Protein Phosphatase 1 Is Targeted to Microtubules by the Microtubule-associated Protein Tau
1998; Elsevier BV; Volume: 273; Issue: 34 Linguagem: Inglês
10.1074/jbc.273.34.21901
ISSN1083-351X
AutoresHong Liao, Yarong Li, David L. Brautigan, Gregg G. Gundersen,
Tópico(s)Alzheimer's disease research and treatments
ResumoPhosphorylation has been implicated in the regulation of microtubule (MT) stability and function by controlling the interactions between MTs and MT-associated proteins. We found previously that protein phosphatase inhibitors selectively break down stable MTs, suggesting that protein phosphatases may be involved in regulating MT stability. To identify the protein phosphatases involved, we examined purified calf brain MTs and found a protein phosphatase activity that copurified with MTs to constant stoichiometry. Western blot analysis and inhibitor profiles demonstrated that the MT-associated phosphatase was a type 1 protein phosphatase (PP1), which we named PP1MT. Recombinant PP1 catalytic subunit (PP1c) did not bind to MTs, whereas PP1MT did bind, suggesting the presence of proteins that target PP1 to MTs. By Sepharose CL-6B chromatography, the phosphatase activity of PP1MT eluted as a large protein complex of ∼400 kDa. High salt (2 m NaCl) treatment followed by CL-6B chromatography dissociated PP1MT into PP1c and the MT-targeting subunit(s). The MT-targeting subunit was shown to be the MT-associated protein tau by PP1 blot overlays and other assays. Also, recombinant tau reconstituted the binding of PP1c to MTs. These results identify PP1 as the first tau binding protein and suggest that tau is a novel PP1-targeting subunit. Phosphorylation has been implicated in the regulation of microtubule (MT) stability and function by controlling the interactions between MTs and MT-associated proteins. We found previously that protein phosphatase inhibitors selectively break down stable MTs, suggesting that protein phosphatases may be involved in regulating MT stability. To identify the protein phosphatases involved, we examined purified calf brain MTs and found a protein phosphatase activity that copurified with MTs to constant stoichiometry. Western blot analysis and inhibitor profiles demonstrated that the MT-associated phosphatase was a type 1 protein phosphatase (PP1), which we named PP1MT. Recombinant PP1 catalytic subunit (PP1c) did not bind to MTs, whereas PP1MT did bind, suggesting the presence of proteins that target PP1 to MTs. By Sepharose CL-6B chromatography, the phosphatase activity of PP1MT eluted as a large protein complex of ∼400 kDa. High salt (2 m NaCl) treatment followed by CL-6B chromatography dissociated PP1MT into PP1c and the MT-targeting subunit(s). The MT-targeting subunit was shown to be the MT-associated protein tau by PP1 blot overlays and other assays. Also, recombinant tau reconstituted the binding of PP1c to MTs. These results identify PP1 as the first tau binding protein and suggest that tau is a novel PP1-targeting subunit. Microtubules (MT) 1The abbreviations used are: MTmicrotubuleMAPmicrotubule-associated proteinPPprotein phosphatasePP1cprotein phosphatase 1 catalytic subunitPAGEpolyacrylamide gel electrophoresisADAlzheimer's diseaseOAokadaic acidCL-Acalyculin-APHFpaired helical filamentPipes1,4-piperazinediethanesulfonic acid.1The abbreviations used are: MTmicrotubuleMAPmicrotubule-associated proteinPPprotein phosphatasePP1cprotein phosphatase 1 catalytic subunitPAGEpolyacrylamide gel electrophoresisADAlzheimer's diseaseOAokadaic acidCL-Acalyculin-APHFpaired helical filamentPipes1,4-piperazinediethanesulfonic acid. are major cytoskeletal elements in eukaryotic cells and play essential roles in cell motility, division and differentiation. The assembly and functions of MTs are dependent on their interactions with microtubule-associated proteins (MAPs) (1Schoenfeld T.A. Obar R.A. Int. Rev. Cytol. 