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• W2043924172 abstract "Hyperphosphorylated forms of the neuronal microtubule (MT)-associated protein tau are major components of Alzheimer's disease paired helical filaments. Previously, we reported that ABαC, the dominant brain isoform of protein phosphatase 2A (PP2A), is localized on MTs, binds directly to tau, and is a major tau phosphatase in cells. We now describe direct interactions among tau, PP2A, and MTs at the submolecular level. Using tau deletion mutants, we found that ABαC binds a domain on tau that is indistinguishable from its MT-binding domain. ABαC binds directly to MTs through a site that encompasses its catalytic subunit and is distinct from its binding site for tau, and ABαC and tau bind to different domains on MTs. Specific PP2A isoforms bind to MTs with distinct affinities in vitro, and these interactions differentially inhibit the ability of PP2A to dephosphorylate various substrates, including tau and tubulin. Finally, tubulin assembly decreases PP2A activity in vitro, suggesting that PP2A activity can be modulated by MT dynamics in vivo. Taken together, these findings indicate how structural interactions among ABαC, tau, and MTs might control the phosphorylation state of tau. Disruption of these normal interactions could contribute significantly to development of tauopathies such as Alzheimer's disease. Hyperphosphorylated forms of the neuronal microtubule (MT)-associated protein tau are major components of Alzheimer's disease paired helical filaments. Previously, we reported that ABαC, the dominant brain isoform of protein phosphatase 2A (PP2A), is localized on MTs, binds directly to tau, and is a major tau phosphatase in cells. We now describe direct interactions among tau, PP2A, and MTs at the submolecular level. Using tau deletion mutants, we found that ABαC binds a domain on tau that is indistinguishable from its MT-binding domain. ABαC binds directly to MTs through a site that encompasses its catalytic subunit and is distinct from its binding site for tau, and ABαC and tau bind to different domains on MTs. Specific PP2A isoforms bind to MTs with distinct affinities in vitro, and these interactions differentially inhibit the ability of PP2A to dephosphorylate various substrates, including tau and tubulin. Finally, tubulin assembly decreases PP2A activity in vitro, suggesting that PP2A activity can be modulated by MT dynamics in vivo. Taken together, these findings indicate how structural interactions among ABαC, tau, and MTs might control the phosphorylation state of tau. Disruption of these normal interactions could contribute significantly to development of tauopathies such as Alzheimer's disease. microtubule microtubule-associated protein protein phosphatase 2A protein phosphatase 1 recombinant tau 1,4-piperazinediethanesulfonic acid 4-morpholinepropanesulfonic acid polyacryl- amide gel electrophoresis The axonal microtubule (MT)1-associated protein (MAP) tau (1Binder L.I. Frankfurter A. Rebhun L.I. J. Cell Biol. 1985; 101: 1371-1378Crossref PubMed Scopus (1213) Google Scholar, 2Weingarten M.D. Lockwood A.H. Hwo S.-Y. Kirschner M.W. Proc. Natl. Acad. Sci. U. S. A. 1975; 72: 1858-1862Crossref PubMed Scopus (2151) Google Scholar) is encoded by one alternatively spliced gene that directs the synthesis of six tau isoforms in human brain (3Goedert M. Spillantini M.G. Jakes R. Rutherford D. Crowther R.A. Neuron. 1989; 3: 519-526Abstract Full Text PDF PubMed Scopus (1780) Google Scholar). The C-terminal half of brain tau encompasses three or four contiguous MT-binding repeats that act synergistically with regions flanking both sides of the repeats to support higher affinity MT binding (4Mandelkow E.-M. Biernat J. Drewes G. Gustke N. Trinczek B. Mandelkow E. Neurobiol. Aging. 