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• W2023265997 abstract "We previously characterized PP1bp134 and PP1bp175, two neuronal proteins that bind the protein phosphatase 1 catalytic subunit (PP1). Here we purify from rat brain actin-cytoskeletal extracts PP1A holoenzymes selectively enriched in PP1γ1 over PP1β isoforms and also containing PP1bp134 and PP1bp175. PP1bp134 and PP1bp175 were identified as the synapse-localized F-actin-binding proteins spinophilin (Allen, P. B., Ouimet, C. C., and Greengard, P. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 9956–9561; Satoh, A., Nakanishi, H., Obaishi, H., Wada, M., Takahashi, K., Satoh, K., Hirao, K., Nishioka, H., Hata, Y., Mizoguchi, A., and Takai, Y. (1998) J. Biol. Chem. 273, 3470–3475) and neurabin (Nakanishi, H., Obaishi, H., Satoh, A., Wada, M., Mandai, K., Satoh, K., Nishioka, H., Matsuura, Y., Mizoguchi, A., and Takai, Y. (1997)J. Cell Biol. 139, 951–961), respectively. Recombinant spinophilin and neurabin interacted with endogenous PP1 and also with each other when co-expressed in HEK293 cells. Spinophilin residues 427–470, or homologous neurabin residues 436–479, were sufficient to bind PP1 in gel overlay assays, and selectively bound PP1γ1 from a mixture of brain protein phosphatase catalytic subunits; additional N- and C-terminal sequences were required for potent inhibition of PP1. Immunoprecipitation of spinophilin or neurabin from crude brain extracts selectively coprecipitated PP1γ1 over PP1β. Moreover, immunoprecipitation of PP1γ1 from brain extracts efficiently coprecipitated spinophilin and neurabin, whereas PP1β immunoprecipitation did not. Thus, PP1A holoenzymes containing spinophilin and/or neurabin target specific neuronal PP1 isoforms, facilitating efficient regulation of synaptic phosphoproteins. We previously characterized PP1bp134 and PP1bp175, two neuronal proteins that bind the protein phosphatase 1 catalytic subunit (PP1). Here we purify from rat brain actin-cytoskeletal extracts PP1A holoenzymes selectively enriched in PP1γ1 over PP1β isoforms and also containing PP1bp134 and PP1bp175. PP1bp134 and PP1bp175 were identified as the synapse-localized F-actin-binding proteins spinophilin (Allen, P. B., Ouimet, C. C., and Greengard, P. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 9956–9561; Satoh, A., Nakanishi, H., Obaishi, H., Wada, M., Takahashi, K., Satoh, K., Hirao, K., Nishioka, H., Hata, Y., Mizoguchi, A., and Takai, Y. (1998) J. Biol. Chem. 273, 3470–3475) and neurabin (Nakanishi, H., Obaishi, H., Satoh, A., Wada, M., Mandai, K., Satoh, K., Nishioka, H., Matsuura, Y., Mizoguchi, A., and Takai, Y. (1997)J. Cell Biol. 139, 951–961), respectively. Recombinant spinophilin and neurabin interacted with endogenous PP1 and also with each other when co-expressed in HEK293 cells. Spinophilin residues 427–470, or homologous neurabin residues 436–479, were sufficient to bind PP1 in gel overlay assays, and selectively bound PP1γ1 from a mixture of brain protein phosphatase catalytic subunits; additional N- and C-terminal sequences were required for potent inhibition of PP1. Immunoprecipitation of spinophilin or neurabin from crude brain extracts selectively coprecipitated PP1γ1 over PP1β. Moreover, immunoprecipitation of PP1γ1 from brain extracts efficiently coprecipitated spinophilin and neurabin, whereas PP1β immunoprecipitation did not. Thus, PP1A holoenzymes containing spinophilin and/or neurabin target specific neuronal PP1 isoforms, facilitating efficient regulation of synaptic phosphoproteins. catalytic subunit of protein phosphatase 1 dithiothreitol phenylmethylsulfonyl fluoride PP1-binding protein catalytic subunit of protein phosphatase 2A polyacrylamide gel electrophoresis glutathioneS-transferase 3-(cyclohexylamino)propanesulfonic acid