The Neuronal Actin-binding Proteins, Neurabin I and Neurabin II, Recruit Specific Isoforms of Protein Phosphatase-1 Catalytic Subunits
2002; Elsevier BV; Volume: 277; Issue: 31 Linguagem: Inglês
10.1074/jbc.m203365200
ISSN1083-351X
AutoresRyan T. Terry-Lorenzo, Leigh Carmody, James W. Voltz, John H. Connor, Li Shi, F. Donelson Smith, Sharon L. Milgram, Roger Colbran, Shirish Shenolikar,
Tópico(s)Cellular transport and secretion
ResumoNeurabins are protein phosphatase-1 (PP1) targeting subunits that are highly concentrated in dendritic spines and post-synaptic densities. Immunoprecipitation of neurabin I and neurabin II/spinophilin from rat brain extracts sedimented PP1γ1 and PP1α but not PP1β. In vitro studies showed that recombinant peptides representing central regions of neurabins also preferentially bound PP1γ1 and PP1α from brain extracts and associated poorly with PP1β. Analysis of PP1 binding to chimeric neurabins suggested that sequences flanking a conserved PP1-binding motif altered their selectivity for PP1β and their activity as regulators of PP1 in vitro. Assays using recombinant PP1 catalytic subunits and a chimera of PP1 and protein phosphatase-2A indicated that the C-terminal sequences unique to the PP1 isoforms contributed to their recognition by neurabins. Collectively, the results from several different in vitro assays established the rank order of PP1 isoform selection by neurabins to be PP1γ1 > PP1α > PP1β. This PP1 isoform selectivity was confirmed by immunoprecipitation of neurabin I and II from brain extracts from wild type and mutant PP1γ null mice. In the absence of PP1γ1, both neurabins showed enhanced association with PP1α but not PP1β. These studies identified some of the structural determinants in PP1 and neurabins that together contribute to preferential targeting of PP1γ1 and PP1α to the mammalian synapse. Neurabins are protein phosphatase-1 (PP1) targeting subunits that are highly concentrated in dendritic spines and post-synaptic densities. Immunoprecipitation of neurabin I and neurabin II/spinophilin from rat brain extracts sedimented PP1γ1 and PP1α but not PP1β. In vitro studies showed that recombinant peptides representing central regions of neurabins also preferentially bound PP1γ1 and PP1α from brain extracts and associated poorly with PP1β. Analysis of PP1 binding to chimeric neurabins suggested that sequences flanking a conserved PP1-binding motif altered their selectivity for PP1β and their activity as regulators of PP1 in vitro. Assays using recombinant PP1 catalytic subunits and a chimera of PP1 and protein phosphatase-2A indicated that the C-terminal sequences unique to the PP1 isoforms contributed to their recognition by neurabins. Collectively, the results from several different in vitro assays established the rank order of PP1 isoform selection by neurabins to be PP1γ1 > PP1α > PP1β. This PP1 isoform selectivity was confirmed by immunoprecipitation of neurabin I and II from brain extracts from wild type and mutant PP1γ null mice. In the absence of PP1γ1, both neurabins showed enhanced association with PP1α but not PP1β. These studies identified some of the structural determinants in PP1 and neurabins that together contribute to preferential targeting of PP1γ1 and PP1α to the mammalian synapse. protein phosphatase-1 protein phosphatase-2A glutathioneS-transferase post-synaptic density protein kinase-A wild type neurabin N-methyl-d-aspartic acid Protein phosphatase-1 (PP1),1 a major eukaryotic protein serine/threonine phosphatase, is encoded by multiple genes in both plants and animals. Disruption of PP1 genes in fungi (1Ohkura H. Kinoshita N. Miyatani S. Toda T. Yanagida M. Cell. 1989; 57: 997-1007Abstract Full Text PDF PubMed Scopus (351) Google Scholar), fruit flies (2Axton