1995; 16: 355-363Crossref PubMed Scopus (244) Google Scholar, 5Goode B.L. Denis P.E. Panda D. Radeke M.J. Miller H.P. Wilson L. Feinstein S.C. Mol. Biol. Cell. 1997; 8: 353-365Crossref PubMed Scopus (233) Google Scholar, 6Gustke N. Trinczek B. Biernat J. Mandelkow E.-M. Mandelkow E. Biochemistry. 1994; 33: 9511-9522Crossref PubMed Scopus (506) Google Scholar). All tau isoforms in human brain contain 21 serine/threonine phosphorylation sites (7Goedert M. Trojanowski J.Q. Lee V.M.-Y. Wasco W. Tanzi R.E. Molecular Mechanisms of Dementia. Humana Press, Inc., Totowa, NJ1997: 199-218Crossref Google Scholar), some of which modulate MT binding of tau (8Illenberger S. Zheng-Fischöfer Q. Preuss U. Stamer K. Baumann K. Trinczek B. Biernat J. Godemann R. Mandelkow E.-M. Mandelkow E. Mol. Biol. Cell. 1998; 9: 1495-1512Crossref PubMed Scopus (268) Google Scholar, 9Biernat J. Gustke N. Drewes G. Mandelkow E.-M. Mandelkow E. Neuron. 1993; 11: 153-163Abstract Full Text PDF PubMed Scopus (634) Google Scholar, 10Leger J. Kempf M. Lee G. Brandt R. J. Biol. Chem. 1997; 272: 8441-8446Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar, 11Xie H. Litersky J.M. Hartigan J.A. Jope R.S. Johnson G.V. Brain Res. 1998; 798: 173-183Crossref PubMed Scopus (60) Google Scholar). Only a few sites on tau are phosphorylated at any moment in normal adults (12Morishima-Kawashima M. Hasegawa M. Takio K. Suzuki M. Yoshida H. Titani K. Ihara Y. J. Biol. Chem. 1995; 270: 823-829Abstract Full Text Full Text PDF PubMed Scopus (524) Google Scholar,13Matsuo E.S. Shin R.-W. Billingsley M. Van de Voorde A. O'Connor M. Trojanowski J.Q. Lee V.M.-Y. Neuron. 1994; 13: 989-1002Abstract Full Text PDF PubMed Scopus (544) Google Scholar). In Alzheimer's disease brain, however, tau is more heavily phosphorylated (12Morishima-Kawashima M. Hasegawa M. Takio K. Suzuki M. Yoshida H. Titani K. Ihara Y. J. Biol. Chem. 1995; 270: 823-829Abstract Full Text Full Text PDF PubMed Scopus (524) Google Scholar, 13Matsuo E.S. Shin R.-W. Billingsley M. Van de Voorde A. O'Connor M. Trojanowski J.Q. Lee V.M.-Y. Neuron. 1994; 13: 989-1002Abstract Full Text PDF PubMed Scopus (544) Google Scholar), due in part to decreased tau phosphatase activity (13Matsuo E.S. Shin R.-W. Billingsley M. Van de Voorde A. O'Connor M. Trojanowski J.Q. Lee V.M.-Y. Neuron. 1994; 13: 989-1002Abstract Full Text PDF PubMed Scopus (544) Google Scholar, 14Goedert M. Jakes R. Qi Z. Wang J.H. Cohen P. J. Neurochem. 1995; 65: 2804-2807Crossref PubMed Scopus (148) Google Scholar). Hyperphosphorylated tau is the principal component of Alzheimer's disease paired helical filaments and neurofibrillar lesions present in several other neurodegenerative disorders (15Spillantini M.G. Goedert M. Trends Neurosci. 1998; 21: 428-433Abstract Full Text Full Text PDF PubMed Scopus (598) Google Scholar) and has very low affinity for MTs (16Busciglio J. Lorenzo A. Yeh J. Yankner B.A. Neuron. 1995; 14: 879-888Abstract Full Text PDF PubMed Scopus (561) Google Scholar, 17Sontag E. Nunbhaki-Craig V. Bloom G.S. Mumby M.C. J. Cell Biol. 1995; 128: 1131-1144Crossref PubMed Scopus (296) Google Scholar). Although non-phosphorylated tau can assemble into paired helical filament-like filaments in vitro (18Goedert M. Jakes R. Spillantini M.G. Hasegawa M. Smith M.J. Crowther R.A. Nature. 1996; 383: 550-553Crossref PubMed Scopus (838) Google Scholar, 19Wilson D.M. Binder L.I. J. Biol. Chem. 1995; 270: 24306-24314Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar, 20Wilson D.M. Binder L.I. Am. J. Pathol. 1997; 150: 2181-2195PubMed Google Scholar, 21Friedhoff P. von Bergen M. Mandelkow E.-M. Davies P. Mandelkow E. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 15712-15717Crossref PubMed Scopus (280) Google Scholar), it is reasonable to hypothesize that changes in tau phosphorylation are decisive events in paired helical filament biogenesis in vivo. To study how tau phosphorylation is regulated, we have been focusing on protein phosphatase 2A (PP2A), a heterotrimeric enzyme that comprises one catalytic C subunit, one non-catalytic A subunit, and one of several structurally distinct, regulatory B subunits (22Kamibayashi C. Estes R. Lickteig R.L. Yang S.-I. Craft C. Mumby M.C. J. Biol. Chem. 1994; 269: 20139-20148Abstract Full Text PDF PubMed Google Scholar). We previously reported that PP2A is likely to be a major tau phosphatase in vivo (23Sontag E. Nunbhakdi-Craig V. Lee G. Bloom G.S. Mumby M.C. Neuron. 1996; 17: 1201-1207Abstract Full Text Full Text PDF PubMed Scopus (350) Google Scholar). Initially, we found that a pool of ABαC, the major PP2A isoform in brain (22Kamibayashi C. Estes R. Lickteig R.L. Yang S.-I. Craft C. Mumby M.C. J. Biol. Chem. 1994; 269: 20139-20148Abstract Full Text PDF PubMed Google Scholar), is associated with MTs in brain and cultured cells (17Sontag E. Nunbhaki-Craig V. Bloom G.S. Mumby M.C. J. Cell Biol. 1995; 128: 1131-1144Crossref PubMed Scopus (296) Google Scholar). Subsequently, we determined that tau binds with high affinity to ABαC and ABβC; less tightly to AB′C; and poorly, if at all, to AC or individual PP2A subunits (23Sontag E. Nunbhakdi-Craig V. Lee G. Bloom G.S. Mumby M.C. Neuron. 1996; 17: 1201-1207Abstract Full Text Full Text PDF PubMed Scopus (350) Google Scholar). Finally, we found that the relative affinities of PP2A isoforms for tau correlated with their tau phosphatase activities, and suppression of PP2A activity in cells stimulated Alzheimer's disease-like phosphorylation of tau and prevented tau from binding MTs (23Sontag E. Nunbhakdi-Craig V. Lee G. Bloom G.S. Mumby M.C. Neuron. 1996; 17: 1201-1207Abstract Full Text Full Text PDF PubMed Scopus (350) Google Scholar). Here, we describe the use of tau deletion mutants, specific PP2A enzymes, and intact and proteolyzed MTs to define binding sites on tau for PP2A, on PP2A for tau and MTs, and on MTs for PP2A. When considered collectively, the results indicate how structural interactions among PP2A, tau, and MTs can control the phosphorylation of tau. The results suggest, moreover, that disruption of the normal interactions could contribute significantly to the development of tauopathies such as Alzheimer's disease. Purified bovine brain or bovine cardiac (24Mumby M.C. Russell K.L. Garrard L.J. Green D.D. J. Biol. Chem. 1987; 262: 6257-6265Abstract Full Text PDF PubMed Google Scholar, 25Kamibayashi C. Estes R. Slaughter C. Mumby M.C. J. Biol. Chem. 1991; 266: 13251-13260Abstract Full Text PDF PubMed Google Scholar) or human recombinant (22Kamibayashi C. Estes R. Lickteig R.L. Yang S.-I. Craft C. Mumby M.C. J. Biol. Chem. 1994; 269: 20139-20148Abstract Full Text PDF PubMed Google Scholar) ABαC (300 nm) in storage buffer (25 mm Tris, 1 mmdithiothreitol, 1 mm EDTA, and 50% glycerol, pH 7.5) was incubated for 15 min on ice in a final volume of 5 μl with a 600 nm concentration of either purified bovine brain tau (26Kim H. Binder L.I. Rosenbaum J. J. Cell Biol. 1979; 80: 266-276Crossref PubMed Scopus (310) Google Scholar) or any of several previously described human recombinant tau (rTau) fragments (27Brandt R. Lee G. J. Biol. Chem. 1993; 268: 3414-3419Abstract Full Text PDF PubMed Google Scholar, 28Carmel G. Mager E.M. Binder L.I. Kuret J. J. Biol. Chem. 1996; 271: 32789-32795Abstract Full Text Full Text PDF PubMed Scopus (367) Google Scholar). We also used one new recombinant tau fragment, rTau9 (see Fig. 1), which was made following the same method used to generate previously produced fragments (27Brandt R. Lee G. J. Biol. Chem. 1993; 268: 3414-3419Abstract Full Text PDF PubMed Google Scholar). In some assays, PP2A was incubated on ice for 15 min with 1–5 μm okadaic acid before adding tau. For competition experiments with the synthetic tau peptide 224KKVAVVRTPPKSP236 (numbered according to the longest isoform of human brain tau (3Goedert M. Spillantini M.G. Jakes R. Rutherford D. Crowther R.A. Neuron. 