dioxigenin glycogen-targeting subunit of protein phosphatase 1 α-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid Phosphorylation/dephosphorylation of synaptic proteins is regulated by neurotransmitters and is a major modulator of synaptic function. For example, Ca2+/calmodulin-dependent protein kinase II and protein phosphatase 1 are two major dendritic proteins that play key roles in hippocampal long-term potentiation and long-term depression, respectively (1Malenka R.C. Cell. 1994; 78: 535-538Abstract Full Text PDF PubMed Scopus (540) Google Scholar). Both of these forms of synaptic plasticity display remarkable synapse-specificity; only synapses stimulated with an appropriate activity pattern undergo long-term potentiation or long-term depression, whereas other synapses in the same postsynaptic neuron remain unaffected. These observations imply that synaptic activity stimulated signal transduction molecules and events are restricted (or targeted) specifically to synapses. While mechanisms for synaptic targeting of neuronal protein kinases are being elucidated (2Klauck T.M. Scott J.D. Cell. Signal. 1995; 7: 747-757Crossref PubMed Scopus (33) Google Scholar,3Strack S. Colbran R.J. J. Biol. Chem. 1998; 273: 20689-20692Abstract Full Text Full Text PDF PubMed Scopus (381) Google Scholar), comparatively little is known about mechanisms for synaptic targeting of protein phosphatases. The monomeric catalytic subunit of protein phosphatase 1 (PP1)1 likely does not represent a significant fraction of the total enzyme in any tissue; diverse tissue-specific PP1-binding proteins (PP1bps) are thought to confer specific regulatory properties and subcellular localization to each cellular PP1 holoenzyme (reviewed in Ref. 4Hubbard M.J. Cohen P. Trends Biochem. Sci. 1993; 18: 172-177Abstract Full Text PDF PubMed Scopus (790) Google Scholar). For example, in skeletal muscle, GM-regulatory subunits target PP1 to glycogen particles and sarcoplasmic reticulum. Phosphorylation of GM by protein kinase A dissociates PP1 and thereby inhibits the dephosphorylation of glycogen-associated substrates. Conversely, phosphorylation of GM by an insulin-sensitive protein kinase activates the associated PP1 and promotes dephosphorylation of substrate proteins associated with the glycogen particle (4Hubbard M.J. Cohen P. Trends Biochem. Sci. 1993; 18: 172-177Abstract Full Text PDF PubMed Scopus (790) Google Scholar). More than 10 proteins that bind the catalytic subunit of PP1 in non-neuronal tissues have been identified (5Shenolikar S. Annu. Rev. Cell Biol. 1994; 10: 55-86Crossref PubMed Scopus (402) Google Scholar). A conserved sequence motif V-X-F/W in PP1bps, often preceded by basic residues, makes important interactions with PP1 (6Johnson D.F. Moorhead G. Caudwell F.B. Cohen P. Chen Y.H. Chen M.X. Cohen P.T. Eur. J. Biochem. 1996; 239: 317-325Crossref PubMed Scopus (127) Google Scholar, 7Egloff M.P. Johnson D.F. Moorhead G. Cohen P.T. Cohen P. Barford D. EMBO J. 1997; 16: 1876-1887Crossref PubMed Scopus (531) Google Scholar, 8Zhao S. Lee E.Y. J. Biol. Chem. 1997; 272: 28368-28372Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar). However, there are undoubtedly additional contacts that play a role in the interaction and in modulating phosphatase activity (see “Discussion”). The four isoforms of PP1 (α, β, γ1, γ2) are expressed in neuronal tissues and exhibit distinct cellular distributions and subcellular localizations (9da Cruz e Silva E.F. Fox C.A. Ouimet C.C. Gustafson E. Watson S.J. Greengard P. J. Neurosci. 1995; 15: 3375-3389Crossref PubMed Google Scholar, 10Ouimet C.C. da Cruz e Silva E.F. Greengard P. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 3396-3400Crossref PubMed Scopus (113) Google Scholar, 11Strack S. Kini S. Ebner F. Wadzinski B.E. Colbran R.J. J. Comp. Neurol. 