J.M. Dombradi V. Cohen P.T. Glover D.M. Cell. 1990; 63: 33-46Abstract Full Text PDF PubMed Scopus (230) Google Scholar), and mice (3Varmuza S. Jurisicova A. Okano K. Hudson J. Boekelheide K. Shipp E.B. Dev. Biol. 1999; 205: 98-110Crossref PubMed Scopus (135) Google Scholar) suggested that PP1 isoforms encoded by individual genes control distinct but overlapping physiological functions. Three mammalian isoforms, PP1α, PP1β, and PP1γ1, are expressed in all tissues (4Shima H. Hatano Y. Chun Y.S. Sugimura T. Zhang Z. Lee E.Y. Nagao M. Biochem. Biophys. Res. Commun. 1993; 192: 1289-1296Crossref PubMed Scopus (106) Google Scholar) with PP1γ2, an alternately spliced product of the PP1γ gene, present predominantly in testes (5Shima H. Haneji T. Hatano Y. Kasugai I. Sugimura T. Nagao M. Biochem. Biophys. Res. Commun. 1993; 194: 930-937Crossref PubMed Scopus (63) Google Scholar, 6Strack S. Kini S. Ebner F.F. Wadzinski B.E. Colbran R.J. J. Comp. Neurol. 1999; 413: 373-384Crossref PubMed Scopus (77) Google Scholar). Immunocytochemistry using isoform-specific antibodies suggested that expression of PP1 isoforms varied in different brain regions where they are also localized to different subcellular compartments (6Strack S. Kini S. Ebner F.F. Wadzinski B.E. Colbran R.J. J. Comp. Neurol. 1999; 413: 373-384Crossref PubMed Scopus (77) Google Scholar, 7da 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). For example, PP1β was the predominant isoform associated with microtubules in the neuronal cell body, whereas PP1γ1 and PP1α were preferentially concentrated in dendritic spines (6Strack S. Kini S. Ebner F.F. Wadzinski B.E. Colbran R.J. J. Comp. Neurol. 1999; 413: 373-384Crossref PubMed Scopus (77) Google Scholar, 8Ouimet C.C. da Cruz e Silva E.F. Greengard P. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 3396-3400Crossref PubMed Scopus (115) Google Scholar). Furthermore, by analyzing endogenous PP1 movement during the cell cycle, Andreassenet al. (9Andreassen P.R. Lacroix F.B. Villa-Moruizz E. Margolis R.L. J. Cell Biol. 1998; 141: 1207-1215Crossref PubMed Scopus (173) Google Scholar) showed that the distribution of PP1 isoforms in cells was highly dynamic. This placed new emphasis on understanding the mechanisms that target individual PP1 isoforms to cellular organelles. Isolation of PP1 bound to skeletal muscle glycogen (10Hubbard M.J. Cohen P. Trends Biochem. Sci. 1993; 18: 172-177Abstract Full Text PDF PubMed Scopus (792) Google Scholar) and myosin (11Dent P. MacDougall L.K. MacKintosh C. Campbell D.G. Cohen P. Eur. J. Biochem. 1992; 210: 1037-1044Crossref PubMed Scopus (49) Google Scholar) established the paradigm that regulatory or targeting subunits bound to PP1 catalytic subunits dictate the subcellular localization, substrate recognition, and hormonal control of PP1. The search for PP1 regulators that control functions as diverse as protein synthesis, gene expression, cell division, and motility has thus far yielded more than 50 PP1-binding proteins (12Cohen P.T. J. Cell Sci. 2002; 115: 241-256Crossref PubMed Google Scholar). Whereas myosin phosphatases from skeletal (11Dent P. MacDougall L.K. MacKintosh C. Campbell D.G. Cohen P. Eur. J. Biochem. 1992; 210: 1037-1044Crossref PubMed Scopus (49) Google Scholar) and cardiac muscle (13Chu Y. Wilson S.E. Schlender K.K. Biochim. Biophys. Acta. 1994; 1208: 45-54Crossref PubMed Scopus (23) Google Scholar) copurified with both PP1α and β (14Moorhead G. MacKintosh C. Morrice N. Cohen P. FEBS Lett. 1995; 362: 101-105Crossref PubMed Scopus (82) Google Scholar), the smooth muscle myosin phosphatase bound exclusively PP1β (15Okubo S. Ito M. Takashiba Y. Ichikawa K. Miyahara M. Shimizu H. Konishi T. Shima H. Nagao M. Hartshorne D.J. Nakano T. Biochem. Biophys. Res. Commun. 