Differential Expression of the B′β Regulatory Subunit of Protein Phosphatase 2A Modulates Tyrosine Hydroxylase Phosphorylation and Catecholamine Synthesis
2006; Elsevier BV; Volume: 282; Issue: 1 Linguagem: Inglês
10.1074/jbc.m607407200
ISSN1083-351X
AutoresAmit Saraf, David M. Virshup, Stefan Strack,
Tópico(s)Parkinson's Disease Mechanisms and Treatments
ResumoTyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine synthesis, is stimulated by N-terminal phosphorylation by several kinases and inhibited by protein serine/threonine phosphatase 2A (PP2A). PP2A is a family of heterotrimeric holoenzymes containing one of more than a dozen different regulatory subunits. In comparison with rat forebrain extracts, adrenal gland extracts exhibited TH hyperphosphorylation at Ser19, Ser31, and Ser40, as well as reduced phosphatase activity selectively toward phosphorylated TH. Because the B′β regulatory subunit of PP2A is expressed in brain but not in adrenal glands, we tested the hypothesis that PP2A/B′β is a specific TH phosphatase. In catecholamine-secreting PC12 cells, inducible expression of B′β decreased both N-terminal Ser phosphorylation and in situ TH activity, whereas inducible silencing of endogenous B′β had the opposite effect. Furthermore, PP2A/B′β directly dephosphorylated TH in vitro. As to specificity, other PP2A regulatory subunits had negligible effects on TH activity and phosphorylation in situ and in vitro. Whereas B′β was highly expressed in dopaminergic cell bodies in the substantia nigra, the PP2A regulatory subunit was excluded from TH-positive terminal fields in the striatum and failed to colocalize with presynaptic markers in general. Consistent with a model in which B′β enrichment in neuronal cell bodies helps confine catecholamine synthesis to axon terminals, TH phosphorylation was higher in processes than in somata of dopaminergic neurons. In summary, we show that B′β recruits PP2A to modulate TH activity in a tissue- and cell compartment specific fashion. Tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine synthesis, is stimulated by N-terminal phosphorylation by several kinases and inhibited by protein serine/threonine phosphatase 2A (PP2A). PP2A is a family of heterotrimeric holoenzymes containing one of more than a dozen different regulatory subunits. In comparison with rat forebrain extracts, adrenal gland extracts exhibited TH hyperphosphorylation at Ser19, Ser31, and Ser40, as well as reduced phosphatase activity selectively toward phosphorylated TH. Because the B′β regulatory subunit of PP2A is expressed in brain but not in adrenal glands, we tested the hypothesis that PP2A/B′β is a specific TH phosphatase. In catecholamine-secreting PC12 cells, inducible expression of B′β decreased both N-terminal Ser phosphorylation and in situ TH activity, whereas inducible silencing of endogenous B′β had the opposite effect. Furthermore, PP2A/B′β directly dephosphorylated TH in vitro. As to specificity, other PP2A regulatory subunits had negligible effects on TH activity and phosphorylation in situ and in vitro. Whereas B′β was highly expressed in dopaminergic cell bodies in the substantia nigra, the PP2A regulatory subunit was excluded from TH-positive terminal fields in the striatum and failed to colocalize with presynaptic markers in general. Consistent with a model in which B′β enrichment in neuronal cell bodies helps confine catecholamine synthesis to axon terminals, TH phosphorylation was higher in processes than in somata of dopaminergic neurons. In summary, we show that B′β recruits PP2A to modulate TH activity in a tissue- and cell compartment specific fashion. Tyrosine hydroxylase (TH) 2The abbreviations used are: TH, tyrosine hydroxylase; CaMKII, calcium/calmodulin-dependent kinase II; DOPA, l-3,4-dihydroxyphenylalanine; ERK, extracellular signal-regulated kinase; GST, glutathione