Secretory Granule to the Nucleus
2009; Elsevier BV; Volume: 284; Issue: 38 Linguagem: Inglês
10.1074/jbc.m109.035782
ISSN1083-351X
AutoresChitra Rajagopal, Kathryn L. Stone, Victor P. Francone, Richard E. Mains, Betty Eipper,
Tópico(s)Lipid Membrane Structure and Behavior
ResumoIntrinsically unstructured domains occur in one-third of all proteins and are characterized by conformational flexibility, protease sensitivity, and the occurrence of multiple phosphorylation. They provide large interfaces for diverse protein-protein interactions. Peptidylglycine α-amidating monooxygenase (PAM), an enzyme essential for neuropeptide biosynthesis, is a secretory granule membrane protein. As one of the few proteins spanning the granule membrane, PAM is a candidate to relay information about the status of the granule pool and conditions in the granule lumen. Here, we show that the PAM cytosolic domain is unstructured. Mass spectroscopy and two-dimensional gel electrophoresis demonstrated phosphorylation at 10–12 sites in the cytosolic domain. Stimulation of exocytosis resulted in coupled phosphorylation and dephosphorylation of specific sites and in the endoproteolytic release of a soluble, proteasome-sensitive cytosolic domain fragment. Analysis of granule-rich tissues, such as pituitary and heart, showed that a similar fragment was generated endogenously and translocated to the nucleus. This multiply phosphorylated unstructured domain may act as a signaling molecule that relays information from secretory granules to both cytosol and nucleus. Intrinsically unstructured domains occur in one-third of all proteins and are characterized by conformational flexibility, protease sensitivity, and the occurrence of multiple phosphorylation. They provide large interfaces for diverse protein-protein interactions. Peptidylglycine α-amidating monooxygenase (PAM), an enzyme essential for neuropeptide biosynthesis, is a secretory granule membrane protein. As one of the few proteins spanning the granule membrane, PAM is a candidate to relay information about the status of the granule pool and conditions in the granule lumen. Here, we show that the PAM cytosolic domain is unstructured. Mass spectroscopy and two-dimensional gel electrophoresis demonstrated phosphorylation at 10–12 sites in the cytosolic domain. Stimulation of exocytosis resulted in coupled phosphorylation and dephosphorylation of specific sites and in the endoproteolytic release of a soluble, proteasome-sensitive cytosolic domain fragment. Analysis of granule-rich tissues, such as pituitary and heart, showed that a similar fragment was generated endogenously and translocated to the nucleus. This multiply phosphorylated unstructured domain may act as a signaling molecule that relays information from secretory granules to both cytosol and nucleus. One-third to one-half of all eukaryotic proteins include a disordered region more than 50 amino acids long (1Dunker A.K. Lawson J.D. Brown C.J. Williams R.M. Romero P. Oh J.S. Oldfield C.J. Campen A.M. Ratliff C.M. Hipps K.W. Ausio J. Nissen M.S. Reeves R. Kang C. Kissinger C.R. Bailey R.W. Griswold M.D. Chiu W. Garner E.C. Obradovic Z. J. Mol. Graph. Model. 