1994; 151: 67-137Crossref PubMed Scopus (98) Google Scholar, 2Hirokawa N. Curr. Opin. 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The Neuronal Cytoskeleton. Wiley-Liss, New York1991: 5-74Google Scholar). Although the mechanisms by which MT function is regulated are not completely understood, phosphorylation of MAPs has been shown to regulate their interactions with MTs (1Schoenfeld T.A. Obar R.A. Int. Rev. Cytol. 1994; 151: 67-137Crossref PubMed Scopus (98) Google Scholar, 2Hirokawa N. Curr. Opin. Cell Biol. 1994; 6: 74-81Crossref PubMed Scopus (342) Google Scholar, 3Mandelkow E.M. Mandelkow E. Curr. Opin. Cell Biol. 1995; 7: 72-81Crossref PubMed Scopus (373) Google Scholar, 4Maccioni R.B. Cambiazo V. Physiol. Rev. 1995; 75: 836-864Crossref Scopus (329) Google Scholar). In general, increased phosphorylation of MAPs reduces the affinity of the MAPs for MTs (11Brion J.P. Guilleminot J. Couchie D. Flament-Durand J. Nunez J. Neuroscience. 1988; 25: 139-146Crossref PubMed Scopus (118) Google Scholar, 12Jameson L. Frey T. Zeeberg B. Dalldorf F. Caplow M. 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In contrast, much less is known about the protein phosphatases involved. However, several lines of evidence suggest that protein phosphatases may be rate-limiting enzymes in the regulation of MAP-MT interactions. In our previous study, phosphatase inhibitors okadaic acid (OA) and calyculin-A (CL-A) were found to induce the complete loss of stable MTs without significantly affecting the level of dynamic MTs in interphase cells (22Gurland G. Gundersen G.G. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 8827-8831Crossref PubMed Scopus (84) Google Scholar). The dose response curves of OA and CL-A in this study suggest that protein phosphatase 1 (PP1) is involved in maintaining stable MTs. Treatment of interphase sea urchin egg extracts with OA induces mitotic-like MT dynamics by eliminating the rescue phase of MT dynamics (23Gliksman N.R. Parsons S.F. Salmon E.D. J. Cell Biol. 1992; 119 (1226): 1271Crossref PubMed Scopus (97) Google Scholar). Incubation of mouse oocytes with OA leads to lengthening of mitotic spindles, disorganization of the metaphase plate, and phosphorylation of proteins associated with MTs (24de Pennart H. Verlhac M. Cibert C. Maria A.S. Maro B. Dev. Biol. 1993; 157: 170-181Crossref PubMed Scopus (26) Google Scholar). These results imply that protein phosphatases are necessary for dephosphorylation of MAPs, resulting in increased MT assembly and stability in vivo. However, the identity of the protein phosphatases involved, their specific in vivo substrates, and how are they regulated in response to internal and external cues are still unknown. PP1 is one of the four major serine/threonine phosphatases found in eukaryotic cells. It plays important roles in diverse cellular activities, such as carbohydrate and lipid metabolism, calcium transport, gating of ionic channels, muscle contraction, nuclear organization, protein synthesis, mitosis, and meiosis (25Bollen M. Stalmas W. Crit. Rev. Biochem. Mol. Biol. 1992; 27: 227-281Crossref PubMed Scopus (260) Google Scholar, 26Shenoliker S. Annu. Rev. Cell Biol. 1994; 10: 55-86Crossref PubMed Scopus (401) Google Scholar, 27Cohen P. Cohen P.T.W. J. Biol. Chem. 1989; 264: 21435-21438Abstract Full Text PDF PubMed Google Scholar, 28Cohen P. Annu. Rev. Biochem. 1989; 58: 453-508Crossref PubMed Scopus (2135) Google Scholar). The fact that numerous and diverse substrates are dephosphorylated by this single enzyme in various subcellular localizations suggests the existence of mechanisms to determine and regulate phosphatase specificity and activity. Emerging evidence has supported a general mechanism whereby PP1 is directed to various substrates by specific targeting subunits that also modulate the enzymatic activity of PP1 toward its substrates (29Hubbard M.J. Cohen P. Trends Biochem. Sci. 1993; 18: 172-177Abstract Full Text PDF PubMed Scopus (785) Google Scholar). Thus, for a particular cellular event regulated by PP1, it is essential to identify the targeting subunit that directs PP1 to protein substrates involved in the event. Several protein kinases and one phosphatase have been found to associate with MTs (30Sontag E. Numbhakdi-Craig V. Bloom G.S. Mumby M.C. J. Cell Biol. 1995; 128: 1131-1144Crossref PubMed Scopus (296) Google Scholar, 31Rubino H.M. Dammerman M. Shafit-Zagardo B. Erlichman J. Neuron. 