetto A.J. Mills J.C. J. Neurosci. 1993; 13: 3669-3680Crossref PubMed Google Scholar). To investigate the expression of endogenous PP2A subunits, PC6-3 cells were treated for several days in the presence or absence of NGF and analyzed by immunoblotting with specific antibodies (17Strack S. Zaucha J.A. Ebner F.F. Colbran R.J. Wadzinski B.E. J. Comp. Neurol. 1998; 392: 515-527Crossref PubMed Scopus (150) Google Scholar). NGF treatment resulted in robust neurite outgrowth and expression of differentiation markers such as VGF and neurofilament heavy chain (Fig. 1 and not shown). Concomitant with differentiation, NGF induced expression of the brain-specific Bβ and Bγ, as well as the brain-enriched B′β isoform of PP2A regulatory subunits. Levels of the catalytic C, the scaffolding A, the Bα and Bδ subunit (detected by an antibody that recognizes both B subunits), and B′α and B′δ remained unchanged. Thus, differentiation of PC6-3 cells is accompanied by the induction of neuronal PP2A holoenzymes. To test whether neuronal PP2A isoforms are involved in the establishment of the neuronal phenotype, PC6-3 cells were transiently transfected with a panel of regulatory subunit expression plasmids or vector alone together with a plasmid encoding GFP to mark transfected cells. Expression was verified by immunoblotting; however, protein levels could not be compared because of a lack of antibodies that recognize all regulatory subunits (not shown). 48 h after transfection, living cells were scored for neurite outgrowth by measuring the area of a rectangle that bounded the cell body and neurites. Transfection with Bγ, but not Bα or Bβ, resulted in the extension of numerous short neurites quantified as an increase of the bounding rectangle area (51 ± 9%,n = 11 experiments, Fig.2 and data not shown). Bγ also enhanced neurite outgrowth in cells treated 24 h after transfection with NGF (46 ± 16%, n = 9 experiments), indicating that NGF and this PP2A subunit act synergistically. NGF-treated cells were also scored for neurite outgrowth by counting cells with neurites longer than twice the diameter of the soma (Fig. 2 C). This method gave similar results as the bounding rectangle method. The bounding area method was used in all subsequent experiments, as it allowed quantification of early morphological changes. Neuronal differentiation of PC12 cells involves a "cell fate" choice characterized by morphological changes (i.e. neuritogenesis) but also cessation of growth, loss of chromaffin cell markers, and synthesis of neuronal proteins. Previous studies with the protein phosphatase inhibitor okadaic acid have shown that PP2A activity is required for neurite maintenance of differentiated neurons (33Chiou J.Y. Westhead E.W. J. Neurochem. 1992; 59: 1963-1966Crossref PubMed Scopus (31) Google Scholar, 34Giasson B.I. Mushynski W.E. J. Neurobiol. 1997; 32: 193-201Crossref PubMed Scopus (16) Google Scholar, 35Merrick S.E. Trojanowski J.Q. Lee V.M. J. Neurosci. 1997; 17: 5726-5737Crossref PubMed Google Scholar). It therefore became important to investigate whether Bγ expression causes neuronal differentiation as opposed to neurite outgrowthper se. To this end, PC6-3 cell lines were created that express FLAG epitope-tagged Bγ under control of a tetracycline-inducible CMV promoter (see "Experimental Procedures"). Several clonal isolates exhibited no detectable leak expression and responded to the addition of the tetracycline analog doxycycline to the medium with Bγ protein levels that were 5–10-fold higher than could be achieved by transient transfection of CMV-promoter driven cDNAs. In agreement with transient transfection studies, doxycycline treatment for 36–48 h resulted in robust neurite outgrowth; however, neurites appeared somewhat shorter than in NGF-treated sister cultures (Fig.3 A). To test whether Bγ causes the growth arrest characteristic of neuronal differentiation, cells lines were incubated for 4 days with doxycycline, NGF, or vehicle alone and assayed for DNA synthesis by pulse-labeling with [3H]thymidine. Cell line 275, which expresses high inducible levels of Bγ, responded to doxycycline or NGF treatment with a ∼50% decrease in [3H]thymidine incorporation (Fig. 3 B). In contrast, cell line 280, which was isolated in parallel but failed to induce Bγ, showed decreased DNA synthesis only when cultured in NGF, demonstrating that doxycycline itself does not slow down growth. Cell line 275 was next analyzed for neuronal protein expression. Doxycycline or NGF but not vehicle treatment for 4 days induced high levels of neurofilament heavy chain protein (Fig.3 C). Bγ must incorporate into the PP2A heterotrimer to escape degradation by the ubiquitin-proteasome pathway (11Strack 