2001; 19: 26-59Crossref PubMed Scopus (1850) Google Scholar). These intrinsically unstructured domains are characterized by low hydrophobicity and high net charge, features that contribute to their flexibility, low secondary structure, and protease sensitivity (2Tompa P. Trends Biochem. Sci. 2002; 27: 527-533Abstract Full Text Full Text PDF PubMed Scopus (1676) Google Scholar). Unstructured domains are common in proteins that play key roles in complex pathways like cell cycle regulation, endocytic trafficking, and control of transcription (2Tompa P. Trends Biochem. Sci. 2002; 27: 527-533Abstract Full Text Full Text PDF PubMed Scopus (1676) Google Scholar, 3Kalthoff C. Alves J. Urbanke C. Knorr R. Ungewickell E.J. J. Biol. Chem. 2002; 277: 8209-8216Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar). Their ability to mediate several low affinity interactions with specific interactors makes them well suited to participation in multistep processes. Intrinsically unstructured domains are often sites of multiple phosphorylation (2Tompa P. Trends Biochem. Sci. 2002; 27: 527-533Abstract Full Text Full Text PDF PubMed Scopus (1676) Google Scholar). The presence of an extended unstructured domain may provide a larger interface for protein-protein interactions, with phosphorylation at multiple sites contributing to increased local order, facilitating cooperativity, and driving specific intermolecular interactions (4Lenz P. Swain P.S. Curr. Biol. 2006; 16: 2150-2155Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). Identification of phosphorylation sites in the unstructured domains of several proteins and mutational analyses have confirmed their functional significance (5Nash P. Tang X. Orlicky S. Chen Q. Gertler F.B. Mendenhall M.D. Sicheri F. Pawson T. Tyers M. Nature. 2001; 414: 514-521Crossref PubMed Scopus (637) Google Scholar, 6Jonker H.R. Wechselberger R.W. Pinkse M. Kaptein R. Folkers G.E. FEBS J. 2006; 273: 1430-1444Crossref PubMed Scopus (27) Google Scholar, 7Hsu J.M. Lee Y.C. Yu C.T. Huang C.Y. J. Biol. Chem. 2004; 279: 32592-32602Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar). Bioinformatic analyses reveal an increase in their prevalence with organism complexity and an association with alternative splicing (8Dunker A.K. Silman I. Uversky V.N. Sussman J.L. Curr. Opin. Struct. Biol. 2008; 18: 756-764Crossref PubMed Scopus (791) Google Scholar). Regulated exocytosis of secretory granules is crucial for maintaining neuronal homeostasis. Very little is known about the feedback mechanisms that signal the status of the secretory granule pool and regulate recycling. This process involves even greater complexity in neurons where the site of release is distant from the cell body. Peptidylglycine α-amidating monooxygenase (PAM), 2The abbreviations used are: PAMpeptidylglycine α-amidating monooxygenasePAM-CDPAM cytosolic domainsf-CDsoluble fragment of cytosolic domainMyc-TMD-CDMyc-tagged transmembrane/cytosolic domainPHMpeptidylglycine α-hydroxylating monooxygenasePALpeptidyl α-hydroxyglycine α-amidating lyasePALmPAL attached to the transmembrane/cytosolic domainMALDI-TOFmatrix-assisted laser desorption ionization time-of-flightTES2-{[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]amino}ethanesulfonic acidCIPcalf intestinal alkaline phosphataseIPGImmobilineTM Dry Strip pH 4–7 isoelectric focusing gelMSmass spectrometryPBSphosphate-buffered saline. a type I integral membrane protein that catalyzes one of the final steps in the biosynthesis of neuropeptides, is composed of two independently folded catalytic domains, a single transmembrane domain and a highly conserved 86-amino acid cytosolic domain whose sequence suggested that it might lack structure. Removal of this cytosolic domain limited the access of PAM to secretory granules and eliminated endocytosis (9Tausk F.A. Milgram S.L. Mains R.E. Eipper B.A. Mol. Endocrinol. 