1989; 3: 631-638Abstract Full Text PDF PubMed Scopus (99) Google Scholar, 32Morishima-Kawashima M. Kosik K.S. Mol. Biol. Cell. 1996; 7: 893-905Crossref PubMed Scopus (144) Google Scholar, 33Ookata K. Hisanaga S. Bulinski J.C. Murofushi H. Aizawa H. Itoh T.J. Hotani H. Okumura E. Tachibana K. Kishimoto T. J. Cell Biol. 1995; 128: 849-862Crossref PubMed Scopus (237) Google Scholar, 34Rattner J.B. Lew J. Wang J.H. Cell Motil. Cytoskelet. 1990; 17: 227-235Crossref PubMed Scopus (70) Google Scholar). In all known cases, the kinases were found to associate with MTs through MAPs. MAP2 was found to bind both the regulatory subunit of Type II cAMP-dependent protein kinase and MAP kinase to MTs (31Rubino H.M. Dammerman M. Shafit-Zagardo B. Erlichman J. Neuron. 1989; 3: 631-638Abstract Full Text PDF PubMed Scopus (99) Google Scholar, 32Morishima-Kawashima M. Kosik K.S. Mol. Biol. Cell. 1996; 7: 893-905Crossref PubMed Scopus (144) Google Scholar), and MAP4 was found to bind cyclin B, which may direct Cdc2 kinase to MTs (33Ookata K. Hisanaga S. Bulinski J.C. Murofushi H. Aizawa H. Itoh T.J. Hotani H. Okumura E. Tachibana K. Kishimoto T. J. Cell Biol. 1995; 128: 849-862Crossref PubMed Scopus (237) Google Scholar). This suggests that regulatory enzymes associated with MTs may use members of the MAP family to mediate their association. This may be a general mechanism that also applies to MT-associated phosphatases, although the mechanism of their association has not been elucidated. In this study, we report that there is a PP1 activity that remains tightly associated with MTs during their biochemical isolation from brain tissue. We provide evidence that this association is mediated by a MT-targeting protein. Using biochemical assays, we show that the MAP tau is predominantly responsible for binding PP1 to MTs. Our results suggest that tau is a novel PP1-targeting subunit and raise the possibility that phosphatases and kinases use different MAPs to associate with MTs. Okadaic acid and CL-A were from LC Laboratories (Woburn, MA). Taxol was obtained from the Drug Synthesis and Chemical Branch of the Division of Cancer Treatment, NCI. All tissue culture media were from Life Technologies, Inc. Sepharose CL-6B, blue dextran, and catalase were obtained from Amersham Pharmacia Biotech. [γ-32P]ATP (3000–6000 Ci/mmol, 5–10 mCi/ml) was purchased from Amersham Pharmacia Biotech. The ECL solution was from Pierce. Unless specified otherwise, all other chemicals were purchased from Sigma. A peptide antibody against PP1 was generated by immunizing rabbits with a synthetic peptide CDLQSMEQIRRIM (residues 180–191 of PP1c with an additional Cys residue at the N terminus for coupling) corresponding to a region conserved among all PP1 isoforms. Rabbit antisera were produced by Pocono Rabbit Farms (Canadensis, PA) by immunizing rabbits with the peptide-keyhole limpet hemocyanin conjugate (50 μg peptide/injection) (35Yoshitake S. Yamada Y. Ishikawa E. Masseyeff R. Eur. J. Biochem. 1979; 101: 395-399Crossref PubMed Scopus (164) Google Scholar). A peptide antibody against PP2A was generated as described previously (36Brautigan D.L. Chen J. Thompson P. Adv. Protein Phosphatases. 1993; 7: 43-60Google Scholar). Tau antibody T46, which reacts with all tau isoforms (37Trojanowski J.Q. Schmidt M.L. Lee V.M.Y. J. Histochem. Cytochem. 