1989; 3: 519-526Abstract Full Text PDF PubMed Scopus (1780) Google Scholar)), 300 nmABαC was first incubated for 15 min with 1 or 10 μmpeptide and then for an additional 15 min in the presence of peptide plus 600 nm tau. After all incubations were completed, samples were applied directly onto pre-cast, nondenaturing 8–25% polyacrylamide gels (Amersham Pharmacia Biotech); subjected to native gel electrophoresis using the Amersham Pharmacia Biotech PhastSystem; and transferred to nitrocellulose for immunoblotting with antibodies to the C or Bα subunits of PP2A (23Sontag E. Nunbhakdi-Craig V. Lee G. Bloom G.S. Mumby M.C. Neuron. 1996; 17: 1201-1207Abstract Full Text Full Text PDF PubMed Scopus (350) Google Scholar). Immunoreactive proteins were detected using enhanced chemiluminescence reagents (ECL, Amersham Pharmacia Biotech). Blots were densitometrically scanned and quantitatively analyzed using a PhosphorImager and ImageQuant software (Molecular Dynamics, Inc.). Purified bovine brain tubulin (29Fullerton A.T. Bau M.Y. Conrad P.A. Bloom G.S. Mol. Biol. Cell. 1998; 9: 2699-2714Crossref PubMed Scopus (29) Google Scholar) at 5 μm (equal to 0.5 mg/ml) was polymerized into MTs by incubation for 10 min at 37 °C in PEM buffer (0.1 m PIPES, pH 6.9, 2 mm EGTA, and 5 mm MgCl2) containing 1 mm GTP and 20 μm Taxol (provided by Nancita Lomax, NCI, National Institutes of Health). When used in PP2A enzymatic assays, MTs were first washed free of GTP by centrifugation at 100,000 ×gmax for 20 min at 30 °C in a Beckman TLA 100.3 rotor, followed by resuspension in PEM buffer lacking GTP, but containing 20 μm Taxol. This step was essential because high levels of free GTP interfered with PP2A enzymatic assays. To remove C-terminal domains of polymerized tubulin, Taxol-stabilized MTs were incubated overnight at 30 °C with ∼1% (w/w) subtilisin (Roche Molecular Biochemicals) (30Paschal B.M. Obar R.A. Vallee R.B. Nature. 1989; 342: 569-572Crossref PubMed Scopus (147) Google Scholar). Proteolysis was then terminated by addition of phenylmethylsulfonyl fluoride to 5 mm. Subtilisin-digested MTs were sedimented for 45 min at 100,000 ×gmax in a Beckman TLA 100.3 rotor, resuspended in PEM buffer containing 2 mm phenylmethylsulfonyl fluoride and 20 μm Taxol, and used immediately for co-sedimentation assays. The extent of MT proteolysis was verified by SDS-polyacrylamide gel electrophoresis (PAGE) as described previously (30Paschal B.M. Obar R.A. Vallee R.B. Nature. 1989; 342: 569-572Crossref PubMed Scopus (147) Google Scholar, 31Melki R. Kerjan P. Waller J. Carlier M.-F. Pantaloni D. Biochemistry. 1991; 30: 11536-11545Crossref PubMed Scopus (55) Google Scholar, 32Serrano L. Montejo de Garcini E. Hernandez M.A. Avila J. Eur. J. Biochem. 1985; 153: 595-600Crossref PubMed Scopus (97) Google Scholar). PP2A and tau were incubated either individually or together for 15–20 min at room temperature with PEM buffer alone or with PEM buffer containing F-actin (33Spudich J.A. Watt S. J. Biol. Chem. 1971; 246: 4866-4871Abstract Full Text PDF PubMed Google Scholar) or intact or subtilisin-digested MTs. The samples were then centrifuged at ∼50,000 × gmax for 20 min at 25 °C in a Beckman TLA 100.3 rotor. Next, the supernatants were collected, and each pellet was resuspended to the starting volume (20–50 μl). The samples were then resolved by SDS-PAGE using 12% polyacrylamide gels, transferred