1992; 6: 2185-2196PubMed Google Scholar, 10Milgram S.L. Mains R.E. Eipper B.A. J. Cell Biol. 1993; 121: 23-36Crossref PubMed Scopus (74) Google Scholar). Radiolabeling studies demonstrated that phosphorylation of PAM-1 was restricted to its cytosolic domain (11Yun H.Y. Milgram S.L. Keutmann H.T. Eipper B.A. J. Biol. Chem. 1995; 270: 30075-30083Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar), and trafficking roles were identified for phosphorylation at two specific residues, Ser937 and Ser949 (12Steveson T.C. Zhao G.C. Keutmann H.T. Mains R.E. Eipper B.A. J. Biol. Chem. 2001; 276: 40326-40337Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar, 13Steveson T.C. Keutmann H.T. Mains R.E. Eipper B.A. J. Biol. Chem. 1999; 274: 21128-21138Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar). peptidylglycine α-amidating monooxygenase PAM cytosolic domain soluble fragment of cytosolic domain Myc-tagged transmembrane/cytosolic domain peptidylglycine α-hydroxylating monooxygenase peptidyl α-hydroxyglycine α-amidating lyase PAL attached to the transmembrane/cytosolic domain matrix-assisted laser desorption ionization time-of-flight 2-{[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]amino}ethanesulfonic acid calf intestinal alkaline phosphatase ImmobilineTM Dry Strip pH 4–7 isoelectric focusing gel mass spectrometry phosphate-buffered saline. C-terminal amidation is the final reaction in the generation of many bioactive peptides, and PAM is one of the small number of secretory granule proteins that span the granule membrane and has been shown to be recycled and reused in secretory granules (14Wasmeier C. Hutton J.C. J. Biol. Chem. 2001; 276: 31919-31928Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar, 15Ferraro F. Eipper B.A. Mains R.E. J. Biol. Chem. 2005; 280: 25424-25435Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar). Biochemical studies demonstrated that PAM was sensitive to the pH changes that occur as granules mature (16Bell-Parikh L.C. Eipper B.A. Mains R.E. J. Biol. Chem. 2001; 276: 29854-29863Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar), and analysis of a cell line in which expression of PAM-1 could be induced suggested an important role for PAM-1 in signaling from the granule lumen to the cytosol (17Ciccotosto G.D. Schiller M.R. Eipper B.A. Mains R.E. J. Cell Biol. 1999; 144: 459-471Crossref PubMed Scopus (54) Google Scholar, 18Alam M.R. Steveson T.C. Johnson R.C. Bäck N. Abraham B. Mains R.E. Eipper B.A. Mol. Biol. Cell. 2001; 12: 629-644Crossref PubMed Scopus (28) Google Scholar). Cytosolic PAM-CD interactors include Kalirin and Trio, Rho GDP/GTP exchange factors that affect cytoskeletal organization (19Alam M.R. Caldwell B.D. Johnson R.C. Darlington D.N. Mains R.E. Eipper B.A. J. Biol. Chem. 1996; 271: 28636-28640Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar, 20Mains R.E. Alam M.R. Johnson R.C. Darlington D.N. Bäck N. Hand T.A. Eipper B.A. J. Biol. Chem. 1999; 274: 2929-2937Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar), and P-CIP2 (KIS, Uhmk1), a protein kinase that binds to and phosphorylates PAM-CD at Ser949, a site known to affect its endocytic trafficking (19Alam M.R. Caldwell B.D. Johnson R.C. Darlington D.N. Mains R.E. Eipper B.A. J. Biol. Chem. 1996; 271: 28636-28640Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). We set out to explore the hypothesis that this key region of PAM was involved in relaying information from the secretory granule lumen. We demonstrate that PAM-CD achieves this by being intrinsically unstructured and acting as a display site for multiple phosphorylation. Mass spectroscopy was used to identify additional sites of phosphorylation, and two-dimensional gel electrophoresis was used to demonstrate regulated multiple site phosphorylation in pituitary cells. A newly developed antibody to the C terminus of PAM was used to identify a soluble fragment of PAM-CD (sf-CD). Formed in response to stimulated secretion, sf-CD was identified in nuclei isolated from both heart atrium and pituitary. Production of sf-CD was limited in cells expressing phosphomimetic mutants of PAM. This study demonstrates that the unstructured domain of PAM acts as a signaling module by utilizing multisite phosphorylation, proteolysis, and nuclear translocation. Recombinant PAM-CD was purified from Escherichia coli as described (21Yun H.Y. Johnson R.C. Mains R.E. Eipper B.A. Arch. Biochem. Biophys. 