1999; 413: 373-384Crossref PubMed Scopus (74) Google Scholar). For example, PP1γ1 is selectively enriched at synapses in cultured rat cortical neurons, whereas PP1β is enriched in the soma (11Strack S. Kini S. Ebner F. Wadzinski B.E. Colbran R.J. J. Comp. Neurol. 1999; 413: 373-384Crossref PubMed Scopus (74) Google Scholar). Immunohistochemical staining of rat brain sections also indicates that PP1α and PP1γ1 are enriched in synaptic layers and dendritic spines (10Ouimet C.C. da Cruz e Silva E.F. Greengard P. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 3396-3400Crossref PubMed Scopus (113) Google Scholar, 11Strack S. Kini S. Ebner F. Wadzinski B.E. Colbran R.J. J. Comp. Neurol. 1999; 413: 373-384Crossref PubMed Scopus (74) Google Scholar), whereas PP1β is relatively enriched in somatic layers (11Strack S. Kini S. Ebner F. Wadzinski B.E. Colbran R.J. J. Comp. Neurol. 1999; 413: 373-384Crossref PubMed Scopus (74) Google Scholar). Although neuronal PP1 inhibitor proteins, such as DARPP-32, have been characterized in detail (reviewed in Ref. 12Greengard P. Nairn A.C. Girault J.A. Ouimet C.C. Snyder G.L. Fisone G. Allen P.B. Fienberg A. Nishi A. Brain Res. Rev. 1998; 26: 274-284Crossref PubMed Scopus (110) Google Scholar), proteins responsible for selective targeting of PP1 isoforms have not been identified. We characterized four PP1bps with molecular masses of 75, 134, 175, and 216 kDa in whole rat forebrain extracts which were enriched in isolated postsynaptic densities (13Colbran R.J. Bass M.A. McNeill R.B. Bollen M. Zhao S. Wadzinski B.E. Strack S. J. Neurochem. 1997; 69: 920-929Crossref PubMed Scopus (44) Google Scholar). Interestingly, PP1bp134 and PP1bp175 exhibited isoform selectivity, binding to the α, γ1, γ2 isoforms better than the β isoform in gel overlays (13Colbran R.J. Bass M.A. McNeill R.B. Bollen M. Zhao S. Wadzinski B.E. Strack S. J. Neurochem. 1997; 69: 920-929Crossref PubMed Scopus (44) Google Scholar). Recent screening of brain cDNA libraries using yeast two-hybrid approaches with a PP1α probe identified two novel neuronal PP1bps, spinophilin, a dendritic spine-localized protein (14Allen P.B. Ouimet C.C. Greengard P. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 9956-9961Crossref PubMed Scopus (382) Google Scholar), and PNUTS, a PP1bp found in the nucleus (15Allen P.B. Kwon Y.G. Nairn A.C. Greengard P. J. Biol. Chem. 1998; 273: 4089-4095Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar). However, there is only limited information available about native spinophilin and PNUTS proteins, and their roles in isoform-selective targeting of PP1 has not been investigated. Here we report the purification and characterization of a novel neuronal PP1 holoenzyme containing PP1γ1 associated with PP1bp134 and PP1bp175, and identify the PP1bps as spinophilin (14Allen P.B. Ouimet C.C. Greengard P. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 9956-9961Crossref PubMed Scopus (382) Google Scholar,17Satoh A. Nakanishi H. Obaishi H. Wada M. Takahashi K. Satoh K. Hirao K. Nishioka H. Hata Y. Mizoguchi A. Takai Y. J. Biol. Chem. 1998; 273: 3470-3475Abstract Full Text Full Text PDF PubMed Scopus (195) Google Scholar) 2Spinophilin was initially identified by yeast two-hybrid screening of a rat brain cDNA library, and was named based on its immunohistochemical enrichment in dendritic spines (14Allen P.B. Ouimet C.C. Greengard P. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 9956-9961Crossref PubMed Scopus (382) Google Scholar). The same protein was independently purified and cloned as an F-actin binding protein and named neurabin II (17Satoh A. Nakanishi H. Obaishi H. Wada M. Takahashi K. Satoh K. Hirao K. Nishioka H. Hata Y. Mizoguchi A. Takai Y. J. Biol. Chem. 1998; 273: 3470-3475Abstract Full Text Full Text PDF PubMed Scopus (195) Google Scholar) based on its homology to neurabin (16Nakanishi H. Obaishi H. Satoh A. Wada M. Mandai K. Satoh K. Nishioka H. Matsuura Y. Mizoguchi A. Takai Y. J. Cell Biol. 1997; 139: 951-961Crossref PubMed Scopus (162) Google Scholar). We elected to use spinophilin to name this protein in the present paper since it was the first published name. 2Spinophilin was initially identified by yeast two-hybrid screening of a rat brain cDNA library, and was named based on its immunohistochemical enrichment in dendritic spines (14Allen P.B. Ouimet C.C. Greengard P. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 9956-9961Crossref PubMed Scopus (382) Google Scholar). The same protein was independently purified and cloned as an F-actin binding protein and named neurabin II (17Satoh A. Nakanishi H. Obaishi H. Wada M. Takahashi K. Satoh K. Hirao K. Nishioka H. Hata Y. Mizoguchi A. Takai Y. J. Biol. Chem. 1998; 273: 3470-3475Abstract Full Text Full Text PDF PubMed Scopus (195) Google Scholar) based on its homology to neurabin (16Nakanishi H. Obaishi H. Satoh A. Wada M. Mandai K. Satoh K. Nishioka H. Matsuura Y. Mizoguchi A. Takai Y. J. Cell Biol. 1997; 139: 951-961Crossref PubMed Scopus (162) Google Scholar). We elected to use spinophilin to name this protein in the present paper since it was the first published name. and neurabin (16Nakanishi H. Obaishi H. Satoh A. Wada M. Mandai K. Satoh K. Nishioka H. Matsuura Y. Mizoguchi A. Takai Y. J. Cell Biol. 1997; 139: 951-961Crossref PubMed Scopus (162) Google Scholar), respectively, two homologous actin-binding proteins. We show that spinophilin and neurabin selectively interact with PP1γ1over PP1β, suggesting that they are at least in part responsible for the enrichment of PP1γ1 at synapses. Forebrains were rapidly dissected following euthanasia, quick-frozen in liquid N2, and then stored at −80 °C until needed. All subsequent procedures were performed at 4 °C. Forebrains were partially thawed in 10 ml per forebrain of Buffer A (10 mm Tris-HCl, pH 7.5, 1 mm EGTA, 1 mm EDTA, 1 mm DTT, 0.2 mm PMSF, 1 mm benzamidine, 40 mg/liter soybean trypsin inhibitor, 10 mg/liter leupeptin) plus 150 mm KCl and then homogenized using a Polytron followed by 15 passes in a motorized Teflon/glass homogenizer. After centrifugation at 35,000 × g for 25 min, the supernatant (S1) was discarded. The pellet (P1) was re-homogenized in 10 ml/forebrain of Buffer B (1 mm Tris-HCl, pH 7.5, 0.5 mm EGTA, 0.5 mm EGTA, 1 mm DTT, 0.2 mm PMSF, 1 mm benzamidine, 20 mg/liter soybean trypsin inhibitor, 5 mg/liter leupeptin) and then dialyzed for 2 h against Buffer B. Similar low ionic strength conditions have been shown previously to destabilize the F-actin cytoskeleton, solubilizing actin-binding proteins (23Levilliers N. Peron-Renner M. Coffe G. Pudles J. Biochimie (Paris). 1984; 66: 531-537Crossref PubMed Scopus (5) Google Scholar). After centrifugation at 100,000 × g for 60 min, the supernatant (S2) was saved and the pellet (P3) was re-homogenized in 10 ml/forebrain of Buffer B containing 1% Triton X-100. After mixing gently for 1 h followed by centrifugation at 100,000 × g for 60 min, the supernatant (S3) was removed and the pellet (P3) was resuspended in Buffer B. All procedures were performed at 4 °C. Combined S2/S3 extracts from 10–20 rat forebrains (see above) were applied to a Fast-Flow Q-Sepharose ion exchange column (100 ml) equilibrated in Buffer C (10 mmHEPES, pH 7.5, 150 mm NaCl, 1 mm EDTA, 1 mm EGTA, 1 mm DTT, 0.1% (v/v) Triton X-100, 0.5 mm benzamidine, 0.1 mm PMSF, 20 mg/liter soybean trypsin inhibitor, and 5 mg/liter leupeptin). The column was sequentially washed with 250 ml of Buffer C and then with 250 ml (each) of Buffer C adjusted to 0.25, 0.40, and 0.5 m NaCl, collecting 20-ml fractions. PP1bps were