1989; 37: 209-215Crossref PubMed Scopus (258) Google Scholar), was a generous gift from Dr. J. Q. Trojanowski (University of Pennsylvania). MTs were prepared from calf brains by 3–4 cycles of warm assembly/cold disassembly as described (38Vallee R.B. Methods Enzymol. 1986; 134: 89-103Crossref PubMed Scopus (137) Google Scholar). MAPs and tubulin were prepared from thrice-cycled MT pellets by DEAE Sephadex A-50 frozen chromatography (38Vallee R.B. Methods Enzymol. 1986; 134: 89-103Crossref PubMed Scopus (137) Google Scholar), frozen in liquid nitrogen, and stored in aliquots at −80 °C. All steps were performed at 4 °C. A Sepharose CL-6B column (0.9 × 90 cm) was equilibrated in PEM buffer (100 mm Pipes, pH 6.9, 1 mm MgCl2, 1 mm EGTA) (low salt) or PEM buffer containing 2m NaCl (high salt). Blue dextran and catalase were used to calibrate the column. For low salt conditions, PP1c or MAPs were applied to the column and eluted with PEM buffer at 0.2 ml/min. Fractions of 0.5 ml were collected. For high salt conditions, PP1c or MAPs were incubated in PEM containing 2 m NaCl overnight at 4 °C and then applied to the column and eluted with PEM containing 2m NaCl. Fractions from the high salt column were dialyzed in PEM buffer overnight using a microdialysis system (Life Technologies, Inc.). Protein concentrations of the column fractions were estimated by A280. Phosphorylase b was purified from rabbit skeletal muscle as described (39Gratecos D. Detwiler T.C. Hurd S. Fisher E.H. Biochemistry. 1977; 16: 4812-4817Crossref PubMed Scopus (62) Google Scholar) and labeled with [γ-32P]ATP (40Brautigan D.L. Shriner C.L. Gruppuso P.A. J. Biol. Chem. 1985; 260: 4295-4302Abstract Full Text PDF PubMed Google Scholar). Phosphatase assays were performed as described (40Brautigan D.L. Shriner C.L. Gruppuso P.A. J. Biol. Chem. 1985; 260: 4295-4302Abstract Full Text PDF PubMed Google Scholar), except that 0.5 mm MnCl was included in the assay buffer. A PEM buffer blank was run in parallel, and the background was subtracted from each sample. One unit of activity is equal to one nmol of phosphate released per min. The inhibitor profile of the MT-associated protein phosphatase was determined by adding inhibitors to the reaction mixture before adding the [32P]phosphorylase a substrate. For experiments with the heat stable inhibitor I2, samples were preincubated for 15 min at 30 °C prior to adding the substrate. I2 was purified as described previously (41Gruppuso P.A. Johnson G.L. Constantinides M. Brautigan D.L. J. Biol. Chem. 1985; 260: 4288-4294Abstract Full Text PDF PubMed Google Scholar). The rabbit skeletal muscle PP1c used for comparison in these experiments was purified to homogeneity as described earlier (42DeGuzman A. Lee E.Y.C. Methods Enzymol. 1988; 159: 356-366Crossref PubMed Scopus (62) Google Scholar). All procedures were performed at room temperature. Samples were first centrifuged at 50,000 ×g for 15 min at room temperature to remove aggregates. MTs were prepared by polymerizing DEAE-purified tubulin with 20 μm taxol as described (43Vallee R.B. Collins C.A. Methods Enzymol. 1986; 134: 116-127Crossref PubMed Scopus (46) Google Scholar). Samples were incubated with taxol-MTs in PEM buffer for 20 min followed by centrifugation at 50,000 × g for 10 min. The supernatants and pellets were recovered, and phosphatase activity was measured. In control experiments, PEM buffer was used instead of MTs to assess nonspecific sedimentation. Fractions from the Sepharose CL-6B column eluted with 2 m NaCl were dialyzed and assayed for their ability to bind recombinant PP1cα to taxol-MTs. Purified recombinant PP1cα was a generous gift of Dr. E. Y. C. Lee (New York Medical School, Valhala, NY) and was prepared as described previously (44Zhang Z. Bai G. Deans-Zirattu S. Browner M.F. Lee E.Y.C. J. Biol. Chem. 1992; 267: 1484-1490Abstract Full Text PDF PubMed Google Scholar). Recombinant PP1c (0.13 μg) was incubated with ∼2 μg of protein from each fraction for 10 min at room temperature. Samples