Neuronal Cdc2-like Protein Kinase (Cdk5/p25) Is Associated with Protein Phosphatase 1 and Phosphorylates Inhibitor-2
2001; Elsevier BV; Volume: 276; Issue: 26 Linguagem: Inglês
10.1074/jbc.m010002200
ISSN1083-351X
AutoresAlka Agarwal-Mawal, Hemant K. Paudel,
Tópico(s)Protein Kinase Regulation and GTPase Signaling
ResumoProtein phosphatase 1 (PP1) is complexed with inhibitor 2 (I-2) in the cytosol. In rabbit muscle extract PP1·I-2 is activated upon preincubation with ATP/Mg. This activation is caused by phosphorylation of I-2 on Thr72 by glycogen synthase kinase 3 (GSK3). We have found that PP1·I-2 in bovine brain extract is also activated upon preincubation with ATP/Mg. However, blocking GSK3 action by LiCl inhibited only ∼29% of PP1 activity and indicated that GSK3 is not the sole PP1·I-2 activator in the brain. When bovine brain extract was analyzed by gel filtration PP1·I-2 and neuronal Cdc2-like protein kinase (NCLK), a heterodimer of Cdk5 and the regulatory p25 subunit, co-eluted as a ∼450-kDa size species. The NCLK from the eluted column fractions bound to PP1-specific microcystin-Sepharose and glutathioneS-transferase (GST)-I-2-coated glutathione-agarose beads. Similarly, PP1 from the eluted column fractions was pulled down with GST-Cdk5-coated glutathione-agarose beads. In vitro, NCLK phosphorylated I-2 on Thr72 and activated PP1·I-2 in an ATP/Mg-dependent manner. NCLK bound to PP1 through its Cdk5 subunit and the PP1 binding region was localized to Cdk5 residues 28–41. Our data demonstrate that in brain extract PP1·I-2 and NCLK are associated within a complex of ∼450 kDa and suggest that NCLK is one of the PP1·I-2-activating kinases in the mammalian brain. Protein phosphatase 1 (PP1) is complexed with inhibitor 2 (I-2) in the cytosol. In rabbit muscle extract PP1·I-2 is activated upon preincubation with ATP/Mg. This activation is caused by phosphorylation of I-2 on Thr72 by glycogen synthase kinase 3 (GSK3). We have found that PP1·I-2 in bovine brain extract is also activated upon preincubation with ATP/Mg. However, blocking GSK3 action by LiCl inhibited only ∼29% of PP1 activity and indicated that GSK3 is not the sole PP1·I-2 activator in the brain. When bovine brain extract was analyzed by gel filtration PP1·I-2 and neuronal Cdc2-like protein kinase (NCLK), a heterodimer of Cdk5 and the regulatory p25 subunit, co-eluted as a ∼450-kDa size species. The NCLK from the eluted column fractions bound to PP1-specific microcystin-Sepharose and glutathioneS-transferase (GST)-I-2-coated glutathione-agarose beads. Similarly, PP1 from the eluted column fractions was pulled down with GST-Cdk5-coated glutathione-agarose beads. In vitro, NCLK phosphorylated I-2 on Thr72 and activated PP1·I-2 in an ATP/Mg-dependent manner. NCLK bound to PP1 through its Cdk5 subunit and the PP1 binding region was localized to Cdk5 residues 28–41. Our data demonstrate that in brain extract PP1·I-2 and NCLK are associated within a complex of ∼450 kDa and suggest that NCLK is one of the PP1·I-2-activating kinases in the mammalian brain. protein phosphatase 1 casein kinase fast protein liquid chromatography high pressure liquid chromatography glycogen synthase kinase 3 glutathione S-transferase inhibitor 1 inhibitor 2 4-morpholinepropanesulfonic acid neuronal cdc2-like protein kinase cAMP-dependent protein kinase polymerase chain reaction dithiothreitol mitogen-activated protein kinase polyacrylamide gel electrophoresis PKA inhibitory peptide Protein phosphatase 1 (PP1)1 is a major Ser/Thr phosphatase involved in the regulation of metabolism, cell cycle, cell signaling, muscle contraction, and gene expression (for reviews see Refs. 1Cohen P. Annu. Rev. Biochem. 1989; 58: 453-508Crossref PubMed Scopus (2146) Google Scholar, 2Lee E.Y.C. Zhang L. Zhao S. Wei Q. Zhang J. Qi Z.Q. Belmonte E.R. Frontiers Biosci. 