S-transferase; HA, hemagglutinin; MBP, myelin basic protein; PKA, protein kinase A; PP, protein phosphatase; RNAi, RNA interference. catalyzes the rate-limiting step in the biosynthesis of catecholamines (dopamine, norepinephrine, and epinephrine) from the amino acid precursor l-tyrosine (1Levitt M. Spector S. Sjoerdsma A. Udenfriend S. J. Pharmacol. Exp. Ther. 1965; 148: 1-8PubMed Google Scholar). The enzyme consists of an N-terminal regulatory domain, a central catalytic domain, and a C-terminal association domain, which mediates tetrameric assembly (2Kumer S.C. Vrana K.E. J. Neurochem. 1996; 67: 443-462Crossref PubMed Scopus (620) Google Scholar, 3Goodwill K.E. Sabatier C. Marks C. Raag R. Fitzpatrick P.F. Stevens R.C. Nat. Struct. Biol. 1997; 4: 578-585Crossref PubMed Scopus (269) Google Scholar). Several kinases regulate TH activity by phosphorylating key serines in the regulatory domain (Ser8, Ser19, Ser31, and Ser40). Best characterized is Ser40 phosphorylation by cyclic AMP-dependent protein kinase (PKA), which markedly enhances TH catalytic activity both in vitro and in vivo by relieving feedback inhibition by the catecholamines (4Daubner S.C. Lauriano C. Haycock J.W. Fitzpatrick P.F. J. Biol. Chem. 1992; 267: 12639-12646Abstract Full Text PDF PubMed Google Scholar, 5Waymire J.C. Craviso G.L. Lichteig K. Johnston J.P. Baldwin C. Zigmond R.E. J. Neurochem. 1991; 57: 1313-1324Crossref PubMed Scopus (53) Google Scholar, 6Lew J.Y. Garcia-Espana A. Lee K.Y. Carr K.D. Goldstein M. Haycock J.W. Meller E. Mol. Pharmacol. 1999; 55: 202-209Crossref PubMed Scopus (44) Google Scholar, 7Haycock J.W. J. Biol. Chem. 1990; 265: 11682-11691Abstract Full Text PDF PubMed Google Scholar, 8Ramsey A.J. Fitzpatrick P.F. 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Sim A.T. Goncalves C.A. Dunkley P.R. Neurochem. Res. 2002; 27: 207-213Crossref PubMed Scopus (31) Google Scholar, 13Berresheim U. Kuhn D.M. Brain Res. 1994; 637: 273-276Crossref PubMed Scopus (21) Google Scholar, 14Haavik J. Schelling D.L. Campbell D.G. Andersson K.K. Flatmark T. Cohen P. FEBS Lett. 1989; 251: 36-42Crossref PubMed Scopus (90) Google Scholar). However, okadaic acid also inhibits the related PP4, PP5, and PP6 catalytic subunits with high affinity (15Honkanen R.E. Golden T. Curr. Med. Chem. 2002; 9: 2055-2075Crossref PubMed Scopus (231) Google Scholar, 16Cohen P.T. Trends Biochem. Sci. 1997; 22: 245-251Abstract Full Text PDF PubMed Scopus (462) Google Scholar), and no information is available regarding the subunit composition of the presumptive TH phosphatase(s). As one of four major groups of serine/threonine phosphatases, PP2A exists predominantly as a heterotrimer of a 36-kDa catalytic or C subunit, a 65-kDa scaffolding or A subunit, and a variable regulatory subunit (17Gallego M. Virshup D.M. Curr. Opin. Cell Biol. 2005; 17: 197-202Crossref PubMed Scopus (134) Google Scholar, 18Janssens V. Goris J. Biochem. J. 2001; 353: 417-439Crossref PubMed Scopus (1549) Google Scholar). Three families of regulatory subunits referred to as B, B′, and B″ associate with the core dimer of A and C subunits. The B family (or PR55) of regulatory subunits consists of four genes (α-δ). The B′ family (or B56/PR61) is encoded by five genes (α-ϵ), whereas the third B″ family consists of three genes (PR72/130, PR59, and PR48). Alternative splicing of several genes further diversifies the variable subunit repertoire. Regulatory subunits determine subcellular localization and substrate specificity and enable PP2A holoenzymes to respond to specific second messengers (19Dagda 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, 20Seeling 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, 21Okamoto 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, 22Firulli 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, 23Ito A. Kataoka T.R. Watanabe M. Nishiyama K. Mazaki Y. Sabe H. Kitamura Y. Nojima H. 