1994; 200: 429-434Crossref PubMed Scopus (55) Google Scholar, 16Alessi D. MacDougall L.K. Sola M.M. Ikebe M. Cohen P. Eur. J. Biochem. 1992; 210: 1023-1035Crossref PubMed Scopus (331) Google Scholar, 17Moorhead G. Johnson D. Morrice N. Cohen P. FEBS Lett. 1998; 438: 141-144Crossref PubMed Scopus (48) Google Scholar). The molecular basis by which the myosin-binding subunits and other regulators selected specific PP1 isoforms remains unknown. PP1 plays a key role in regulating synaptic transmission in mammalian neurons (18Winder D.G. Sweatt J.D. Nat. Rev. Neurosci. 2001; 2: 461-474Crossref PubMed Scopus (283) Google Scholar). PP1γ1 and PP1α, but not PP1β, were enriched in dendritic spines (6Strack S. Kini S. Ebner F.F. Wadzinski B.E. Colbran R.J. J. Comp. Neurol. 1999; 413: 373-384Crossref PubMed Scopus (77) Google Scholar, 8Ouimet C.C. da Cruz e Silva E.F. Greengard P. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 3396-3400Crossref PubMed Scopus (115) Google Scholar), where they associated with the actin-rich structure known as the post-synaptic density (PSD) (19Terry-Lorenzo R.T. Inoue M. Connor J.H. Haystead T.A. Armbruster B.N. Gupta R.P. Oliver C.J. Shenolikar S. J. Biol. Chem. 2000; 275: 2439-2446Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). Recent studies (20Muly E.C. Greengard P. Goldman-Rakic P.S. J. Comp. Neurol. 2001; 440: 261-270Crossref PubMed Scopus (24) Google Scholar) also suggested a heterogeneity of spines that contained either PP1α alone or both PP1α and PP1γ1. The neuronal actin-binding proteins, neurabin I and neurabin II/spinophilin, were also localized in PSD and bind PP1 (19Terry-Lorenzo R.T. Inoue M. Connor J.H. Haystead T.A. Armbruster B.N. Gupta R.P. Oliver C.J. Shenolikar S. J. Biol. Chem. 2000; 275: 2439-2446Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar, 21McAvoy T. Allen P.B. Obaishi H. Nakanishi H. Takai Y. Greengard P. Nairn A.C. Hemmings H.C., Jr. Biochemistry. 1999; 38: 12943-12949Crossref PubMed Scopus (74) Google Scholar, 22Allen P.B. Ouimet C.C. Greengard P. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 9956-9961Crossref PubMed Scopus (393) Google Scholar). Thus, neurabins were excellent candidates for recruiting specific PP1 isoforms to the synapse. Prior studies showed that anti-neurabin immune complexes from rat brain contained PP1γ1 but excluded PP1β (23MacMillan L.B. Bass M.A. Cheng N. Howard E.F. Tamura M. Strack S. Wadzinski B.E. Colbran R.J. J. Biol. Chem. 1999; 274: 35845-35854Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar). Far Westerns of brain extracts using recombinant PP1 catalytic subunits as probes suggested that neurabins displayed a significant preference for PP1γ1 and PP1α over PP1β (24Colbran 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 (46) Google Scholar). Our studies highlighted a domain in both neurabins that displayed PP1 isoform selectivity similar to the native proteins. Subsequent analyses of chimeric neurabins focused attention on sequences in neurabin I that regulated PP1 activity and determined PP1β binding. Mutation of the PP1 catalytic subunit also suggested that its C terminus participated in neurabin binding. Finally, neurabin complexes from tissues of a PP1γ null mouse confirmed their preference for PP1γ1 and PP1α over PP1β. These studies provided the first experimental evidence for structural contributions from both PP1 and its regulator, neurabin, in selective targeting of PP1γ1 and PP1α to the neuronal actin cytoskeleton. A monoclonal antibody against neurabin I (dilutions used for Western blotting are in parentheses) (1:250) and a pan anti-PP1 antibody (1:2000) were obtained from Transduction Laboratories. The rabbit polyclonal antibody against neurabin II/spinophilin (1:500) was described previously (65Oliver C.J. Terry-Lorenzo R.T. Elliott E. Bloomer W.A.C. Barutigan D.L. Colbran R.J. Shenolikar S. Mol. Cell Biol. 2002; 22: 4690-4701Crossref PubMed Scopus (104) Google Scholar). Rabbit and sheep polyclonal antibodies against PP1β (1:1000) and PP1γ1 (1:1000) were provided by Brian Wadzinski, Vanderbilt University (25Strack S. Barban M.A. Wadzinski B.E. Colbran R.J. J. Neurochem. 