were preclarified before incubation as described above. Taxol-MTs (20 μg) were then added to the mixture and incubated for 20 min. Supernatants and pellets were prepared by centrifugation (50,000 ×g for 10 min) and then assayed for phosphatase activity. The pellets were solubilized in PEM buffer before assay. To measure the activation of PP1c activity by column fractions, PP1c was incubated for 10 min with each column fraction that was used in the MT targeting assay and then assayed directly for phosphatase activity. The activity of PP1c alone was used as a base line to calculate the magnitude of activation by each column fraction. The phosphatase activity in each MT pellet in the targeting assay was then normalized by the level of activation in the absence of MTs. Protein samples were run on 4–14% SDS-PAGE and, transferred to a nitrocellulose membrane, and blots were stained by Ponceau Red. The blot was blocked with NET buffer (150 mm NaCl, 50 mm Tris, pH 7.5, 5 mm EDTA, 0.05% Triton, 2.5 g/liter gelatin) containing 1% bovine serum albumin at room temperature for > 1 h. For blot overlays with PP1c, the membrane was incubated with 1.8 μg/ml of recombinant PP1c in NET buffer for 2 h at room temperature followed by extensive washing with NET buffer. The membrane was then incubated with rabbit PP1 peptide antibody (at 1:2000 dilution) in NET buffer for ∼2 h. The blot was washed and incubated in goat anti-rabbit horseradish peroxidase (Cappel, Organon Teknik Co.) at 1:20,000 dilution in NET buffer followed by detection with ECL solution. For Western blot analysis, blots were blocked with NET buffer containing 1% bovine serum albumin and incubated with the primary antibodies indicated under “Results.” Blots were then developed with horseradish peroxidase-conjugated secondary antibodies and ECL. Sf9 cells were infected by recombinant tau baculovirus (provided by Dr. T. Frappier) as described (45Knops J. Kosik K.S. Lee G. Pardee J.D. Cohen G.L. McConlogue L. J. Cell Biol. 1991; 114: 725-733Crossref PubMed Scopus (234) Google Scholar). After 2 days of infection, the cells were collected, washed twice in phosphate-buffered saline buffer containing proteinase inhibitors chymostatin, leupeptin, antipain, and pepstatin (each at 0.1 mg/ml) and phenylmethylsulfonyl fluoride (0.2 mm), resuspended in lysis buffer (50 mm Pipes, pH 6.9, 50 mm β-glycerol phosphate, 1 mm EGTA, 0.5 mm MgCl2, phenylmethylsulfonyl fluoride, and chymostatin, leupeptin, antipain, and pepstatin), and then sonicated. NaCl and β-mercaptoethanol were added to the cell extract at final concentrations of 0.4 m and 10 mm, respectively. The mixture was then boiled for 5 min and centrifuged at 30,000 × g for 15 min. The supernatant containing heat stable tau was dialyzed in PEM buffer and stored at −80 °C. The sample was > 90% tau as judged by quantitative SDS-PAGE. To identify protein phosphatases that might be involved in regulating MT stability, preparations of MTs purified from calf brain homogenates were obtained by cycles of warm assembly and cold disassembly using the standard protocol (38Vallee R.B. Methods Enzymol. 1986; 134: 89-103Crossref PubMed Scopus (137) Google Scholar). After three or four cycles, the preparation yielded about 80% assembly competent tubulin and 20% presumptive MAPs. Using [32P]phosphorylase, a standard substrate for both PP1 and PP2A, we detected a phosphatase activity that co-assembled with MTs throughout four cycles of MT polymerization/depolymerization (TableI). Approximately 5% of the total soluble phosphatase activity in brain extracts cosedimented with MTs during the first cycle. With additional cycles, about half of this activity was lost (compare activity in P1 with that of P2–P4), presumably because of the loss of phosphatases loosely associated with MTs or with contaminants that were removed in subsequent cycles. Nonetheless, 1–2% of the total extract activity cycled with