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DeJohn M.M. Miller D.W. Young A.B. Vrana K.E. Cha J.H. Brain Res. Mol. Brain Res. 2003; 119: 28-36Crossref PubMed Scopus (53) Google Scholar, 29Damier P. Hirsch E.C. Agid Y. Graybiel A.M. Brain. 1999; 122: 1437-1448Crossref PubMed Scopus (1332) Google Scholar). Here, we show that the neuron-enriched PP2A/B′β heterotrimer dephosphorylates TH and inhibits its activity. We also provide evidence that tissue- and cell compartment-specific expression of B′β modulates TH phosphorylation locally, thereby defining sites of elevated catecholamine synthesis. B′β Antibody Generation—To generate antisera specific to B′β, the synthetic peptide CPLQRLTPQVAASGGQS from the extreme C terminus of B′β was coupled to maleimide-activated keyhole limpet hemocyanin (Pierce). Antiserum was raised by Covance Research Products (Princeton, NJ) and was affinity purified by coupling the same peptide to Affi-Gel 10 (Bio-Rad). Specificity was confirmed in heterologous expression experiments. Other Reagents—Rabbit polyclonal antibodies recognizing total TH and Ser19-phosphorylated TH were provided by John Haycock (Louisiana State University (30Haycock J.W. Anal. Biochem. 1989; 181: 259-266Crossref PubMed Scopus (40) Google Scholar, 31Haycock J.W. Lew J.Y. Garcia-Espana A. Lee K.Y. Harada K. Meller E. Goldstein M. J. Neurochem. 1998; 71: 1670-1675Crossref PubMed Scopus (60) Google Scholar)). The pGEX-2T plasmid expressing glutathione S-transferase (GST)-TH (regulatory domain residues 31-164) was a gift from Drs. Lily Moy and Li-Huei Tsai (Harvard University, Cambridge, MA (32Moy L.Y. Tsai L.H. J. Biol. Chem. 2004; 279: 54487-54493Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar)). Plasmids expressing HA-tagged, human B′(B56) subunits and FLAG-tagged rat B′β and Bγ subunits were described previously (33McCright 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, 34Strack S. J. Biol. Chem. 2002; 277: 41525-41532Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar, 35Van Kanegan M.J. Adams D.G. Wadzinski B.E. Strack S. J. Biol. Chem. 2005; 280: 36029-36036Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar). The following reagents were obtained commercially: mouse TH antibody (Immunostar, Hudson, WI); mouse synaptotagmin antibody (Developmental Studies Hybridoma Bank, University of Iowa); mouse bassoon antibody (Nventa, San Diego, CA); mouse FLAG tag antibody (M2) and its agarose conjugate (Sigma); extracellular signal regulated kinase (ERK) 1/2 (Santa Cruz Biotechnology, Santa Cruz, CA); rabbit phospho-Ser40 TH, phospho-(Thr/Tyr) ERK1/2 and mouse HA tagdirected antibodies (Cell Signaling Technologies, Beverly, CA); rabbit phospho-Ser31 TH antibody (Chemicon, Temecula, CA); microcystin-agarose (Upstate Biotechnology, Inc., Lake Placid, NY); okadaic acid (EMD Biosciences, Darmstadt, Germany); and l-[1-14C] tyrosine (Moravek Biochemicals, Brea, CA). Generation of Tetracycline-inducible Overexpression and Knock-down Cell Lines—Tetracycline-inducible (T-Rex system; BD Biosciences) PC6-3 cell lines stably expressing FLAG epitope-tagged PP2A regulatory subunits and PC6-3 cells with inducible, RNA interference (RNAi)-mediated knock-down of endogenous PP2A subunits were generated as described previously (34Strack S. J. Biol. Chem. 2002; 277: 41525-41532Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). The B′β-silencing short hairpin (sh) RNA targets nucleotides 436-456 of the rat coding sequence (GenBank™ accession number AY251278). Inducible cell lines were cultured (37 °C, 5% CO2) on collagen-coated plates in RPMI 1640 medium with 10% horse serum and 5% fetal bovine serum containing 2 μg/ml blasticidin and 200 μg/ml of either hygromycin (overexpression) or G418 (RNAi). B′β and Bγ overexpression and silencing was induced by the addition