1999; 4: 270-285Crossref PubMed Google Scholar). PP1 is a 37-kDa catalytic subunit bound to two types of regulatory subunits: a targeting subunit and an inhibitory subunit. Targeting subunits confer substrate specificity and localize PP1 to various subcellular compartments. Inhibitory subunits suppress PP1 activity. There are three PP1 inhibitory subunits: inhibitor 1 (I-1), DARPP-32, and inhibitor 2 (I-2) (1Cohen P. Annu. Rev. Biochem. 1989; 58: 453-508Crossref PubMed Scopus (2146) Google Scholar, 2Lee E.Y.C. Zhang L. Zhao S. Wei Q. Zhang J. Qi Z.Q. Belmonte E.R. Frontiers Biosci. 1999; 4: 270-285Crossref PubMed Google Scholar, 3Oliver C.J. Shenolikar S. Frontiers Biosci. 1998; 3: 961-972Crossref PubMed Google Scholar). I-1 and DARPP-32 require phosphorylation for PP1 inhibitory activity, whereas nonphosphorylated I-2 inhibits PP1. These inhibitors are phosphorylated in response to many extracellular stimuli and allow PP1 to respond to various growth factors and hormones (3Oliver C.J. Shenolikar S. Frontiers Biosci. 1998; 3: 961-972Crossref PubMed Google Scholar). In rabbit skeletal muscle extract PP1 is found in both particulate and cytosolic fractions. PP1 in the particulate fraction is active, whereas in the cytosolic fraction it is inactive (1Cohen P. Annu. Rev. Biochem. 1989; 58: 453-508Crossref PubMed Scopus (2146) Google Scholar, 2Lee E.Y.C. Zhang L. Zhao S. Wei Q. Zhang J. Qi Z.Q. Belmonte E.R. Frontiers Biosci. 1999; 4: 270-285Crossref PubMed Google Scholar). The inactive cytosolic enzyme, a PP1·I-2 complex, is activated upon incubation with ATP/Mg and is hence called ATP/Mg-dependent PP1 (1Cohen P. Annu. Rev. Biochem. 1989; 58: 453-508Crossref PubMed Scopus (2146) Google Scholar). An activating factor named Fa is necessary for ATP/Mg-dependent activation of PP1·I-2. Fa has been identified to be glycogen synthase kinase 3 (GSK3) (4Woodgett J.R. Cohen P. Biochim. Biophys. Acta. 1984; 788: 339-347Crossref PubMed Scopus (164) Google Scholar, 5Yang S.D. Vandenheede J.R. Goris J. Merlevede W. J. Biol. Chem. 1980; 255: 11759-11767Abstract Full Text PDF PubMed Google Scholar, 6Hemmings B.A. Yellowless D. Kernohan J.C. Cohen P. Eur. J. Biochem. 1981; 119: 443-451Crossref PubMed Scopus (184) Google Scholar). The ATP/Mg-dependent activation is due to the phosphorylation of I-2 within the PP1·I-2 complex by GSK3. Nonphosphorylated I-2 suppresses PP1 activity within the PP1·I-2 complex. GSK3 phosphorylates I-2 on Thr72 and relieves PP1 from I-2 inhibition (4Woodgett J.R. Cohen P. Biochim. Biophys. Acta. 1984; 788: 339-347Crossref PubMed Scopus (164) Google Scholar, 5Yang S.D. Vandenheede J.R. Goris J. Merlevede W. J. Biol. Chem. 1980; 255: 11759-11767Abstract Full Text PDF PubMed Google Scholar, 6Hemmings B.A. Yellowless D. Kernohan J.C. Cohen P. Eur. J. Biochem. 1981; 119: 443-451Crossref PubMed Scopus (184) Google Scholar, 7Yang S.D. Vandenheede J.R. Merlevede W. J. Biol. Chem. 1981; 256: 10231-10234Abstract Full Text PDF PubMed Google Scholar, 8DePaoli-Roach A.A. J. Biol. Chem. 1984; 259: 12144-12152Abstract Full Text PDF PubMed Google Scholar, 9Park I.K. Roach P. Bondor J. Fox S.P. DePaoli-Roach A.A. J. Biol. Chem. 1994; 269: 944-954Abstract Full Text PDF PubMed Google Scholar). Even though GSK3 is a well-characterized PP1·I-2-activating kinase, several reports suggest that other kinases also phosphorylate I-2 and activate PP1·I-2 (10Chan C.P. McNally S.J. Krebs E.G. Fischer E.H. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 6257-6261Crossref PubMed Scopus (60) Google Scholar, 11Wang Q.M. Guan K.L. Roach P.J. DePaoli-Roach A.A. J. Biol. Chem. 1995; 270: 18352-18358Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar, 12Puntoni F. Villa-Moruzzi E. Biochem. Biophys. Res. Commun. 