to nitrocellulose, and immunoblotted with antibodies to the C subunit of PP2A or with the Tau-1 (1Binder L.I. Frankfurter A. Rebhun L.I. J. Cell Biol. 1985; 101: 1371-1378Crossref PubMed Scopus (1213) Google Scholar) or Tau-5 (28Carmel G. Mager E.M. Binder L.I. Kuret J. J. Biol. Chem. 1996; 271: 32789-32795Abstract Full Text Full Text PDF PubMed Scopus (367) Google Scholar) antibodies. Radiolabeled soluble tau (∼5 mol of phosphate/mol of protein) was produced by incubating bovine brain tau with protein kinase A (Sigma) in the presence of 20 μm [γ-32P]ATP, 10 mmMgCl2, 10 mm dithiothreitol, and 10 μm cAMP and then purifying phosphorylated tau as described previously (23Sontag E. Nunbhakdi-Craig V. Lee G. Bloom G.S. Mumby M.C. Neuron. 1996; 17: 1201-1207Abstract Full Text Full Text PDF PubMed Scopus (350) Google Scholar). Radiolabeled, MT-bound tau was obtained by incubating radiolabeled soluble tau with Taxol-assembled MTs, centrifuging MTs for 10 min in a Beckman TLA 100.3 rotor at ∼50,000 × gmax, and resuspending MTs in GTP-free PEM buffer. For the experiment shown in the upper panel of Fig. 6, ∼1000 cpm each of radiolabeled soluble and MT-bound tau were incubated for 5–45 min at 30 °C with 14 nm ABαC, and dephosphorylation of tau was halted at various time points by addition of 3× sample buffer for SDS-PAGE. For the experiment shown in the lower panel of Fig. 6, 200 nm radiolabeled soluble tau was mixed with 40 nm ABαC and 0–10 μm tubulin that had been polymerized in the presence of Taxol. The samples were incubated at 30 °C for 15 min, after which tau dephosphorylation was terminated by addition of 3× sample buffer for SDS-PAGE. The samples were resolved by SDS-PAGE using 12% polyacrylamide gels, and32P incorporation into tau was measured on dried gels using a PhosphorImager. Purified ABαC, AC, or C subunits (25 nm) in storage buffer were incubated for 15 min with either polymerized or dimeric tubulin (20 μm) in a final volume of 50 μl of phosphatase assay buffer (20 mm MOPS, 0.02% β-mercaptoethanol, and 0.25 mg/ml bovine serum albumin, pH 7.0). The reactions were performed in 96-well U-bottom microtiter plates. Green reagent (BIOMOL Research Labs, Inc.) was used in a quantitative colorimetric assay for free phosphate (Pi) released after 30-min incubations at room temperature. Pi levels were determined by measuring A620 nm according to the manufacturer's instructions. Control wells containing only tubulin, MTs, or buffer alone, but no phosphatases, were used to determine the background values of Pi, and the PP2A activities reported here were background-corrected. ABαC, AC, or C subunits were incubated for 15 min with or without 5 μmpolymerized or dimeric tubulin. The samples were then incubated for 5 min at 30 °C with a 100 μm concentration of either of two substrates: radiolabeled phosphorylated myosin light chain (24Mumby M.C. Russell K.L. Garrard L.J. Green D.D. J. Biol. Chem. 1987; 262: 6257-6265Abstract Full Text PDF PubMed Google Scholar) or the synthetic phosphopeptide RRREEE(pT)EEE (Biosynthesis Inc.). Dephosphorylation of myosin light chain was assayed by measuring the release of 32Pi as described previously (17Sontag E. Nunbhaki-Craig V. Bloom G.S. Mumby M.C. J. Cell Biol. 1995; 128: 1131-1144Crossref PubMed Scopus (296) Google Scholar). Dephosphorylation of the phosphopeptide was determined by measuring the release of Pi using the colorimetric assay described above for tubulin. Dephosphorylation of polymerized or dimeric tubulin by PP2A enzymes represented, at most, ∼4% of the total phosphatase activity measured for phosphorylated myosin light chain or the phosphopeptide. To localize the binding site on tau for ABαC, a gel mobility shift assay (23Sontag E. Nunbhakdi-Craig V. Lee G. Bloom G.S. Mumby M.C. Neuron. 