of doxycycline (1 μg/ml) for 2-4 days prior to analysis. Control cultures received 0.1% ethanol vehicle. Tissue and Cell Extract Preparation, Purification, and Immunoblotting—Six weeks or older male Sprague-Dawley rats were euthanized by swift decapitation according to protocols approved by the Institutional Animal Care and Use Committee at the University of Iowa. Tissues dissected within 5 min of euthanasia were flash frozen on dry ice and stored at -70 °C until use. For total extract preparation, frozen tissues were pulverized and homogenized in ∼10 volumes of SDS sample buffer supplemented with 0.5 μm microcystin-LR and 2 mm EDTA to inhibit phosphatases. Homogenates were sonicated to shear DNA and cleared by centrifugation (15 min at 20,000 × g). For microcystin-agarose affinity purification and in vitro phosphatase assays, the tissues were homogenized in 3-4 volumes of buffer containing 1% Triton X-100, and 150 mm NaCl, 20 mm Tris, pH 7.5, 1 mm EDTA, 1 mm EGTA, 1 mm phenylmethylsulfonyl fluoride, 1 mm benzamidine, and 1 μg/ml leupeptin. Insoluble debris was removed by centrifugation. Affinity purification of PP1 and PP2A holoenzymes on microcystin-agarose was carried out as described (36Strack 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). For immunoblot analysis, PC6-3 cells were harvested by adding SDS sample buffer plus 0.5 μm microcystin-LR, 2 mm EDTA directly to the culture plate. Homogenates were sonicated and cleared as above. Protein concentrations were determined by a dot blot assay, and quantitative immunoblotting was performed as described (35Van Kanegan M.J. Adams D.G. Wadzinski B.E. Strack S. J. Biol. Chem. 2005; 280: 36029-36036Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar), normalizing phospho-TH to total TH signals. Control experiments showed that signal intensities scaled linearly with the amount of lysate loaded. Phosphatase Assay and Immunoprecipitation—Partially dephosphorylated myelin basic protein (Sigma) or GST-TH was phosphorylated at 5 μg/μl by PKA catalytic subunit (0.25 units/μl; Sigma) for 1 h at 30 °C in buffer containing 1 mm ATP, 50 μCi of [γ-32P]ATP, 50 mm Tris, pH 7.5, 10 mm MgCl2, 2mm dithiothreitol, 1 mm EGTA, and 0.01% Triton X-100 (19Dagda 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). Immunoisolation of PP2A holoenzymes containing FLAG-tagged B′β and Bγ and radioactive phosphatase assays were performed as described previously (19Dagda 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). Trials varying the enzyme concentration established the linear range of the assay (<20% of total 32P released). TH Activity Assay—An in situ dopamine biosynthesis assay, which monitors 14CO2 production following the conversion of l-[1-14C] tyrosine to dopamine, was carried out as described (37Cheah T.B. Bobrovskaya L. Goncalves C.A. Hall A. Elliot R. Lengyel I. Bunn S.J. Marley P.D. Dunkley P.R. J. Neurosci. Methods. 1999; 87: 167-174Crossref PubMed Scopus (21) Google Scholar) with modifications. Briefly, PC6-3 cells growing in 24-well plates (4-6 wells/condition) were treated with doxycycline for 3 days to induce either overexpression or RNAi-mediated silencing of PP2A regulatory subunits. After replacing the medium with Hanks' balanced salt solution containing 1 μCi/ml of l-[1-14C]tyrosine, the wells were immediately sealed with a small chimney capped with a rubber stopper and fitted with a plastic cup containing 300 μ l of 1 m NaOH to absorb the 14CO2 released by the cells. After 1 h, NaOH was subjected to liquid scintillation counting, subtracting blank values of wells without cells. Immunofluorescence—The brains of adult male Sprague-Dawley rats were fixed by transcardial perfusion with 4% paraformaldehyde in phosphate-buffered saline and cryoprotected in 30% sucrose, and free-floating 40-μm sagittal sections were cut on a cryostat. Dissociated rat hippocampal cultures were prepared from embryonic day 18 pups as described (38Lim I.A. Merrill M.A. Chen Y. Hell J.W. Neuropharmacology. 