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). The percentage of endogenous A and C subunits that associates with inducibly expressed Bγ was determined by quantitative immunoprecipitation. PP2A holoenzymes containing Bγ were estimated to comprise 5% of the total PP2A pool in the inducible PC6-3 line 275 (Fig. 3 D). Thus, incorporation of Bγ into a minor pool of PP2A holoenzymes is sufficient for NGF-independent neuronal differentiation of PC6-3 cells. It is well established that activation of the MAP kinase signaling cascade is both necessary and sufficient for neuronal differentiation of PC12 cells (26Cowley S. Paterson H. Kemp P. Marshall C.J. Cell. 1994; 77: 841-852Abstract Full Text PDF PubMed Scopus (1854) Google Scholar). This also holds for the PC12-derived PC6-3 cell line, because the MEK inhibitor U0126 blocks NGF-induced neurite outgrowth (Fig.5 B), and transfection of a constitutively active MEK1 mutant promotes neuritogenesis, growth arrest, and neurofilament expression (data not shown). Because of the pivotal role of this signaling cascade in neuronal differentiation, a possible activation of the MAP kinase signaling by Bγ was investigated. PC6-3 cells were transiently transfected with plasmids encoding different PP2A regulatory subunits or empty vector in combination with reporter plasmids that read out ERK-dependent activation of the transcription factor Elk1 (Elk1 PathDetect Trans-reporter assay, Stratagene). Transfection of Bγ, but not any other regulatory subunit tested, resulted in robust activation of the MAP kinase reporter (Fig.4 A, 15.1 ± 2.6-fold activation, n = 13 experiments). Activation was not only subunit- but also cell line-specific, because Bγ had no effect on ERK activity in HEK293 and COS-M6 cells even though Bγ could be expressed to significantly higher levels in these non-neuronal cell lines (Fig. 4 B). MAP kinase activation by Bγ in PC6-3 cells was comparable to stimulation with low (∼ 1 ng/ml) NGF concentrations, and a combination of both treatments resulted in more than additive induction of Elk1-mediated luciferase activity (Fig.4 C). Fig. 4 D illustrates this effect over a range of NGF concentrations. Bγ transfection caused a leftward shift of the NGF dose-response curve, enhancing MAP kinase signaling from 2-fold at saturating NGF concentrations to 12-fold in the absence of NGF in this experiment.Figure 4Bγ activates MAP kinase signaling. PC6-3 (A–D), HEK293 (B), and COS-M6 (B) cells were cotransfected with the indicated expression vectors and plasmids that report ERK/MAP kinase activity by luciferase expression and then assayed 36–48 h later for reporter activity. PC6-3 cells in C and D were treated for 6 h with the indicated NGF concentrations prior to the assay. Shown are the means ± S.E. of normalized activities from triplicate transfections of a representative experiment. For the experiment in B, cells transfected with empty vector (−) or Bγ (+, duplicate loading) were also analyzed for FLAG- Bγ, PP2A catalytic (C) subunit, and B-Raf expression by immunoblotting. E, PC6-3 line 275 was treated for 24 h in the presence of vehicle (−) or doxycycline (+Dox, 1 μg/ml) to induce Bγ expression. Cells were then stimulated for 5 min with the indicated concentrations of NGF and analyzed for ERK phosphorylation by immunoblotting total lysates for phosphorylated (pERK1, pERK2) and total ERK1/2. The inset shows a representative blot of cells treated without NGF. ERK phosphorylation was quantified by densitometry as the ratio of phosphoreactivity to total immunoreactivity normalized to control. The means ± standard deviation of duplicate pERK2 determinations from a representative experiment are plotted. ERK1 phosphorylation followed an almost identical dose response. F, PC6-3 line 275 was induced to express Bγ by the addition of doxycycline (1 μg/ml) or treated with NGF (20 ng/ml) for 9–96 h. Total lysates were immunoblotted for the indicated proteins, and ERK1/2 phosphorylation was quantified as described for panel E (average of duplicate determinations from a representative experiment). Significant increases over control: *, p < 0.05, **,p < 0.005.View Large Image Figure ViewerDownload (PPT) Tetracycline-inducible PC6-3 cells were used to investigate whether Bγ activates MAP kinase signaling by stimulating ERK phosphorylation of activation loop residues (Thr-202 and Tyr-204 in human ERK1). Cells treated for 24 h in the absence or presence of doxycycline to induce Bγ expression were challenged for 5 min with increasing concentrations of NGF, and ERK1/2 phosphorylation was assayed by immunoblotting lysates with a phosphorylation-state specific antibody (Fig. 4 E). Paralleling the MAP kinase reporter assays in Fig. 4 D, the data demonstrate that B