1995; 207: 732-739Crossref PubMed Scopus (23) Google Scholar). PP1 is highly expressed in brain (1Cohen P. Annu. Rev. Biochem. 1989; 58: 453-508Crossref PubMed Scopus (2146) Google Scholar). An earlier study found that most of the PP1 in brain extract is inactive and requires incubation with ATP/Mg to become active (13Yang S.D. Fong Y.-L. J. Biol. Chem. 1985; 260: 13464-13470Abstract Full Text PDF PubMed Google Scholar). The purified enzyme is a PP1·I-2 complex, which is activated upon incubation with ATP/Mg in the presence of muscle GSK3. It was suggested that brain ATP/Mg-dependent PP1 is regulated in a manner similar to its muscle counterpart, via phosphorylation of I-2 (13Yang S.D. Fong Y.-L. J. Biol. Chem. 1985; 260: 13464-13470Abstract Full Text PDF PubMed Google Scholar). A type of Fa activity was partially purified from porcine brain extract (13Yang S.D. Fong Y.-L. J. Biol. Chem. 1985; 260: 13464-13470Abstract Full Text PDF PubMed Google Scholar), but the identity of this activity has remained unknown. Thus, until now it has not been clear as to which kinase activates PP1·I-2 in the brain. Neuronal Cdc2-like protein kinase (NCLK) is a heterodimer of cyclin-dependent protein kinase 5 (Cdk5) and a neuronal-specific p25 regulatory subunit (reviewed in Ref. 14Lew J. Qi Z. Huang Q.-Q. Paudel H. Matsuura I. Matsushita M. Zhu X. Wang J.H. Neurobiol. Aging. 1995; 16: 263-268Crossref PubMed Scopus (39) Google Scholar). Cdk5, a member of the cyclin-dependent protein kinase family, is widely expressed in various tissues and cell lines (15Meyerson M. Enders G.H. Wu C.L. Su L.K. Gorka C. Nelson C. Harlow E. Tsai L.-H. EMBO J. 1992; 11: 2909-2917Crossref PubMed Scopus (784) Google Scholar). However, its kinase activity is detected only in terminally differentiated neurons where it is associated with a p25 subunit (16Lew J. Beaudette K. Litwin C.M.E. Wang J.H. J. Biol. Chem. 1992; 267: 13383-13390Abstract Full Text PDF PubMed Google Scholar). p25 is a proteolytic fragment of a 35-kDa protein and is expressed only in neurons (17Tsai L.-H. Delalle I. Caviness Jr., V.S. Chae T. Harlow E. Nature. 1994; 371: 419-423Crossref PubMed Scopus (808) Google Scholar). NCLK is involved in brain development, neurite outgrowth, cell migration, cell signaling, microtubule dynamics regulation, and Alzheimer's disease pathogenesis (18Ohshima T. Ward J.M. Huh C.-G. Longenecker G. Veeranna Pant H.C. Brady R.O. Martin L.J. Kulkarni A.B. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 11173-11178Crossref PubMed Scopus (806) Google Scholar, 19Nlkolic M. Dudek H. Kwon Y.T. Ramos Y.F.M. Tsai L.-H. Gene Dev. 1996; 10: 816-825Crossref PubMed Scopus (529) Google Scholar, 20Chae T. Kwon Y.T. Bronson R. Dikkes P. Li E. Tsai L.-H. Neuron. 1996; 18: 29-42Abstract Full Text Full Text PDF Scopus (662) Google Scholar, 21Sobue K. Agarwal-Mawal A. Li W. Sun W. Miura Y Paudel H.K. J. Biol. Chem. 2000; 275: 16673-16680Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar, 22Paudel H.K. Lew J. Ali Z. Wang J.H. J. Biol. Chem. 1993; 268: 23512-23518Abstract Full Text PDF PubMed Google Scholar, 23Patrick G.N. Zukerberg L. Nikolic M. de la Monte S. Dikkes P Tsai L.-H. Nature. 