2003; 45: 738-754Crossref PubMed Scopus (57) Google Scholar), plated onto poly d-lysine coated 2-well chamber slides (Nalge Nunc, Naperville, IL), and fixed with 4% paraformaldehyde after 17 days in culture. After blocking in 2% normal donkey serum, sections and cultures were incubated (16 h, 4 °C) with a combination of the following primary antibodies: mouse anti-TH (1:1600 dilution), rabbit anti-phospho-Ser19 TH (1:1200), mouse anti-synaptotagmin (1:20), mouse anti-bassoon (1:1000), and rabbit anti-B′β (1:300). Bound primary antibodies were detected with AlexaFluor 488-conjugated anti-rabbit IgG (Molecular Probes, Eugene, OR) and Cy3-conjugated anti-mouse IgG (Jackson ImmunoResearch, PA) and visualized with a digital camera-equipped epifluorescence microscope. Total and phospho-Ser19 TH immunofluorescence signals were digitally quantified after outlining cell bodies and processes using ImageJ software. Correlation of TH Dephosphorylation and PP2A/B′β Expression in Tissues—We examined expression and phosphorylation of TH in a panel of rat tissues by immunoblotting with total and phospho-specific TH antibodies. As expected, TH was detected only in forebrain and adrenal gland. Unexpectedly, Ser40-phosphorylated TH was strikingly enriched in adrenal gland compared with forebrain (Fig. 1A, top panel). In addition to the PKA site, TH phosphorylation of the CaMKII site Ser19 and the ERK/Cdk5 site Ser31 was also substantially higher in adrenal gland (Fig. 1B). The simplest explanation for this result is that adrenal gland has less TH-directed phosphatase activity than brain tissue. Speculating that a brain-specific PP2A regulatory subunit could be responsible for the comparative hypophosphorylation of TH in this tissue, we explored the expression pattern of different PP2A subunits by immunoblotting tissue extract after affinity purification with microcystin-agarose. Microcystin binds with high affinity to catalytic subunits of PP1 and PP2A and microcystin affinity resins have been used to document the specificity of PP2A regulatory subunit antibodies (36Strack 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, 39Strack S. Kini S. Ebner F.F. Wadzinski B.E. Colbran R.J. J. Comp. Neurol. 1999; 413: 373-384Crossref PubMed Scopus (77) Google Scholar). In agreement with previous results (36Strack 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, 39Strack S. Kini S. Ebner F.F. Wadzinski B.E. Colbran R.J. J. Comp. Neurol. 1999; 413: 373-384Crossref PubMed Scopus (77) Google Scholar), the core scaffolding and catalytic (A and C) subunits and Bα/δ regulatory subunits (detected with a pan B antibody) were ubiquitously expressed (Fig. 1A, bottom panel). In contrast, one of the five B′ subunit family members, B′β was almost exclusively localized to forebrain and cerebellum. The enrichment of B′β protein in the brain is in accordance with earlier mRNA expression studies (40McCright B. Virshup D.M. J. Biol. Chem. 1995; 270: 26123-26128Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar, 41Martens E. Stevens I. Janssens V. Vermeesch J. Gotz J. Goris J. Van Hoof C. J. Mol. Biol. 2004; 336: 971-986Crossref PubMed Scopus (47) Google Scholar). To test the hypothesis that increased TH phosphorylation in adrenal gland is due to the absence of a specific TH phosphatase such as PP2A/B′β, we compared phosphatase activities in forebrain and adrenal gland extracts, using either a PKA-phosphorylated GST-TH fusion protein (regulatory domain residues 31-164 (32Moy L.Y. Tsai L.H. J. Biol. Chem. 2004; 279: 54487-54493Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar)) or myelin basic protein (MBP) as substrates. Forebrain extracts were able to dephosphorylate 32P-labeled TH much more readily than