most abundant in the 0.4m NaCl eluate, coeluting with PP1 activity and immunoreactivity, and were precipitated from pooled Q-Sepharose fractions by slowly adding ammonium sulfate (313 g/liter). After stirring for 30 min, precipitated proteins were collected by centrifugation. The precipitate was redissolved in ×0.1 the original volume of Buffer B and 2-ml aliquots were size-fractionated on a FPLC Superdex 200 column (100 ml) equilibrated in Buffer C, collecting 2 ml fractions at 1 ml/min. PP1 activity and immunoreactivity eluted from Superdex 200 in two peaks (400–600 kDa, ≈100 kDa), and PP1bps were mostly associated with the larger PP1 species (13Colbran R.J. Bass M.A. McNeill R.B. Bollen M. Zhao S. Wadzinski B.E. Strack S. J. Neurochem. 1997; 69: 920-929Crossref PubMed Scopus (44) Google Scholar). The 400–600-kDa pools from 2 or 3 Superdex 200 columns were applied directly to a 1-ml FPLC Mono Q (0.5 ml/min) or a 8-ml Source Q (4 ml/min) ion exchange column equilibrated in Buffer C; the columns were eluted with a NaCl gradient, collecting 1- or 8-ml fractions, respectively. PP1bp134, PP1bp175, and PP1 co-eluted with a peak of PP1 activity at 0.3–0.4m NaCl (13Colbran R.J. Bass M.A. McNeill R.B. Bollen M. Zhao S. Wadzinski B.E. Strack S. J. Neurochem. 1997; 69: 920-929Crossref PubMed Scopus (44) Google Scholar). Peak fractions were pooled and mixed overnight with microcystin-Sepharose (0.5 ml) (generous gift of Dr. C. Macintosh, Dundee) equilibrated in Buffer C. After washing with 0.5 mNaCl in Buffer C (10 ml), bound proteins were eluted with 3m NaSCN, collecting 0.5-ml fractions. Fractions containing PP1bp134, PP1bp175, and PP1 were pooled, concentrated in Centricon 30 devices, and then dialyzed against 10 mm HEPES, pH 7.5, 30% (v/v) glycerol, 0.1 m NaCl, 1 mm EDTA, 1 mm DTT, 0.1% Triton X-100. Samples were analyzed for PP1bps by DIG-PP1 overlay assay, and for the presence of protein phosphatase catalytic subunits by immunoblotting, as described (13Colbran R.J. Bass M.A. McNeill R.B. Bollen M. Zhao S. Wadzinski B.E. Strack S. J. Neurochem. 1997; 69: 920-929Crossref PubMed Scopus (44) Google Scholar). Briefly, samples were resolved by SDS-PAGE, electroblotted to nitrocellulose membranes, and then probed with either 2.5–10 nm digoxigenin-conjugated PP1γ1 or with antibodies specific to the indicated proteins (see below). Blots were developed using alkaline phosphatase-conjugated secondary antibodies and colorimetric reagents. Protein phosphatase activities were measured essentially as described (18Strack S. Barban M.A. Wadzinski B.E. Colbran R.J. J. Neurochem. 1997; 68: 2119-2128Crossref PubMed Scopus (259) Google Scholar). During purification from brain extracts, PP1 activity is defined as glycogen phosphorylasea phosphatase activity measured in the presence of 2.5 nm okadaic acid. Similar definitions were used previously to define PP1 activity in brain extracts (18Strack S. Barban M.A. Wadzinski B.E. Colbran R.J. J. Neurochem. 1997; 68: 2119-2128Crossref PubMed Scopus (259) Google Scholar, 28Sim A.T. Ratcliffe E. Mumby M.C. Villa-Moruzzi E. Rostas J.A. J. Neurochem. 1994; 62: 1552-1559Crossref PubMed Scopus (47) Google Scholar). Although 2.5 nm okadaic acid is generally considered to block >90% of PP2A-like activities, certain forms of PP2A may have reduced sensitivity to okadaic acid (22Wadzinski B.E. Wheat W.H. Jaspers S. Peruski Jr., L.F. Lickteig R.L. Johnson G.L. Klemm D.J. Mol. Cell. Biol. 1993; 13: 2822-2834Crossref PubMed Scopus (283) Google Scholar). Thus, poorly defined PP2A-like species may contribute to our operationally defined PP1 activity, particularly at earlier stages of the purification. The activity of recombinant PP1γ1 (bacterially expressed; generous gift of Dr. E. Y. Lee) was determined in the absence of okadaic acid and in the