1999; 402: 615-622Crossref PubMed Scopus (1310) Google Scholar, 24Paudel H.K. J. Biol. Chem. 1997; 272: 28328-28334Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). Herein we show that NCLK is complexed with PP1·I-2 in brain extract, phosphorylates I-2 on Thr72, and activates PP1·I-2. Our data suggest that NCLK is one of the kinases that activate PP1·I-2 in the central nervous system. Human I-2 cDNA plasmid in pT7T3D-pac vector (American Type Culture Collection, Manassas, VA) was subcloned into two different bacterial expression vectors: pET-9a (Promega, Madison, WI) and pGEX-6P-1 (Amersham Pharmacia Biotech, Baied'Urfe, Quebec, Canada). To subclone into the pET-9a vector Pfu DNA polymerase catalyzed 30 cycles polymerase chain reaction (PCR) was carried out using I-2 cDNA as the template and forward (5′-AAAAAA CAT ATG GCG GCC TCG ACG G-3′) and reverse (5′-AAA AAA GGA TCC CTA TGA ACT TCG TAA TTT GTT TTG-3′) primers. The PCR condition was 95 °C for 1 min, 60 °C for 1 min, and 72 °C for 2 min. A final 7-min extension at 72 °C was followed by a 10-min incubation with Taq polymerase (Promega) at 72 °C. The PCR product was ligated into a pGEM-T Easy vector (Promega) and amplified. The I-2 cDNA insert from the pGEM-T Easy vector was excised by NdeI/BamHI and ligated into a NdeI/BamHI cloning site of the pET-9a vector. To subclone into pGEX-6P-1 vector, PCR was carried out using forward primer 5′-AAA AAA GGA TCC ATG GCG GCC TCG ACG G-3′ containing a BamHI site (underlined) and reverse primer 5′-AAA AAA GAA TTC CTA TGA ACT TCG TAA TTT GTT TTG-3′ containing an EcoRI site (underlined) as above, except Taq polymerase and pGEM-T Easy vector steps were excluded. The PCR product was excised withBamHI/EcoRI and ligated into aBamHI/EcoRI cloning site of the pGEX-6P-1 vector. Three forward primers, F1 (5′-AAA AAA GGA TCC ATG CAG AAA TAC GAG A-3′), F2 (5′-AAA AAA GGA TCC GGT GTG CCG AGT TCC GCC-3′), and F3 (5′-AAA AAA GGA TCC ATC GTG GCT CTG AAA C-3′), each containing a BamHI site (underlined) and two reverse primers R1 (5′-AAA AAA GTC GAC CTA GGC GGA ACT CGG CAC-3′), and R2 (5′-AAA AAA GTC GAC CTA GGG CGG ACA GAA GT-3′), each containing an SalI site (underlined) were used in the following combination to generate three cdk5 deletion mutants: cdk5-(1–48) (F1 and R1), cdk5-(42–292) (F2 and R2), and cdk5-(28–292) (F3 and R2). All PCR conditions were the same as described above except for using human cdk5 cDNA (15Meyerson M. Enders G.H. Wu C.L. Su L.K. Gorka C. Nelson C. Harlow E. Tsai L.-H. EMBO J. 1992; 11: 2909-2917Crossref PubMed Scopus (784) Google Scholar) as the template and omitting the Taqpolymerase and pGEM-T Easy vector steps. Each PCR product was excised with BamHI/SalI and ligated into theBamHI/SalI cloning site of the pGEX-6P-1 vector. All recombinant plasmids were transfected into DH5α first followed by BL21(DE3) Escherichia coli cells. All cDNA clones were confirmed by nucleotide sequencing. I-2 was purified from bacterial culture medium as described previously (25Huang H.-b. Horiuchi A. Watanabe T. Shih S.-R. Tsay H.-J. Li H.-C. Greengard P. Nairn A.C. J. Biol. Chem. 1999; 274: 7870-7878Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar) with some modifications. Overnight bacterial culture (10 ml) was diluted 100-fold in fresh medium and incubated with vigorous shaking at 37 °C. When theA600 of the medium reached ∼0.6, isopropyl-β-d-thiogalactoside was added to a final concentration of 1 mm. Shaking then continued for another 3 h at 37 °C. The medium was centrifuged, and the bacterial pellet was suspended in 100 ml of cold buffer A (20 mmTris-HCl (pH 7.5), 0.2 mm EDTA, and 0.1% β-mercaptoethanol) and protease inhibitor mixture (2 mmphenylmethylsulfonyl fluoride, 1 μg/ml aprotinin, 1 μg/ml leupeptin, 1 μg/ml pepstatin, and 5 mg/ml benzamidin). The suspension was subjected to freezing and thawing three times and then was sonicated three times, for 30 s each, using a probe sonicator. The sonicated suspension was centrifuged at 2.7 × 104 ×g for 20 min at 4 °C, and the supernatant was heated in a boiling water bath for 15 min. The heated sample was centrifuged, and the supernatant was loaded onto a DEAE-Sephacel column (2.3 × 23 cm) pre-equilibrated with buffer B (20 mm Tris-HCl (pH 7.5), 0.2 mm EDTA, and 1 mm DTT). The column was washed with buffer B until the effluent