adrenal gland extracts, with an activity ratio of 3.2 ± 0.7 (n = 4, p < 0.01) in extract dilutions matched for PP2A catalytic subunit content (Fig. 1C). The higher phosphatase activity in brain was specific for the [32P]TH substrate, because forebrain and adrenal gland extracts dephosphorylated [32P]MBP to the same extent. In combination, these data suggest that tissue-specific expression of PP2A/B′β inhibits TH phosphorylation. PP2A/B′β Mediates TH Dephosphorylation in Cells—To directly examine the modulation of TH by different PP2A heterotrimeric complexes, we turned to PC12 pheochromocytoma cells as a model system, specifically the PC6-3 subline (42Pittman R.N. Wang S. DiBenedetto A.J. Mills J.C. J. Neurosci. 1993; 13: 3669-3680Crossref PubMed Google Scholar). Derived from rat adrenal chromaffin cells, PC12 and PC6-3 cells abundantly express TH and synthesize and secrete catecholamines (43Markey K.A. Kondo H. Shenkman L. Goldstein M. Mol. Pharmacol. 1980; 17: 79-85PubMed Google Scholar, 44Greene L.A. Tischler A.S. Proc. Natl. Acad. Sci. U. S. A. 1976; 73: 2424-2428Crossref PubMed Scopus (4873) Google Scholar). Treatment of PC6-3 cells with the PP2A inhibitor okadaic acid promoted dose-dependent TH hyperphosphorylation at Ser40 (Fig. 2A). Shown for a single concentration, 250 nm, which inhibits PP2A-like phosphatases, but not PP1, PP2B, or PP2C (15Honkanen R.E. Golden T. Curr. Med. Chem. 2002; 9: 2055-2075Crossref PubMed Scopus (231) Google Scholar), okadaic acid increased TH phosphorylation at all three physiologically relevant sites (Ser19, Ser31, and Ser40; Fig. 2B), confirming PP2A or a related enzyme as the predominant TH phosphatase (7Haycock J.W. J. Biol. Chem. 1990; 265: 11682-11691Abstract Full Text PDF PubMed Google Scholar, 12Leal R.B. Sim A.T. Goncalves C.A. Dunkley P.R. Neurochem. Res. 2002; 27: 207-213Crossref PubMed Scopus (31) Google Scholar, 13Berresheim U. Kuhn D.M. Brain Res. 1994; 637: 273-276Crossref PubMed Scopus (21) Google Scholar, 14Haavik J. Schelling D.L. Campbell D.G. Andersson K.K. Flatmark T. Cohen P. FEBS Lett. 1989; 251: 36-42Crossref PubMed Scopus (90) Google Scholar). Because tissue expression of B′β correlated with TH dephosphorylation (Fig. 1), we investigated TH phosphorylation in PC6-3 cells that transiently expressed different HA-tagged B′ subunit isoforms to similar levels (Fig. 2C). Transfection with B′β and to a lesser extent B′α, a PP2A subunit mostly found in striated muscle (40McCright B. Virshup D.M. J. Biol. Chem. 1995; 270: 26123-26128Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar, 41Martens E. Stevens I. Janssens V. Vermeesch J. Gotz J. Goris J. Van Hoof C. J. Mol. Biol. 2004; 336: 971-986Crossref PubMed Scopus (47) Google Scholar), blunted phospho-Ser40 TH immunoreactivity, whereas forced expression of B′ isoforms B′γ and B′δ had no significant effect (Fig. 2, C and D). To study the regulation of TH by PP2A in a homogenous cell population, we generated stable PC6-3 cell lines expressing FLAG epitope-tagged regulatory subunits under the control of a tetracycline/doxycycline-inducible cytomegalovirus promoter. Overexpressed regulatory subunits appear to associate with a pool of free PP2A core dimer and do not cause compensatory changes in the levels of other PP2A subunits (19Dagda 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, 34Strack S. J. Biol. Chem. 2002; 277: 41525-41532Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). In particular, B′β and Bγ, a neuron-specific member of the B family of PP2A regulatory subunits, immunoprecipitated with equivalent amounts of the catalytic subunit (see Fig. 5A), indicating efficient incorporation into the PP2A holoenzyme. Inducible expression of the B′β regulatory subunit significantly reduced TH phosphorylation at Ser40, Ser31, and Ser19 (Fig. 3, A and C), whereas phosphorylation of ERK1/2 was