MTs to constant stoichiometry from the second to fourth cycles. The phosphatase activity was also detected in DEAE-purified MAP fractions and could be dissociated quantitatively from MTs by 0.3 msalt treatment, which is a characteristic of conventional MAPs (data not shown).Table IAssociation of protein phosphatase activity with MTs during assembly/disassembly cyclesSampleProteinPhosphatase ActivityConcentrationTotalYieldActivityTotalYieldmg/mlmg%unit/mgunit%HSS10.778508.3100542541100P110.017598.922.821285P29.28519.16.10.95aConstant specific activity in P2–P4.4931.16P38.393824.490.89aConstant specific activity in P2–P4.3400.8P48.32305.13.590.87aConstant specific activity in P2–P4.2660.6Following preparation of a brain extract (HSS), MTs were cycled four times between an assembled polymeric state and a disassembled soluble state. P1–P4 are sequential MT pellets that were resolubilized prior to assay. Protein concentration in each fraction was measured, and total protein was from one preparation using six calf brains. Equal amounts of protein from each fraction were assayed for phosphatase activity as described under “Experimental Procedures.” One unit of activity is equal to 1 nmol of phosphate released per min. Similar results were observed in three separate preparations.a Constant specific activity in P2–P4. Open table in a new tab Following preparation of a brain extract (HSS), MTs were cycled four times between an assembled polymeric state and a disassembled soluble state. P1–P4 are sequential MT pellets that were resolubilized prior to assay. Protein concentration in each fraction was measured, and total protein was from one preparation using six calf brains. Equal amounts of protein from each fraction were assayed for phosphatase activity as described under “Experimental Procedures.” One unit of activity is equal to 1 nmol of phosphate released per min. Similar results were observed in three separate preparations. To determine whether PP1 or PP2A were present in the cycled MT preparations and hence might be responsible for the phosphatase activity, we probed Western blots of the fractions from the MT assembly/disassembly cycles using peptide-specific antibodies to either PP1 or PP2A. As shown in Fig.1 A, a single band of ∼37 kDa, which comigrated with bacterially expressed recombinant PP1c, was detected in MT pellet fractions (P1–P4) probed by PP1 antibody. The levels of PP1 judged by the immunoreactive bands in the last three pellet fractions (P2–P4) were similar, which is consistent with the constant stoichiometry of phosphatase activity associated with MTs (Table I). Accordingly, there is little detectable PP1 in supernatant fractions in the last two cycles (S3 and S4). PP1 was also detected in the MAP fraction purified by DEAE-Sephadex chromatography of MT proteins cycled three times (Fig. 1 A). In contrast, PP2A was detected in the first MT pellet fraction but in decreasing amounts in subsequent MT pellets so that by the third or fourth cycle, no PP2A was detected in MT pellets (Fig. 1 B). No significant amount of PP2A was detected in the DEAE-purified MAP fraction (Fig. 1 B). Also, no substantial phosphatase activity was detected in purified MAPs when p-nitrophenyl phosphate, a preferred substrate for PP2A but a much less sensitive substrate for PP1 (46Li H.C. Hsiao K.J. Sampathkumar S. J. Biol. Chem. 1979; 254: 3368-3374Abstract Full Text PDF PubMed Google Scholar), was used to assay phosphatase activity (data not shown). No increase of activity was observed when the PP2B activator Ca2+ was included in the phosphatase assay (data not shown) (26Shenoliker S. Annu. Rev. Cell Biol. 1994; 10: 55-86Crossref PubMed Scopus (401) Google Scholar). These results suggest that PP1, but not PP2A or PP2B, may be responsible for the phosphorylase phosphatase activity that coassembles with MTs to constant stoichiometry. To demonstrate that the MT-associated phosphatase