1997; 68: 2119-2128Crossref PubMed Scopus (264) Google Scholar). Two PP1α-specific antibodies (1:1000) generated in rabbits were provided by Emma Villa-Morruzzi, University of Pisa, Italy (26Villa-Moruizz E. Puntoni F. Marin O. Int. J. Biochem. Cell Biol. 1996; 28: 13-22Crossref PubMed Scopus (27) Google Scholar), and Angus C. Nairn, Rockefeller University (8Ouimet C.C. da Cruz e Silva E.F. Greengard P. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 3396-3400Crossref PubMed Scopus (115) Google Scholar). The plasmid, pGEX-5X-2 (Amersham Biosciences), containing a cDNA encoding amino acids 354–494 of rat neurabin II (NrbII) fused to glutathioneS-transferase (GST) was described previously (27Smith F.D. Oxford G.S. Milgram S.L. J. Biol. Chem. 1999; 274: 19894-19900Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar). The cDNA encoding amino acids 374–516 of rat neurabin I (NrbI) were PCR-cloned with the following DNA oligonucleotide primers: 5′-ACCTGGATCCACGCGTCTGCTTC-3′ (NrbI primer 1) and 5′-AGAATTCTTATATGCCAAGCCCATCCTC-3′ (NrbI primer 2) by using full-length neurabin I cDNA as template and subcloned intoBamHI and EcoRI sites of pGEX-5X-2. A similar approach was also used to clone this cDNA into the NcoI and HindIII sites of pRSET-B (Invitrogen) that expressed hexahistidine-tagged neurabin. To generate NrbI/NrbII chimeras, two different strategies were used. The cDNAs encoding GST-NrbCH3, NrbI-(374–443), were PCR-amplified using NrbI primer 1 and 5′-CAACCCGGGAATTTCCGAG-3′. This introduced 5′-BamHI and 3′-SmaI restriction sites. NrbII-(435–494) was generated from an NrbII cDNA using 5′-GATTCCCGGGCTTTCAGAGGAAG-3′ and 5′-GTGTCAGAGGTTTTCACCGTC-3′, a primer derived from the pGEX vector. This introduced 5′-SmaI and 3′-NotI sites in NrbII fragment. Following digestion with appropriate restriction enzymes, the two PCR products were ligated together into the BamHI and NotI sites in pGEX-5X-2. Other chimeras were generated using the following strategy. For GST-NrbCH4, the NrbII fragment was PCR-amplified using 5′-CGTGGGATCCAGGCGTCGTCAGTG-3′ (NrbII primer 1) and 5′-CAAGCCAACAATCTCCACACACCCTG-3′ that yielded a 5′-BamHI site and a 3′-NrbI overhang. The NrbI fragment was PCR-amplified using 5′-GTGTGGAGATTGTTGGCTTGCCGCAAG-3′ and the NrbI primer 2 to yield a cDNA with 5′-NrbII overhang and 3′-EcoRI site. The two PCR products were combined and used in a second PCR with NrbII primer 1 and NrbI primer 2. Following digestion with BamHI andEcoRI, the chimeric PCR product was subcloned into pGEX-5X-2. Other primers used to generate the chimeras shown in Fig.4 A are as follows. At site 1, 5′-GGTGCTGAAATGGATCTTCCTATTTGCTGGGATTTCTTCCTC-3′ and 5′-CCCAGCAAATAGGAAGATCCATTTCAGCACCGC-3′ produced NrbCH1; 5′-CAACTAAACTTAATTTTCCGGCTCGGGGCTG-3′ and 5′-CCCGAGCCGGAAAATTAAGTTTAGTTGTGCTCCGATTAAGG-3′ were used for NrbCH2. At site 3, 5′-CATCTTCTTCATCGTCCTCTGTGCCCGTCTCTC-3′ and 5′-GAGAGACGGGCACAGAGGACGATGAAGAAGATG-3′ produced NrbCH5; 5′-TCCCCCTCATCCTGCTCTTCCAGGGCACTG-3′ and 5′-GCCCTGGAAGAGCAGGATGAGGGGGACGACAG-3′ yielded NrbCH6. All cDNAs were verified by direct DNA sequencing. pRSET-B encoding hexahistidine-NrbI-(374–516) was transformed into BL21(DE3)pLysSEscherichia coli (Stratagene). Bacteria were grown in LB media to an A 600 = 0.6, and protein expression was induced by addition of 1 mmisopropyl-1-thio-β-d-galactopyranoside to the media and further incubation for 2 