1996; 17: 1201-1207Abstract Full Text Full Text PDF PubMed Scopus (350) Google Scholar) coupled with immunoblotting (see “Experimental Procedures”) was used to monitor binding of ABαC to 13 different rTau proteins, all but one of which (rTau9) have been previously described (27Brandt R. Lee G. J. Biol. Chem. 1993; 268: 3414-3419Abstract Full Text PDF PubMed Google Scholar, 28Carmel G. Mager E.M. Binder L.I. Kuret J. J. Biol. Chem. 1996; 271: 32789-32795Abstract Full Text Full Text PDF PubMed Scopus (367) Google Scholar). These recombinant proteins are derived from adult (rTau1–rTau6) or fetal (rTau7–rTau13) isoforms of human brain tau. The largest recombinant tau that was used, rTau1, contains four MT-binding repeats (four-repeat tau) and two 29-mer N-terminal inserts. As shown in Fig.1, each of the other rTau proteins contained one or more unique deletions. Their N- and C-terminal amino acids and the boundaries of their deletions are numbered relative to the amino acid sequence of the largest isoform of brain tau (3Goedert M. Spillantini M.G. Jakes R. Rutherford D. Crowther R.A. Neuron. 1989; 3: 519-526Abstract Full Text PDF PubMed Scopus (1780) Google Scholar), which is equivalent to rTau1. Fig. 1 summarizes the results of the binding assays in which ABαC and the pertinent rTau proteins were used at 300 and 600 nm, respectively. Densitometry of the resulting immunoblots was used to estimate the percentage of PP2A that was bound to each rTau protein. Maximal binding of ABαC (≥95%) was observed for every rTau protein that contains all four MT-binding repeats plus extensive sequence contiguous with the N terminus of the repeats. Included in this group are rTau1 and rTau2, which do not have any C-terminal deletions, and rTau6 and rTau8, which are missing part or all of the C-terminal 45 amino acid residues of native tau. A modest decrease in binding (to ∼80%) was observed for rTau7, which is equivalent to the fetal isoform of brain tau (3Goedert M. Spillantini M.G. Jakes R. Rutherford D. Crowther R.A. Neuron. 1989; 3: 519-526Abstract Full Text PDF PubMed Scopus (1780) Google Scholar), contains only three MT-binding repeats (three-repeat tau), and lacks the two N-terminal inserts. A similar level of ABαC binding (∼75%) was observed for rTau5, the C terminus of which is in the middle of the third MT-binding repeat, but contains no other deletions relative to rTau1. By comparison, rTau4, which contains a large internal deletion and includes just part of the last MT-binding repeat, was able to bind only ∼45% of ABαC. The minimal protein that retained the ability to bind ABαC (∼38%) was rTau12, which lacks all four MT-binding repeats, but, near its C terminus, contains a proline-rich sequence that has MT-binding activity independent of the repeats (4Mandelkow E.-M. Biernat J. Drewes G. Gustke N. Trinczek B. Mandelkow E. Neurobiol. Aging. 1995; 16: 355-363Crossref PubMed Scopus (244) Google Scholar, 5Goode B.L. Denis P.E. Panda D. Radeke M.J. Miller H.P. Wilson L. Feinstein S.C. Mol. Biol. Cell. 1997; 8: 353-365Crossref PubMed Scopus (233) Google Scholar). In stark contrast, rTau13, which lacks residues 221–242 of rTau12, but is otherwise identical, failed to bind any ABαC. Deletion of the N-terminal 29-mer inserts (rTau8) or residues 84–161 (rTau2), which include part of the second N-terminal repeat, did not impair binding of four-repeat tau to ABαC. Other deletions located N-terminal to the MT-binding repeats yielded demonstrable, albeit modest effects. A slight reduction in binding (to ∼90%) was observed for rTau3, which contains all four MT-binding repeats, but lacks most of the proline-rich MT-binding domain located immediately N-terminal to those repeats. Likewise, binding to ABαC correlated roughly with protein length for the three-repeat proteins (rTau9, rTau10, and rTau11) that have extensive N-terminal deletions. Taken together, these data demonstrate that the overall PP2A-binding region on tau encompasses the MT-binding repeats and a short sequence N-terminal to the repeats. It is thus indistinguishable, within the limits of experimental resolution, from the MT-binding region on tau (4Mandelkow E.