A280was less than 0.1 and then eluted with a linear gradient of 0–0.5m NaCl in buffer B. Fractions containing I-2 were pooled, dialyzed against cold buffer B for 4 h, and then loaded onto an Affi-Gel blue (Bio-Rad, Mississauga, Ontario, Canada) column (3 × 10 cm) previously equilibrated in buffer B. The column was washed and then eluted with a 250-ml linear gradient of NaCl (0–1 m) in buffer B. Fractions containing I-2 were combined and dialyzed against buffer A for 4 h and loaded onto a Q-Sepharose (Amersham Pharmacia Biotech) column (1.5 × 5 cm) pre-equilibrated in buffer A. After washing the column with ∼50 ml of buffer A, the column-bound I-2 was eluted with a 50-ml linear gradient of NaCl (0–0.8m) in buffer A. Fractions containing I-2 were pooled and concentrated by dialysis against Aquacide III (Calbiochem, San Diego, CA). The concentrated sample was dialyzed against buffer A, and stored frozen at −80 °C until used. NCLK (16Lew J. Beaudette K. Litwin C.M.E. Wang J.H. J. Biol. Chem. 1992; 267: 13383-13390Abstract Full Text PDF PubMed Google Scholar) and phosphorylase kinase (26Paudel H.K. J. Biol. Chem. 1997; 272: 1777-1785Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar) were purified from fresh bovine brain and rabbit skeletal muscle extracts, respectively. PP1α was a generous gift from Dr. E. Y. C. Lee (New York Medical College). Phosphorylase b (Sigma-Aldrich Ltd., Oakville, Ontario, Canada) was phosphorylated by phosphorylase kinase using [γ-32P]ATP (26Paudel H.K. J. Biol. Chem. 1997; 272: 1777-1785Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). Monoclonal antibodies against PP1 and I-2 were obtained from Transduction Laboratories (Lexington, KY) and Upstate Biotechnology (Lake Placid, NY), respectively. Polyclonal antibodies against the Cdk5 subunit of NCLK, MAPK (which cross-reacts with both brain-specific p43erk1 and p42erk2), casein kinase 1 (CK1), and casein kinase 2 (CK2) have been described previously (27Huang K. Paudel H.K. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 5824-5829Crossref PubMed Scopus (46) Google Scholar). Polyclonal antibody against the C-terminal region of GSK3β was prepared at Zymed Laboratories Inc. (San Francisco, CA). GSK3α was purchased from Upstate Biotechnology. Stephane Richard (Lady Davis Institute, Montreal) provided purified CK2. Various GST fusion proteins were purified from respective bacterial lysates as described (21Sobue K. Agarwal-Mawal A. Li W. Sun W. Miura Y Paudel H.K. J. Biol. Chem. 2000; 275: 16673-16680Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). The making of the NCLK synthetic peptide substrate has been described previously (22Paudel H.K. Lew J. Ali Z. Wang J.H. J. Biol. Chem. 1993; 268: 23512-23518Abstract Full Text PDF PubMed Google Scholar). cAMP-dependent protein kinase (PKA) substrate Leu-Arg-Arg-Ala-Ser-Leu-Gly (Kemptide) and inhibitory peptide PKI were obtained from Sigma-Aldrich Ltd. The GSK3 peptide substrate KRREILSRRPSYR (28Wang Q.M. Fiol C.J. DePaoli-Roach A.A. Roach P. J. Biol. Chem. 1994; 269: 14566-14574Abstract Full Text PDF PubMed Google Scholar) was synthesized at the peptide synthesis facility of the University of Calgary. This peptide was phosphorylated by PKA, and the phosphorylated peptide was purified by HPLC. The PP1·I-2 complex was reconstituted from PP1α and I-2 as described previously (8DePaoli-Roach A.A. J. Biol. Chem. 1984; 259: 12144-12152Abstract Full Text PDF PubMed Google Scholar) in a 0.1-ml reconstitution mixture containing 50 mm Tris-HCl (pH 7.4), 0.1 mm EDTA, 0.1 mm DTT, 0.1 mg/ml PP1, and 0.1 mg/ml I-2. The mixture was incubated at 30 °C for 60 min and loaded onto an Affi-Gel Blue column (∼1 ml) pre-equilibrated with 50 mm Tris-HCl (pH 7.4), 0.1 mm DTT, and 0.1 mm EDTA. The column was washed