h at 30 °C. Following the lysis of bacterial cells using sonication, hexahistidine fusion protein was purified on Ni+-nitrilotriacetic acid-agarose according to the manufacturer's instructions (Qiagen). The pGEX-5X-2 plasmids were transformed into BL21-Codon Plus-RIL (Stratagene), and the bacteria were grown in LB media toA 600 = 0.6. Protein expression was induced by the addition of 0.1 mmisopropyl-1-thio-β-d-galactopyranoside to the media and continued growth for 6 h at 24 °C. Cells were lysed by two passages through a French press (at 1000 pounds/square inch), and GST fusion proteins were affinity-purified using glutathione-Sepharose according to the manufacturer's instructions (AmershamBiosciences). Rat brain was obtained from Pel-Freeze, and brain and testes from +/+ and PP1γ −/− mice were provided by Susan Varmuza, University of Toronto (3Varmuza S. Jurisicova A. Okano K. Hudson J. Boekelheide K. Shipp E.B. Dev. Biol. 1999; 205: 98-110Crossref PubMed Scopus (135) Google Scholar). The tissue was homogenized in 50 mm Tris-HCl, pH 7.5, containing 5 mm EDTA, 5 mm EGTA, 10 mm NaCl, 1% w/v deoxycholate, 1 mm phenylmethylsulfonyl fluoride, 1 mm benzamidine, and 1 μg/ml each of aprotinin, leupeptin, and pepstatin using a Dounce homogenizer. Following centrifugation at 100,000 × g for 60 min, the supernatant was dialyzed against 100 volumes of 50 mm Tris-HCl, pH 7.5, 1 mm EDTA, 1 mm EGTA, 10 mm NaCl, 0.1 mm phenylmethylsulfonyl fluoride, and 0.1 mmbenzamidine for 3 h with one change of buffer. The lysates were used either immediately or frozen in liquid N2 and stored at −80 °C. For immunoprecipitation, anti-NrbI or NrbII antibody (10 μl) was mixed with rat brain (2.5 mg of total protein) or mouse brain lysate (4 mg of total protein) for 1 h at 4 °C. A 1:1 slurry of protein A-agarose (Bio-Rad) and protein G-Sepharose 4B (Sigma) (25-μl total bed volume) was added, and the mixture was incubated for 1 h. The beads were washed 4 times with NETN-250 (250 mm NaCl, 1 mm EDTA, 10 mm Tris-HCl, pH 7.5, and 0.5% v/v Nonidet P-40), and the bound proteins were eluted using 25 μl of SDS sample buffer prior to SDS-PAGE. For sedimentation or pulldown assays, glutathione-Sepharose (25-μl bed volume) was equilibrated with Tris-buffered saline. GST-neurabin proteins were added, and the volume was adjusted to 300 μl with Tris-buffered saline prior to incubation for 1 h at 4 °C. The beads were washed 2 times with Tris-buffered saline, and rat brain lysate (5 mg total protein) was added and the mixture incubated for a further 1 h at 4 °C. The beads were washed 4 times with NETN-250, and the bound proteins were eluted using 25 μl of SDS sample buffer prior to SDS-PAGE on 12% (w/v) acrylamide gels. Coomassie Blue staining verified the loading of GST-neurabins on beads, and the specific PP1 isoform sedimented was quantified by immunoblotting and densitometry using Densitometer SI and ImageQuant software (Amersham Biosciences). Affinity-purified sheep IgGs specific for PP1β or PP1γ1 (23MacMillan L.B. Bass M.A. Cheng N. Howard E.F. Tamura M. Strack S. Wadzinski B.E. Colbran R.J. J. Biol. Chem. 1999; 274: 35845-35854Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar) were covalently coupled to Affi-Gel 15 resin (≈1 mg of IgG per ml) as described by the manufacturer (Bio-Rad). The anti-β and anti-γ1 resins (0.1 ml packed beads) were incubated at 4 °C for 4 h with 0.25 ml of a rat brain protein phosphatase catalytic subunit preparation containing a mixture of PP1 and PP2A catalytic subunits in 2.5 ml of 50 mm Tris-HCl, pH 7.5, 0.2 m NaCl, 0.1% Triton X-100, 1 mmdithiothreitol, 2 mm MnCl2 (Buffer A), and 0.25 mg/ml bovine serum albumin. The resin was washed 4 times with 5 ml of Buffer A, and bound PP1s were eluted sequentially with 3 mmagnesium chloride (3× 0.3-ml aliquots) and then 3 msodium isothiocyanate (3× 0.3-ml aliquots), both in Buffer A. The eluted samples were dialyzed separately against 20 mmTris-HCl, pH 7.5, 1 mm dithiothreitol, 0.1 mmEGTA, 2 mm MnCl2, 10% glycerol before storage at −80 °C. By using affinity-purified rabbit antibodies to specific PP1 isoforms (6Strack S. Kini S. Ebner F.F. Wadzinski B.E. Colbran R.J. J. Comp. Neurol. 