presence of 0.2 mm MnCl2. About 80 μg of purified PP1A holoenzyme was solubilized in SDS and fractionated by polyacrylamide gel electrophoresis (mini-gels 0.75-mm thick, 3-cm wide lane; 7.5% acrylamide). The gel was electroblotted to polyvinylidene difluoride membrane (Immobilon P, Millipore) for 3 h at 1 A (4 °C) in 10 mm CAPS, pH 11, containing 10% (v/v) methanol. Two widely separated vertical strips (2 mm each) were excised from the lane and analyzed by DIG-PP1 overlay to identify PP1bps, and the remaining membrane was stained with Coomassie Blue. The stained band corresponding to PP1bp134 (estimated 5–10 μg of protein) was excised, washed thoroughly in water, reduced, and alkylated before being incubated with 0.2 μg endo-Lys-C (15 h at 37 °C). Solubilized proteolytic fragments of PP1bp134 were fractionated by reversed-phase high performance liquid chromatography (C18 column) using a gradient of acetonitrile in 0.05% trifluoroacetic acid buffer. Three discretely resolved peptide peaks (detected by absorbance at 214 nm) were subjected to Edman degradation using a Procise 492 protein sequencer (PE Biosystems). Double stranded DNA fragments of spinophilin and neurabin were generated by reverse transcriptase-polymerase chain reaction (Promega Access) using rat brain total RNA. Oligonucleotide primer pairs (spinophilin: 5′-CATTTCAGCACCGCACCGATC and 5′-CCAGCGCCCTTTCTCCTGCTC; neurabin: 5′-GAGGGCTCCCAGCAGAGTAGG and 5′-CACTTCCGGTACTGGCACAGC; 5′-GTCCAAGGCCTGCAAGTTCGG and 5′-GCACACTCCACTCATGGACGG) were designed based on sequences available in the data bases (spinophilin, AF016252; neurabin, U72994). The polymerase chain reaction products served as templates for the synthesis of 32P-labeled DNA probes (Stratagene Prime-It II), which were used to screen a rat brain cDNA library in λZAPII by hybridization, according to the manufacturer's instructions (Stratagene). Hybridizing cDNA clones were plaque-purified, excised, and transferred to pBluescript II SK(+), and their identity confirmed by partial sequencing. Full-length cDNAs were assembled from partial clones using restriction sites. Full-length spinophilin and neurabin cDNAs, with or without an N-terminal Myc epitope tag (MEQKLISEEDL) (19Evan G.I. Lewis G.K. Ramsay G. Bishop J.M. Mol. Cell. Biol. 1985; 5: 3610-3616Crossref PubMed Scopus (2165) Google Scholar), were subcloned into a pCMV4 eukaryotic expression vector containing the cytomegalovirus promoter (generous gift of Drs. M. Wilson and L. E. Limbird, Vanderbilt University). Automated DNA sequencing (Vanderbilt Center for Molecular Neuroscience) confirmed the complete nucleotide sequences of these constructs. HEK293 cells were transfected by calcium phosphate precipitation (13.6 μg of DNA/100-mm dish) essentially as described (20Lovinger D.M. J. Pharmacol. Exp. Ther. 1995; 274: 164-172PubMed Google Scholar). Cells were scraped into lysis buffer (50 mm Tris-HCl, pH 7.5, 150 mm NaCl, 1 mm EDTA, 1 mm DTT, 0.5% Triton X-100, 0.1 mm PMSF, 1 mm benzamidine, 20 mg/liter soybean trypsin inhibitor, and 5 mg/liter leupeptin; 1.25 ml/100-mm dish), probe-sonicated (2 × 20-s bursts), and then centrifuged (100,000 × g, 30 min) to generate soluble cell extracts. Extracts were either used immediately, or stored at −80 °C until required. Regions of cDNAs encoding the indicated residues of spinophilin and neurabin were either isolated by polymerase chain reaction using primers incorporating appropriate restriction sites, or obtained by digestion at naturally occurring restriction sites, and then inserted into pGEX-2T or -4T prokaryotic expression vectors (Amersham Pharmacia Biotech). Bacteria (DH5α) harboring the expression vector were induced with 0.5 mmisopropyl-1-thio-β-d-galactopyranoside for 3–5 h and GST fusion proteins were isolated from soluble extracts using