is indeed PP1, the inhibitor profile of phosphatase activity in the fourth cycle MT pellet (P4) was characterized. OA inhibits phosphatases at different concentrations with an IC50 value of 0.1–1 nmfor PP2A, ∼50 nm for PP1, and >1 μm for PP2B and PP2C (48Bialojan C. Takai A. Biochem. J. 1988; 256: 283-290Crossref PubMed Scopus (1493) Google Scholar). As shown in Fig.2 A, the dose response curve of the MT-associated phosphatase to OA was nearly identical to that measured for PP1c purified from skeletal muscle with an IC50 of about 50–60 nm (49Ishihara H Martin B.L. Brautigan D.L. Karaki H. Ozaki H. Kato Y. Fusetani N. Watabe S. Hashimoto K. Uemura D. Hartshorne D.J. Biochem. Biophys. Res. Commun. 1989; 159: 871-877Crossref PubMed Scopus (907) Google Scholar). Similarly, the dose response curve of the MT-associated phosphatase in P4 toward CL-A closely resembled that of PP1c with an IC50 of ∼10 nm (Fig. 2 B) (49Ishihara H Martin B.L. Brautigan D.L. Karaki H. Ozaki H. Kato Y. Fusetani N. Watabe S. Hashimoto K. Uemura D. Hartshorne D.J. Biochem. Biophys. Res. Commun. 1989; 159: 871-877Crossref PubMed Scopus (907) Google Scholar). The greater sensitivity of the MT-associated phosphatase to CL-A compared with OA, is a characteristic of PP1 but not PP2A phosphatases (49Ishihara H Martin B.L. Brautigan D.L. Karaki H. Ozaki H. Kato Y. Fusetani N. Watabe S. Hashimoto K. Uemura D. Hartshorne D.J. Biochem. Biophys. Res. Commun. 1989; 159: 871-877Crossref PubMed Scopus (907) Google Scholar). Moreover, both PP1c and the MT-associated phosphatase were inhibited by heparin and protamine, two specific inhibitors of PP1 but activators for PP2A (50Pelech S. Cohen P. Eur. J. Biochem. 1985; 148: 245-251Crossref PubMed Scopus (71) Google Scholar). Most definitively, I2, a specific and diagnostic protein inhibitor of PP1, inhibited the activities of both the MT-associated phosphatase and PP1c to >90% (Fig. 2 C). Taken together, the above results clearly indicate that the protein phosphatase that tightly associates with MTs through multiple cycles of assembly/disassembly is exclusively a PP1-type protein phosphatase. We therefore named it PP1MT. To test whether PP1MT binds directly to MTs or indirectly through another protein, the MT binding abilities of PP1MT in a crude MAP fraction and purified, recombinant PP1c were compared in a MT cosedimentation assay. Recombinant PP1c alone did not cosediment with MTs, whereas PP1MT nearly quantitatively cosedimented with MTs (TableII). Neither PP1MT nor PP1c sedimented by themselves. This result suggests that unlike PP1MT, PP1c cannot directly bind to MTs.Table IIMT binding of PP1MT and PP1cProtein phosphatase activitySupernatantPelletunits%units%PP1c8.2960.34PP1c + MT8.5970.33MAPs8950.45MAPs + MT2.1246.576MAPs were eluted from brain MT preparation by 0.3 m NaCl. Before being used in MT cosedimentation assay, MAPs were diluted with PEM buffer to reduce the salt concentration to 0.1 m. MAPs or recombinant PP1c containing an equivalent amount of phosphatase activity were used in MT cosedimentation assay. The percentage of phosphatase activity in supernatant or pellet was calculated against the total phosphatase activity used in each assay. Similar results were observed in three experiments. Open table in a new tab MAPs were eluted from brain MT preparation by 0.3 m NaCl. Before being used in MT cosedimentation assay, MAPs were diluted with PEM buffer to reduce the salt concentration to 0.1 m. MAPs or recombinant PP1c containing an equivalent amount of phosphatase activity were used in MT cosedimentation assay. The percentage of phosphatase activity in supernatant or pellet was calculated against the total phosphatase activity used in each assay. Similar results were observed in three experiments. There are three possible explanations for how PP1MTassociates with MTs. One is that PP1c bin
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