1999; 413: 373-384Crossref PubMed Scopus (77) Google Scholar) and recombinant PP1 isoforms as standards, the purity of the immunopurified rat brain PP1 isoforms was verified, and protein concentration was estimated (0.5–2.5 μg/ml). PP1 catalytic subunit was purified from rabbit skeletal muscle according to DeGuzman and Lee (28DeGuzman A. Lee E.Y. Methods Enzymol. 1988; 159: 356-368Crossref PubMed Scopus (62) Google Scholar), and purified PP2A catalytic subunit was provided by Brian Wadzinski, Vanderbilt University. Human PP1α in pK233 vector was a gift from Richard Honkanen, University of South Alabama College of Medicine (29Walsh A.H. Cheng A. Honkanen R.E. FEBS Lett. 1997; 416: 230-234Crossref PubMed Scopus (198) Google Scholar). PP1β in pET30 vector was provided by Masumi Eto and David Brautigan, University of Virginia, Charlottesville. PP1γ1 in pTACTAC was a gift from Ernest Y. C. Lee, New York Medical College (30Zhang A.J. Bai G. Deans-Zirattu S. Browner M.F. Lee E.Y. J. Biol. Chem. 1992; 267: 1484-1490Abstract Full Text PDF PubMed Google Scholar). CRHM2, a chimera of residues 1–273 of human PP1α fused to amino acids, 267–309, from the bovine PP2ACα catalytic subunit, has been described previously (29Walsh A.H. Cheng A. Honkanen R.E. FEBS Lett. 1997; 416: 230-234Crossref PubMed Scopus (198) Google Scholar). All recombinant phosphatases were expressed in E. coliBL21(DE3) (Stratagene) and purified as described previously (31Connor J.H. Kleeman T. Barik S. Honkanen R.E. Shenolikar S. J. Biol. Chem. 1999; 274: 22366-22372Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). Fractions containing phosphorylase phosphatase activity were pooled, dialyzed overnight against 50 mm Tris-HCl, pH 7.5, 1 mm MnCl2, 0.1% β-mercaptoethanol, and 50% glycerol, and stored at −20 °C. Phosphorylase bobtained from Calzyme Laboratories, Inc., was phosphorylated using phosphorylase kinase (Invitrogen) and [γ-32P]ATP (specific activity 3000 Ci/mmol, PerkinElmer Life Sciences) and used as substrate in a protein phosphatase activity as described in Shenolikar and Ingebritsen (32Shenolikar S. Ingebritsen T.S. Methods Enzymol. 1984; 107: 102-129Crossref PubMed Scopus (75) Google Scholar). GST-GluR1 C-terminal tail (provided by Michael Ehlers, Duke University (33Roche K.W. O'Brien R.J. Mammen A.L. Bernhardt J. Huganir R.L. Neuron. 1996; 16: 1179-1188Abstract Full Text Full Text PDF PubMed Scopus (666) Google Scholar)) was phosphorylated by purified PKA (bovine heart) in the presence of [γ-32P]ATP, and the unincorporated 32P was removed by dialysis. Assays with PP1 and PP2A were performed for 10 min at 37 °C in 50 mmTris-HCl, pH 7.5, 1 mg/ml bovine serum albumin, 1 mm EDTA, and 0.1% β-mercaptoethanol using 20 μm32P-labeled phosphorylase a or 0.2 μm GluR1 as substrates and restricted to a maximum of 20% release of [32P]phosphate to ensure linearity. Three major PP1 isoforms, PP1α, PP1β, and PP1γ1, are expressed in the mammalian brain. Purification of neurabin I (NrbI) and neurabin II/spinophilin (NrbII) from rat brain showed preferential association with PP1γ1 and not PP1β (23MacMillan L.B. Bass M.A. Cheng N. Howard E.F. Tamura M. Strack S. Wadzinski B.E. Colbran R.J. J. Biol. Chem. 1999; 274: 35845-35854Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar). Neurabin association with PP1α was not analyzed. To analyze neurabin-PP1 complexes from