-M. Biernat J. Drewes G. Gustke N. Trinczek B. Mandelkow E. Neurobiol. Aging. 1995; 16: 355-363Crossref PubMed Scopus (244) Google Scholar, 5Goode B.L. Denis P.E. Panda D. Radeke M.J. Miller H.P. Wilson L. Feinstein S.C. Mol. Biol. Cell. 1997; 8: 353-365Crossref PubMed Scopus (233) Google Scholar, 6Gustke N. Trinczek B. Biernat J. Mandelkow E.-M. Mandelkow E. Biochemistry. 1994; 33: 9511-9522Crossref PubMed Scopus (506) Google Scholar). Interestingly, lower affinity binding of ABαC can be achieved by proteins that contain only the extreme N-terminal (rTau12) or C-terminal (rTau4) part of the overall binding region on tau for ABαC. In addition, the binding affinity of PP2A for tau increases with the number of MT-binding repeats present in tau. It is also important to note that inclusion of 1 μm okadaic acid in the binding reactions completely suppressed PP2A activity, but had no effect on the extent of PP2A interaction with tau (data not shown). To seek further evidence that sequences on tau immediately N-terminal to the MT-binding repeats actually bind ABαC, the synthetic peptide224KKVAVVRTPPKSP236, which corresponds to a portion of this region, was tested for its ability to compete with native tau for binding to ABαC. The sequence of this portion of tau is invariant among all isoforms of human, rat, mouse, and bovine tau. Bovine brain ABαC (300 nm) was preincubated first with 1 or 10 μm peptide, after which bovine brain tau (600 nm) or an equivalent volume of buffer was added. After an additional 15-min incubation, binding of ABαC to tau was monitored by native gel electrophoresis and immunoblotting with an anti-Bα antibody (23Sontag E. Nunbhakdi-Craig V. Lee G. Bloom G.S. Mumby M.C. Neuron. 1996; 17: 1201-1207Abstract Full Text Full Text PDF PubMed Scopus (350) Google Scholar). As shown in Fig. 2, 10 μm (but not 1 μm) peptide partially inhibited binding of PP2A to tau. In addition, 10 μmpeptide induced a shift in the electrophoretic mobility of PP2A on native gels, consistent with the formation of an ABαC-peptide complex. Our previous finding that a pool of ABαC copurifies with MTs in vitro and is associated with MTs in cells (17Sontag E. Nunbhaki-Craig V. Bloom G.S. Mumby M.C. J. Cell Biol. 1995; 128: 1131-1144Crossref PubMed Scopus (296) Google Scholar) raised the question of how ABαC, and possibly other PP2A isoforms as well, might bind to MTs. The results presented in Figs. 1 and 2 strongly imply that tau cannot serve as a MT-anchoring protein for PP2A because they show that ABαC and MTs bind to the same region on tau. To determine whether PP2A might bind directly to MTs, a series of MT co-sedimentation assays were performed with various purified PP2A enzymes. As shown in Fig.3 (upper panel), when bovine brain or human recombinant ABαC heterotrimers were mixed with MTs and centrifuged, ∼50% of PP2A was recovered in the MT pellets under the experimental conditions utilized. The possibility that ABαC may have nonspecifically co-sedimented with MTs under these conditions was assessed in parallel control experiments, in which MTs were omitted or F-actin was substituted for MTs. ABαC proteins did not sediment in either of these cases, emphasizing the specificity of the direct interaction between ABαC and MTs. In addition, 0.5 m NaCl was found to prevent co-sedimentation of PP2A with MTs, indicating that ionic