with the equilibration buffer and fractions (0.5 ml each) were collected. Fractions were analyzed by SDS-PAGE. The reconstituted PP1·I-2 complex was recovered in flow-through fractions. I-2, PP1, GSK3, and CK2 concentrations were determined by Bio-Rad protein assay (Bio-Rad laboratories, Mississauga, Ontario, Canada) using bovine serum albumin as the standard. Concentrations of phosphorylase kinase and phosphorylase b were determined spectrophotometrically as described previously (26Paudel H.K. J. Biol. Chem. 1997; 272: 1777-1785Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). The concentration of NCLK is based on its activity (22Paudel H.K. Lew J. Ali Z. Wang J.H. J. Biol. Chem. 1993; 268: 23512-23518Abstract Full Text PDF PubMed Google Scholar). Concentration of PKI was based on dry weight. Concentrations of NCLK and GSK3 peptide substrates were determined by amino acid analysis. Microcystin pull-down assay was carried out by mixing 50 μl of microcystin-Sepharose (Upstate Biotechnology) beads pre-equilibrated in 50 mmTris-HCl (pH 7.5), 0.1 mm EDTA, 5 mmMnCl2, 15 mm β-mercaptoethanol, 0.05% Nonidet P-40 with 0.2 ml of combined gel filtration fractions from Fig.1 A. The mixture was incubated at 4 °C overnight with end-over-end shaking. The incubated mixture was centrifuged, and the recovered beads were washed three times with 0.5 ml of 20 mm Tris-HCl (pH 7.5), 0.5 mm EDTA, 0.2m NaCl, and 0.05% of Tween 20. The washed beads were mixed with 50 μl of SDS-PAGE sample buffer, boiled, and centrifuged, and 10 μl of the supernatant was analyzed by immunoblot analysis using the indicated antibody. To perform the GST pull-down assay 50 μl of glutathione-agarose beads (Sigma-Aldrich Ltd.) coated with the indicated GST fusion protein were mixed with 150 μl of the indicated protein solution and incubated overnight with end-over-end shaking at 4 °C. Incubated beads were washed four times with 20 mmTris-HCl (pH 7.5), 0.1 mm EDTA, 0.2 m NaCl, and 0.05% Tween 20. The washed beads were mixed with 50 μl of SDS-PAGE sample buffer, boiled, and centrifuged, and 10 μl of the supernatant was analyzed by immunoblot analysis using the indicated antibody. ATP/Mg-dependent PP1 activity was assayed as described previously (9Park I.K. Roach P. Bondor J. Fox S.P. DePaoli-Roach A.A. J. Biol. Chem. 1994; 269: 944-954Abstract Full Text PDF PubMed Google Scholar) except that the assay mixture also contained okadaic acid (Sigma-Aldrich Ltd.) (to inhibit PP2A) and PP2B inhibitor cypermethrin (Calbiochem). Samples were preincubated at 30 °C for 15 min in a mixture containing 50 mm Tris-HCl (pH 7.4), 0.1 mm EDTA, 1 mm EGTA, 0.2% β-mercaptoethanol, 0.2 mm ATP, and 5 mmMgCl2. The assay was initiated by the addition of 10 μl of the preincubated sample to a vial containing 30 μl of the rest of the assay components. The final concentrations of the various components in the assay were 50 mm Tris-HCl (pH 7.4), 0.2% β-mercaptoethanol, 5 mm caffeine, 0.5 mmMnCl2, 10 μm[32P]phosphorylase, 5 nm okadaic acid, 40 pm cypermethrin, 1 mm EGTA, and carryover preincubation mixture components. After 20 min at 30 °C, 10 μl of 50% trichloroacetic acid was added to the assay mixture. The assay mixture was cooled on ice for 10 min and centrifuged for 5 min using a bench top centrifuge. The supernatant (20 μl) was withdrawn, spotted onto a filter paper, and counted in a liquid scintillation counter to determine the amount of 32Pi released. NCLK, GSK3, and PKA activities were assayed by using their respective peptide substrates (27Huang K. Paudel H.K. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 5824-5829Crossref PubMed Scopus (46) Google Scholar). I-2 phosphorylation by NCLK was carried out in a reaction mixture containing 25 mm Hepes (pH 7.2), 0.1 mm EDTA, 1 mm DTT, 0.5 mm[γ-32P]ATP, 10 mm MgCl2, 0.5 mg/ml I-2, and 400 units/ml NCLK. Phosphorylation was initiated by the addition of NCLK to the mixture containing the rest of the assay components. After indicated time points at 30 °C aliquots were withdrawn, mixed with an equal volume of SDS-PAGE sample buffer, and electrophoresed on a 12% SDS-gel. The gel was stained and destained, and the I-2 band in the gel was sliced out and counted in a liquid scintillation counter to determine the amount of radioactivity that was incorporated. I-2 phosphorylation by CK2 was carried out as above by using 10 μg/ml CK2 in the phosphorylation mixture. Over 4 h, CK2 incorporated 2.8 mol of phosphate/mol of I-2. CK2-phosphorylated I-2 was desalted by a Sephadex G-25 column and used to generate the data shown in Fig. 4. All procedures were performed at 4 °C. Fresh bovine brain (0.5 kg) was homogenized for 1 min in 1 liter of buffer C (20 mm MOPS (pH 7.4), 50 mm β-glycerophosphate, 1 mmEDTA, 1 mm DTT, and 15 mm MgCl2), which contained protease inhibitor mixture. The homogenate was centrifuged at 104 × g for 30 min, and the supernatant was centrifuged at 105 × g for 45 min. The resulting clear supernatant was loaded onto a DEAE-Sephacel column (2.5 × 45 cm) previously equilibrated with buffer C. The flow-through fraction containing PP1 activity was loaded onto an SP-Sepharose (Amersham Pharmacia Biotech) column (1 × 60 cm) pre-equilibrated with buffer C. The column was washed with buffer C and was eluted with 400 ml of a linear NaCl gradient (0–0.5 m) in buffer C. Fractions containing PP1 activity were pooled, dialyzed against buffer C, concentrated by dialysis against Aquacide III to ∼8 ml, and chromatographed through an FPLC Superose 12 gel filtration column (Amersham Pharmacia Biotech) column (2 × 70 cm) equilibrated and eluted with 20 mm MOPS (pH 7.4), 1 mm EDTA, 1 mm DTT, and 0.15 m NaCl. Fractions 41–43, containing PP1 activity, were combined. A portion of the combined fractions was used to generate Figs. 1 B and 2, and the rest was loaded onto a hydroxylapatite column (1 × 15 cm) pre-equilibrated with buffer C. The column was washed with ∼40 ml of buffer C and eluted with a 50-ml linear gradient (0–0.5 m) of K2HPO4 (pH 7.4) in buffer C. I-2 was phosphorylated for 16 h by NCLK in a 0.7-ml reaction mixture containing 25 mm Hepes (pH 7.2), 0.1 mm EDTA, 1 mm EGTA, 10 mm NaF, 0.5 mm[γ-32P]ATP, 10 mm MgCl2, 1 mg/ml I-2, and 400 units/ml NCLK. Phosphorylated I-2 was desalted through a Sephadex G-25 column, lyophilized, dissolved in 0.2 ml of 50 mm NH4HCO3 (pH 8.0) containing 50 μg/ml trypsin, and incubated at 37 °C for 16 h. The incubated tryptic digest was separated by a HPLC reverse phase column previously equilibrated with 0.1% trifluoroacetic acid as described (26Paudel H.K. J. Biol. Chem. 1997; 272: 1777-1785Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar) using 0–40% acetonitrile gradient in 50 min. Radioactive fractions were lyophilized, dissolved in 50 mmNH4HCO3 containing 50 μg/ml of chymotrypsin, and incubated for 16 h at 37 °C. The chymotryptic digest was lyophilized, dissolved in 25 mm Hepes (pH 7.2), 1 mm EDTA, 1 mm EGTA, and loaded onto a DEAE-Sephacel column (∼2 ml) previously equilibrated in 25 mm Hepes (pH 7.2). The column was washed, and bound peptide was eluted with 0.3 m NaCl in the equilibration buffer. Fractions containing radioactivity were lyophilized, dissolved in 25 mm Hepes (pH 7.2), 1 mm EDTA, 1 mmEGTA, and chromatographed through an HPLC column as described above. Only one radioactive peptide was recovered. This peptide was sequenced at the Department of Biochemistry and Microbiology, University of Victoria by using a gas-phase micro-sequencer. In a previous study PP1·I-2 was found to be the predominant form of PP1 in brain extract (13Yang S.D. Fong Y.