rat brain, we immunoprecipitated NrbI and NrbII from brain lysates by using antibodies specific for each neurabin isoform (Fig.1). Immunoprecipitates with either antibody contained both NrbI and NrbII, consistent with the heterodimerization of these proteins (23MacMillan L.B. Bass M.A. Cheng N. Howard E.F. Tamura M. Strack S. Wadzinski B.E. Colbran R.J. J. Biol. Chem. 1999; 274: 35845-35854Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar, 65Oliver C.J. Terry-Lorenzo R.T. Elliott E. Bloomer W.A.C. Barutigan D.L. Colbran R.J. Shenolikar S. Mol. Cell Biol. 2002; 22: 4690-4701Crossref PubMed Scopus (104) Google Scholar). Immunoblotting with PP1 isoform-specific antibodies showed that the anti-NrbI and NrbII immunoprecipitates contained PP1γ1 and PP1α but not PP1β. This suggested that PP1β was selectively excluded from neuronal complexes containing neurabins. To investigate PP1 recognition by the individual neurabins, we expressed recombinant neurabin polypeptides in E. coli as proteins fused to GST; these constructs lacked the actin-binding and the C-terminal dimerization domains. The GST fusion proteins affinity-purified on glutathione-Sepharose were utilized in sedimentation assays or pulldowns from rat brain lysates (Fig.2 A). Immunoblotting showed that GST-NrbI-(374–516), GST-NrbI-(436–479), GST-NrbII-(354–494), and GST-NrbII-(427–470) did not associate with endogenous brain neurabins (data not shown). In contrast, all four peptides bound PP1. The larger polypeptides, GST-NrbI-(374–516) and GST-NrbII-(354–494), bound both PP1α and PP1γ1, whereas only the NrbI fusion protein bound significant amounts of PP1β. Equivalent amounts of the smaller GST-NrbI-(436–479) and GST-NrbII-(427–470) polypeptides showed a reduced binding to PP1α and PP1γ1 and failed to bind any detectable PP1β. We utilized laser densitometry to quantify PP1 binding by recombinant neurabins in several independent experiments (Fig.2 B). The data are presented as percentage of each PP1 isoform present in the input lysate (5 mg of total protein) that was sedimented by a fixed amount of GST fusion (20 μg). This emphasized the greater efficiency of GST-NrbI-(374–516) and GST-NrbII-(354–494) to sediment PP1γ1 (15% of total) and PP1α (∼9% of total). By comparison, GST-NrbI-(374–516) bound 3% of total PP1β in the input lysate, and GST-NrbII-(354–494) bound only 0.6% of PP1β. These data also showed that the smaller neurabin peptides were 5–10-fold weaker in binding PP1γ1 and PPα and demonstrated no PP1β binding. This suggested that the central PP1-binding domain in NrbI and NrbII contained some of the structural determinants that dictated the preference of native neurabins for PP1γ1 and PP1α over PPβ. Previous studies (21McAvoy T. Allen P.B. Obaishi H. Nakanishi H. Takai Y. Greengard P. Nairn A.C. Hemmings H.C., Jr. Biochemistry. 1999; 38: 12943-12949Crossref PubMed Scopus (74) Google Scholar,22Allen P.B. Ouimet C.C. Greengard P. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 9956-9961Crossref PubMed Scopus (393) Google Scholar) showed that recombinant neurabin peptides, like many other PP1 regulators, inhibited the in vitro activity of purified rabbit skeletal muscle PP1 catalytic subunit using phosphorylasea as substrate; skeletal muscle contains multiple PP1 isoforms but is enriched in PP1β (34Alessi D.R. Street A.J. Cohen P. Cohen P.T. Eur. J. Biochem. 