interactions are important for association of the enzyme with MTs. As was observed for the binding of ABαC to tau, inclusion of 5 μm okadaic acid in the assays completely suppressed PP2A activity, but had no effect on the extent of interaction of ABαC with MTs (data not shown). In the next series of experiments, increasing concentrations of ABαC were incubated with MTs and then centrifuged to generate MT-bound (pellet) and unbound (supernatant) fractions. Fig. 3 (lower panel) shows that ABαC co-pelleted with MTs in a concentration-dependent manner. The highest concentration of ABαC that we were able to use for these experiments (1 μm) was not sufficient for saturation binding to ∼5 μm assembled tubulin (see Fig. 4). Nevertheless, ∼40% of 1 μm ABαC bound to ∼5 μm assembled tubulin, indicating that MTs must be able to accommodate >1 ABαC heterotrimer/12.5 tubulin dimers. Furthermore, because ∼80% of ABαC bound to MTs when the total concentration of ABαC was 0.1 μm (Fig. 3, lower panel), the binding of ABαC to MTs must be tight. Efforts to use Scatchard analysis to determine saturation binding and a dissociation constant more accurately were unsuccessful for ABαC and other forms of PP2A (Figs.3 (lower panel) and 4) because the Scatchard plots could not be described accurately by simple linear equations (data not shown). To assess whether ABαC is the only form of PP2A that can bind MTs, we compared the behavior of distinct PP2A enzymes in the MT co-sedimentation assay. As shown in Fig.4, all enzymatically active proteins tested, including the ABβC and AB′C holoenzymes, the AC dimer, and the catalytic C subunit, were able to bind MTs to some extent. However, distinct PP2A isoforms appeared to have distinct affinities for MTs because at fixed molar concentrations of PP2A and polymerized tubulin, the ratio of MT-bound to soluble enzyme varied considerably among the phosphatases tested. Based on the results presented in Fig. 4, the ability of PP2A to bind to MTs can be ranked as follows: ABαC > AC > ABβC > C > AB′C. When actin filaments were substituted for microtubules, virtually none of the PP2A enzymes pelleted, demonstrating that their binding to microtubules was specific (data not shown). The fact that the monomeric C subunit co-sedimented with MTs indicates that it contains a binding site for MTs. However, our findings suggest that the presence of A and B subunits modulates interactions of the catalytic C subunit with MTs and that each type of B subunit does so in its own unique way. As reported previously for binding of various forms of PP2A to tau (23Sontag E. Nunbhakdi-Craig V. Lee G. Bloom G.S. Mumby M.C. Neuron. 1996; 17: 1201-1207Abstract Full Text Full Text PDF PubMed Scopus (350) Google Scholar), the ABαC heterotrimer bound more tightly to MTs than any of the other forms of PP2A that were assayed. In addition, we found that neither ABαC nor AC forms detectable complexes with unpolymerized tubulin during nondenaturing gel electrophoresis (data not shown), implying that PP2A can efficiently interact with tubulin only when the tubulin has polymerized. Binding to MTs of several MAPs such tau and MAP2 can be partially inhibited by prior exposure of either unassembled (32Serrano L. Montejo de Garcini E. Hernandez M.A. Avila J. Eur. J. Biochem. 1985; 153: 595-600Crossref PubMed Scopus (97) Google Scholar) or polymerized (30Paschal B.M. Obar R.A. Vallee R.B. Nature. 1989; 342: 569-572Crossref PubMed Scopus (147) Google Scholar, 31Melki R. Kerjan P. Waller J. Carlier M.-F. Pantaloni D. Biochemistry. 19" @default.
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• W2043924172 title "Molecular Interactions among Protein Phosphatase 2A, Tau, and Microtubules" @default.
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