unaffected. In contrast, induction of Bγ did not alter TH phosphorylation at any site but increased ERK1/2 phosphorylation as previously reported (34Strack S. J. Biol. Chem. 2002; 277: 41525-41532Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). Neither total TH nor total ERK levels were altered by inducible PP2A regulatory subunit expression.FIGURE 3Inducible expression and silencing of B′β controls TH phosphorylation in PC6-3 cells. A, PC6-3 cells inducibly expressing (↑) FLAG-tagged B′β or Bγ subunits were treated for 2 d with either vehicle (-) or doxycycline (dox., +), and lysates were immunoblotted as indicated. B, PC6-3 cells inducibly expressing short hairpin RNA to silence (↓)B′β were cultured for 3 days with or without dox., and lysates were probed with the indicated antibodies. C, summary of inducible overexpression and knock-down experiments (means ± S.E. of phospho-TH normalized to total TH signals and expressed relative to vehicle-treated cells). *, p < 0.05; **, p < 0.01; ***, p < 0.001 by Student's t test.View Large Image Figure ViewerDownload Hi-res image Download (PPT) To complement these forced expression experiments, clonal PC6-3 cell lines were generated that inducibly express B′β-directed short hairpin RNA to silence the endogenous protein (35Van Kanegan M.J. Adams D.G. Wadzinski B.E. Strack S. J. Biol. Chem. 2005; 280: 36029-36036Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar, 45Strack S. Cribbs J.T. Gomez L. J. Biol. Chem. 2004; 279: 47732-47739Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar). Doxycycline treatment for 3-4 days decreased B′β subunit levels to 56 ± 1% (n = 4) but had no effect on TH, ERK1/2, and PP2A/B pan levels (Fig. 3B and data not shown). Silencing of endogenous B′β was associated with a significant increase in TH phosphorylation at Ser40 and Ser19 (Fig. 3, B and C). Remarkably, even though B′β overexpression decreased Ser31 phosphorylation, inducible B′β knock-down had no effect on phosphorylation at that site (Fig. 3, A, and C). It is possible that PP2A/B′β dephosphorylates Ser31 with higher efficiency, so that the ∼50% of B′β remaining after doxycycline treatment (Fig. 3B) is enough to modulate Ser31 but not Ser19 and Ser40 phosphorylation. In aggregate, these experiments establish PP2A/B′β as the major phosphatase that mediates TH dephosphorylation in PC6-3 cells. PP2A/B′β Modulates TH Activity—TH phosphorylation by PKA at Ser40 stimulates TH activity and catecholamine synthesis (46Dunkley P.R. Bobrovskaya L. Graham M.E. von Nagy-Felsobuki E.I. Dickson P.W. J. Neurochem. 2004; 91: 1025-1043Crossref PubMed Scopus (377) Google Scholar). To demonstrate that PP2A/B′β can also modulate TH activity, we performed TH activity assays, measuring the release of 14CO2 from cells incubated with l-[1-14C]tyrosine (Fig. 4A). The amount of CO2 produced during the conversion of l-3,4-dihydroxyphenylalanine (DOPA) to dopamine by DOPA decarboxylase reflects TH activity, because TH is the rate-limiting enzyme in this biosynthetic cascade (37Cheah T.B. Bobrovskaya L. Goncalves C.A. Hall A. Elliot R. Lengyel I. Bunn S.J. Marley P.D. Dunkley P.R. J. Neurosci. Methods. 1999; 87: 167-174Crossref PubMed Scopus (21) Google Scholar). The contribution of other metabolic pathways involving tyrosine was negligible during the 1-h assay, because preincubation of PC6-3 cells with 100 μm of the DOPA decarboxylase inhibitor carbidopa completely blocked 14CO2 production (data not shown). Inducible overexpression of B′β but not Bγ resulted in a robust (>50%) inhibition of 14CO2 release, closely paralleling the decrease in TH phosphorylation at Ser40 (compare Figs. 4B and 3C). Conversely, inducible RNAi of B′β significantly increased 14CO2 release (>60%; compare Figs. 4C and 3C). Global inhibition of PP2A-like phosphatases with oka
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