1993; 213: 1055-1066Crossref PubMed Scopus (167) Google Scholar, 35Barker H.M. Brewis N.D. Street A.J. Spurr N.K. Cohen P.T. Biochim. Biophys. Acta. 1994; 1220: 212-218Crossref PubMed Scopus (66) Google Scholar). As shown in Fig.3 A, GST-NrbI-(374–516) and GST-NrbII-(354–494) inhibited the phosphorylase phosphatase activity of purified rabbit muscle PP1 catalytic subunit with IC50values of 15 and 70 nm, respectively. In contrast, GST-NrbI-(374–516) had no effect on the activity of a structurally related protein phosphatase, PP2A, even at 1,000-fold higher concentration. Substitution of the core PP1-binding motif, KIKF, with alanines to produce GST-NrbI-(374–516), AAAA, abolished the ability of neurabin to inhibit PP1 activity in vitro. Peptides that disrupt PP1 binding to targeting subunits modified AMPA currents in striatal neurons (36Yan Z. Hsieh-Wilson L. Feng J. Tomizawa K. Allen P.B. Fienberg A.A. Nairn A.C. Greengard P. Nat. Neurosci. 1999; 2: 13-17Crossref PubMed Scopus (247) Google Scholar), suggesting that a PP1 complex dephosphorylated the α-amino-5-hydroxy-3-methyl-4-isoxazole propionate (AMPA) receptor. Mutation of the mouse NrbII gene abolished NrbII expression and impaired AMPA receptor regulation (37Feng J. Yan Z. Ferreira A. Tomizawa K. Liauw J.A. Zhuo M. Allen P.B. Ouimet C.C. Greengard P. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 9287-9292Crossref PubMed Scopus (325) Google Scholar) consistent with a deficit in GluR1 (an AMPA receptor subunit) phosphatase activity. Thus, we analyzed the dephosphorylation of the cytoplasmic C-terminal tail of the GluR1 subunit fused to GST that was phosphorylated in vitro by PKA (Fig. 3 B). Both GST-NrbI-(374–516) and GST-NrbII-(354–494) inhibited dephosphorylation of GST-GluRI by skeletal muscle PP1, although requiring higher concentrations than those needed to inhibit its phosphorylase phosphatase activity (Fig. 3 A). PP1 binding was essential as the mutant GST-NrbI-(374–516; AAAA) failed to inhibit GluRI dephosphorylation by PP1. At similar concentrations, GST-NrbII-(354–494) also failed to inhibit PP2A-mediated dephosphorylation of GST-GluRI. These data suggested that the interaction of neurabins with PP1 via the KIXF motif regulated its activity against neuronal substrates. To analyze the neurabin sequences that mediated PP1 binding and regulation, we generated chimeric neurabins that exchanged regions of NrbI and NrbII, schematically shown in Fig.4 A. NrbI and NrbII sequences C-terminal to site 1 are highly conserved, whereas those N-terminal to this site diverge significantly. Two other sites, 2 and 3, were selected to narrow down the functional domains. PP1 pulldowns from rat brain lysates (Fig. 4 B) and inhibition of skeletal muscle PP1 (Fig. 4, C and D) were used to characterize the chimeric neurabins. Focusing on the nearly 5-fold difference in PP1β binding shown by GST-NrbI-(374–516) compared with GST-NrbII-(354–494) (Fig. 2), we noted that three chimeras, NrbCH1, NrbCH3, and NrbCH6, showed a dose-dependent association with PP1β, similar to GST-NrbI-(374–516) (Fig. 2), whereas NrbCH2, NrbCH4, and NrbCH5, like GST-NrbII-(354–494), bound little or no PP1β. This highlighted the region between sites 2 and 3 in NrbI (dashed box in Fig.4 A) necessary for effective PP1β recognition. This region was largely eliminated in the shorter peptides, GST-NrbI-(436–479) and GST-NrbII-(427–470), that both failed to bind PP1β. Compared with other recombinant neurabins, NrbCH2 and NrbCH4 were significantly attenuated in their binding to PP1α and PP1γ1 in the pulldown assays. Although the reason for this deficiency is not clear, it emphasizes that proper arrangement of neurabin KIXF and N-terminal regions may be crucial for effective PP1 binding. We also analyzed the neurabin chimeras in protein phosphatase assays, which are more readily quantified than the pulldowns and detected both PP1 activity and binding. We utilized a phosphorylase phosphatase assay in the presence of physiological salt concentrations (100–150 mm KCl or NaCl) shown previously to enhance differences in the properties of modified PP1 re
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