glutathione-agarose essentially according to the directions of the manufacturer. A GST-GM fusion protein containing residues 1–240 of the glycogen targeting subunit of PP1 (21Wu J. Liu J. Thompson I. Oliver C.J. Shenolikar S. Brautigan D.L. FEBS Lett. 1998; 439: 185-191Crossref PubMed Scopus (41) Google Scholar) was generously provided by R. Terry and Dr. S. Shenolikar (Duke University). Protein concentrations were determined using the Bio-Rad protein assay with bovine serum albumin as standard; in PP1 inhibition assays, concentrations refer to estimates of the full-length fusion protein by Coomassie Blue staining following SDS-PAGE. Rabbit antibodies raised against divergent sequences from the C termini of PP1 isoforms were described previously (11Strack S. Kini S. Ebner F. Wadzinski B.E. Colbran R.J. J. Comp. Neurol. 1999; 413: 373-384Crossref PubMed Scopus (74) Google Scholar, 13Colbran R.J. Bass M.A. McNeill R.B. Bollen M. Zhao S. Wadzinski B.E. Strack S. J. Neurochem. 1997; 69: 920-929Crossref PubMed Scopus (44) Google Scholar). Similar antibodies were raised against the identical peptides in sheep. The rabbit and sheep antibodies exhibited similar isoform-specificity in immunoblotting experiments using either whole antiserum or affinity purified antibodies: antibodies raised to PP1β and PP1γ1 sequences were specific for the corresponding isoform, whereas the antibodies raised to a PP1α sequence recognized all PP1 isoforms and are designated pan-PP1 antibody (11Strack S. Kini S. Ebner F. Wadzinski B.E. Colbran R.J. J. Comp. Neurol. 1999; 413: 373-384Crossref PubMed Scopus (74) Google Scholar) (data not shown). PP2A catalytic subunit antibodies were either from Transduction Laboratories (mouse monoclonal) or were raised against a PP2AC peptide sequence (PNVTRRTPDYFL) in sheep. Sheep PP2AC antibodies exhibited similar specificity to previously described rabbit antibodies against the same peptide (22Wadzinski B.E. Wheat W.H. Jaspers S. Peruski Jr., L.F. Lickteig R.L. Johnson G.L. Klemm D.J. Mol. Cell. Biol. 1993; 13: 2822-2834Crossref PubMed Scopus (283) Google Scholar) (data not shown). Antibodies to the Myc epitope tag (purified by ammonium sulfate precipitation from mouse ascites fluid) were a generous gift from Drs. J. Flick and R. Wisdom (Vanderbilt University). Rabbit antisera were raised to GST fusion proteins containing divergent regions of either spinophilin (residues 286–390) or neurabin (residues 146–453). Rabbits were given an intranodal injection of 0.2 mg of fusion protein in Freund's complete adjuvant, followed after 21 days by a 0.2-mg intranodal boost in Freund's incomplete adjuvant. Animals were first boosted with 0.1 mg of fusion protein in incomplete adjuvant at subcutaneous and intramuscular sites 14 days after the initial injection, and subsequently at 20-day intervals. All the studies in this manuscript utilize whole antiserum from the first or second bleed, performed 10 days after booster injections. Rabbit injections and bleedings were performed by Upstate Biotechnology. Native brain protein phosphatase catalytic subunits were separated from their associated subunits and proteins as follows. Fresh rat forebrain S1 extract (180 ml) (see above) was quick-frozen and stored at −80 °C until used. The extract was slowly thawed and then 313 g/liter ammonium sulfate were slowly added and the extract was stirred at 4 °C for 30 min. Precipitated proteins were collected by centrifugation for 30 min at 28,000 × g, and then resuspended in 20 ml of Buffer X (20 mm Tris-HCl, pH 7.5, 1 mm DTT, 0.1 mm EGTA, 2 mmMgCl2, 0.5 mm benzamidine, 0.1 mmPMSF, 20 mg/liter soybean trypsin inhibitor, 5 mg/liter leupeptin) containing 10% glycerol using a Polytron homogenizer. Room temperature ethanol (100 ml; 95%) containing 1 mm PMSF was added and the