-L. J. Biol. Chem. 1985; 260: 13464-13470Abstract Full Text PDF PubMed Google Scholar). To learn more about brain PP1·I-2, we chromatographed a fresh bovine brain extract through a DEAE-Sephacel column then an SP-Sepharose column. The effluent containing ATP/Mg-dependent PP1 activity was then analyzed by an FPLC Superose 12 gel filtration column. PP1 activity eluted from the gel filtration column as a broad peak (Fig.1 A). Immunoblot analyses of various gel filtration fractions (by using anti-PP1 or anti-I-2 antibodies) indicated that both PP1 and I-2 were present in column fractions containing PP1 activity (data not shown). The gel filtration analysis indicated that the molecular size of PP1·I-2 in Fig.1 A (fraction 42) is ∼450 kDa. Because the molecular size of PP1·I-2 is ∼70 kDa, these observations corroborate previous reports (2Lee E.Y.C. Zhang L. Zhao S. Wei Q. Zhang J. Qi Z.Q. Belmonte E.R. Frontiers Biosci. 1999; 4: 270-285Crossref PubMed Google Scholar) and indicate that PP1·I-2 is complexed with other protein (s) in the brain. As observed previously (13Yang S.D. Fong Y.-L. J. Biol. Chem. 1985; 260: 13464-13470Abstract Full Text PDF PubMed Google Scholar), we found that PP1 has low activity in brain extract, is complexed with I-2, and is activated by preincubation with ATP/Mg (data not shown). These observations are consistent with a previous report (13Yang S.D. Fong Y.-L. J. Biol. Chem. 1985; 260: 13464-13470Abstract Full Text PDF PubMed Google Scholar) and indicate that the PP1·I-2-activating factor is present in brain. An immunoblot analysis using anti-GSK3 antibody showed that GSK3 is present in Fig. 1 A column fractions (data not shown). To examine if GSK3 is responsible for ATP/Mg-dependent PP1·I-2 activation, we combined gel filtration fractions 41–43 containing peak PP1 activity from Fig.1 A. We preincubated an aliquot from the combined fractions with ATP/Mg the in the presence of the GSK3 inhibitor LiCl (29Klein P.S. Melton D.A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 8455-8459Crossref PubMed Scopus (2072) Google Scholar) to block GSK3 action and assayed PP1 activity. Surprisingly, PP1 activity was only ∼29% inhibited (Fig. 1 B), although a kinase assay confirmed that LiCl had completely suppressed GSK3 activity in our samples (data not shown). These data suggested that the brain contains a kinase other than GSK3 that phosphorylates I-2 and activates PP1. To identify the PP1·I-2-activating kinase we included 2 mm Ca2+-chelator EGTA, or 50 μmPKA inhibitory peptide (PKI) in the preincubation mixture and assayed the Fig. 1 A combined column fractions. PP1 activity was approximately the same in samples preincubated in the presence of EGTA, PKI, or buffer control (data not shown). These data indicated that LiCl-insensitive ATP/Mg-dependent PP1 activation in Fig.1 B could not be due to the involvement of PKA or any Ca2+-dependent kinases (protein kinase C, calmodulin-dependent kinase 2, or phosphorylase kinase). MAPK phosphorylates I-2 in vitro (11Wang Q.M. Guan K.L. Roach P.J. DePaoli-Roach A.A. J. Biol. Chem. 1995; 270: 18352-18358Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). To determine if MAPK is a PP1·I-2-activating kinase, we analyzed Fig. 1 A column fractions by immunoblot analysis using anti-MAPK antibody. Our antibody that detected nanograms of MAPKs (p43erk1 and p42erk2) in brain extract failed to show any immunoreactivity (data not shown). By similar immunoblot analyses we determined that CK1 and CK2 were also absent in Fig. 1 Acolumn fractions (data not shown). These observations indicated that MAPK, CK1, and CK2 are not responsible for activating PP1·I-2 in Fig.1 B. I-2 Thr72 is follo
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