suspension was then immediately centrifuged for 5 min at 28,000 × g. The pellet was extracted with 2 × 20-ml aliquots of Buffer X by homogenization using a Polytron. Ammonium sulfate (430 g/liter) was added to the pooled solubilized proteins, and precipitated proteins were collected by centrifugation (35,000 ×g for 20 min). The pellet was redissolved in 10 ml of Buffer X and then dialyzed overnight against Buffer X. After dialysis, the preparation was clarified at 35,000 × g for 20 min and then stored at −80 °C in aliquots. To estimate concentrations of PP1β and PP1γ1 in this preparation, aliquots were immunoblotted in parallel with known amounts of recombinant PP1 isoforms using isoform-specific antibodies. In two independent experiments, the concentrations were estimated at 1–1.5 μm and 0.5–0.75 μm for PP1β and PP1γ1, respectively (total protein concentration 2.96 mg/ml). Approximately 8 μg of the indicated GST fusion proteins were mixed for 1 h at 4 °C with 50 μl of crude protein phosphatase catalytic subunit mixture (see above) in 14 ml of binding buffer (50 mmTris-HCl, pH 7.5, 0.2 m NaCl, 0.1% Triton X-100, 0.25 mg/ml bovine serum albumin). The final concentration of GST fusion protein was about 17 nm; PP1β and PP1γ1were present at 2–5 nm each, together with an unknown concentration of PP1α. About 20 μl of a 50:50 slurry of glutathione-agarose was added and the incubation was continued overnight at 4 °C. The resin was sedimented, washed with 4 × 5-ml aliquots of binding buffer, and then transferred to a fresh microcentrifuge tube. To concentrate protein phosphatases that did not associate with GST fusion proteins, supernatants from glutathione-agarose sedimentation were incubated for 2 h at 4 °C with 20 μl of a 50:50 slurry of microcystin-agarose (Upstate Biotechnology Inc.), and the resin was then sedimented. Proteins associated with glutathione-agarose and with microcystin-agarose were solubilized by boiling in SDS-PAGE sample buffer and aliquots were analyzed by immunoblotting. Combined S2/S3 rat forebrain extracts or HEK293 cell soluble extracts (see above) were diluted to 1 mg/ml protein in IP Buffer (50 mm Tris-HCl, pH 7.5, 0.15m NaCl, 0.5% Triton X-100) and 0.5-ml aliquots were precleared using 20 μl of a 1:1 slurry of protein A-Sepharose or GammaBind Plus Sepharose (Amersham Pharmacia Biotech). The supernatant was mixed with 5 μl of the indicated rabbit or sheep antiserum (or preimmune serum) or mouse ascites fluid for 1 h at 4 °C. After addition of 20 μl of protein A-Sepharose (rabbit or mouse antibodies) or GammaBind Plus Sepharose (sheep antibodies), respectively, incubations were continued overnight at 4 °C. Resin was collected by microcentrifugation, and then washed with at least 4 × 1 ml of IP Buffer; during the last wash resin was transferred to a new microcentrifuge tube. Immune complexes were solubilized in SDS-PAGE sample buffer. Aliquots of immune complexes and immune supernatants, as well as an aliquot of the initial extract, were analyzed by immunoblotting. In order to identify previously characterized PP1bps (13Colbran R.J. Bass M.A. McNeill R.B. Bollen M. Zhao S. Wadzinski B.E. Strack S. J. Neurochem. 1997; 69: 920-929Crossref PubMed Scopus (44) Google Scholar), we undertook their purification from rat forebrain extracts. This paper focuses on PP1bp134 and PP1bp175, which were partially solubilized by re-homogenization of an" @default.
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• W2023265997 title "Brain Actin-associated Protein Phosphatase 1 Holoenzymes Containing Spinophilin, Neurabin, and Selected Catalytic Subunit Isoforms" @default.
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• W2023265997 doi "https://doi.org/10.1074/jbc.274.50.35845" @default.
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