1993; 301: 77-84Crossref PubMed Scopus (34) Google Scholar), with an additional step of purification by reverse phase chromatography on a C-18 μBondapak column (Waters) equilibrated with 0.1% trifluoroacetic acid and eluted with a gradient to 40% acetonitrile, 0.1% trifluoroacetic acid over 60 min; purity as assessed by SDS-PAGE was >99%; MALDI-TOF analysis revealed that the purified protein terminated at Ser961. PAM-CD-3P and PAM-CD-5P (residues 896–976) with phosphomimetic Ser to Asp and Thr to Glu mutations at positions 932, 937, and 945 (3P) and also at 946 and 949 (5P) were expressed as glutathione S-transferase fusion proteins in pGEX-6P2 (Amersham Biosciences). Purification was accomplished using glutathione-Sepharose 4B resin equilibrated with 50 mm Tris-HCl, 150 mm NaCl, pH 7.4, and phenylmethylsulfonyl fluoride (0.3 mg/ml). The bound glutathione S-transferase fusion proteins were cleaved with PreScission protease (200 μg of protein, 8 units of enzyme, 24 h, 4 °C in 50 mm NaH2PO4, 150 mm NaCl, 1 mm dithiothreitol, 1 mm EDTA). When expressed as a glutathione S-transferase-PAM-CD fusion protein, purification yielded intact PAM-CD. Circular dichroism spectra were recorded at 20 °C using a Jasco J-715 spectropolarimeter (Jasco, Easton, MD). Purified PAM-CD* (20 μm) in 1 mm sodium phosphate, pH 7.4, was used. Spectra were the average of 10 scans acquired using a scan rate of 20 nm/min and a response time of 8 s. The background signal of the buffer was subtracted from the spectra. Protein concentration was determined by measuring absorbance at 280 nm. Stably transfected AtT-20 cells expressing PAM-1 (22Milgram S.L. Johnson R.C. Mains R.E. J. Cell Biol. 1992; 117: 717-728Crossref PubMed Scopus (103) Google Scholar), PAM-1/899 (10Milgram S.L. Mains R.E. Eipper B.A. J. Cell Biol. 1993; 121: 23-36Crossref PubMed Scopus (74) Google Scholar), PAM-1/TS/DD (12Steveson T.C. Zhao G.C. Keutmann H.T. Mains R.E. Eipper B.A. J. Biol. Chem. 2001; 276: 40326-40337Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar), PAM-1/S937D (13Steveson T.C. Keutmann H.T. Mains R.E. Eipper B.A. J. Biol. Chem. 1999; 274: 21128-21138Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar), or Myc-TMD-CD (23El Meskini R. Galano G.J. Marx R. Mains R.E. Eipper B.A. J. Biol. Chem. 2001; 276: 3384-3393Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar) were grown in Dulbecco's modified Eagle's medium/F-12 with 25 mm Hepes, 10% NuSerum, 10% fetal bovine serum, 0.5 mg/ml G418, 100 units/ml penicillin, and 100 μg/ml streptomycin. Cells were passaged weekly and maintained for less than 20 passages, since changes in phosphorylation pattern and a loss of stimulated secretion were observed in cells maintained in culture for longer times. Anterior pituitary cultures were prepared from ex-pregnant rats as described (15Ferraro F. Eipper B.A. Mains R.E. J. Biol. Chem. 2005; 280: 25424-25435Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar). Plastic dishes (12-well) were coated with protamine and NuSerum. Dissociated cells (1 pituitary/well) were plated in Dulbecco's modified Eagle's medium/F-12 (Invitrogen or Mediatech) containing 25 mm HEPES, 10% NuSerum, 10% fetal bovine serum, penicillin/streptomycin, and 10 μm cytosine arabinoside. The next day, the serum-containing medium was replaced with complete serum-free medium (Dulbecco's modified Eagle's medium/F-12 (1:1), 25 mm HEPES, pH 7.4, penicillin/streptomycin, insulin/transferrin/selenium, 1 mg/ml fatty acid-free bovine serum albumin) and cytosine arabinoside (10 μm) and maintained in this medium until harvesting. Cells were maintained for a total of 3 days in vitro. PAM-1 AtT-20 cells and anterior pituitary cultures were rinsed with complete serum-free medium prior to stimulation with complete serum-free medium containing 1 μm phorbol myristate acetate along with 12.5 nm calyculin A (Sigma) for 1 h and, when indicated, 2 μm MG132 (Sigma) for 3 h. Myc-TMD-CD cells were also treated with 0.5 mm cyclic 8-bromo-AMP for 1 h. Cells were extracted with 20 mm NaTES, 10 mm mannitol (TM; pH 7.4), 50 mm NaF, 5 mm EDTA at 4 °C containing protease inhibitors (0.3 mg/ml phenylmethylsulfonyl fluoride, 50 μg/ml lima bean trypsin inhibitor, 2 μg/ml leupeptin, 16 μg/ml benzamidine, and 2 μg/ml pepstatin), 200 μm sodium orthovanadate, and 12.5 nm calyculin A. Extracts were passed 10 times through a 25-gauge needle and centrifuged at 1000 × g for 5 min to remove cell debris and nuclei. Membranes were prepared by centrifugation at 430,000 × g for 15 min. Pellets were resuspended in TM with 1% Triton X-100 (Pierce SurfActs), pH 7.4 (TMT), allowed to tumble at 4 °C for 30 min, and clarified by centrifugation at 10,000 × g for 10 min. Protein concentrations were determined using the bicinchoninic acid assay with bovine serum albumin as the standard (Pierce), and samples were subjected to SDS-PAGE and Western blot analysis (15Ferraro F. Eipper B.A. Mains R.E. J. Biol. Chem. 2005; 280: 25424-25435Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar). Where indicated, membrane protein extracts (150 μg) were treated with calf intestinal alkaline phosphatase (CIP; 30 units; New England BioLabs) at 37 °C for 20 min in 0.5% TMT, 10 mm MgCl2 with protease inhibitors. Immunoprecipitated PAM-1 prepared from AtT-20 extracts received a second addition of CIP and an additional 20-min incubation. Solubilized membrane proteins were precipitated with two volumes of 100% ethanol at −20 °C overnight. The protein pellet obtained by centrifugation at 10,000 × g for 30 min at 4 °C was resuspended in 10 μl of 2% ASB-14 in TM (Sigma) and sonicated. Rehydration buffer (7 m urea, 2 m thiourea, 2% ASB-14, 0.5% ampholytes 4–7 (Invitrogen), 100 mm dithiothreitol, 0.001% bromphenol blue) was added and mixed for 1 h at room temperature. The sample was loaded into Zoom IPGRunner cassettes (Invitrogen), and an ImmobilineTM Dry Strip pH 4–7 isoelectric focusing gel (IPG) (Amersham Biosciences) was placed into the well. Rehydration of IPG strips was carried out overnight, after which the strips were transferred to IPGPhor coffins. Isoelectric focusing was carried out at 20 °C using an IPGPhor electrophoresis unit (Amersham Biosciences) as follows: 200 V, 30 min; 500 V, 30 min; 1000 V, 30 min; ramp to 4000 V over 30 min; 4000 V for 12,000 V-h). After focusing, the IPG strip was incubated for 15 min with 1% dithiothreitol in equilibration buffer (6 m urea, 50 mm Tris, pH 8.8, 30% glycerol, 2% SDS), followed by a 15-min alkylation with 2% iodoacetamide in equilibration buffer. IPG strips were then placed into the IPG wells of 4–20% Zoom gels (Invitrogen) and sealed in place with 0.5% agarose with bromphenol blue to be fractionated. After fractionation by SDS-PAGE, proteins were electroblotted to polyvinylidene difluoride membranes (Millipore). Antigen-antibody complexes were visualized using horseradish peroxidase-conjugated secondary antibody and Super Signal West Pico chemiluminescence substrate (Pierce). To reprobe with a different antibody, membranes were stripped by incubation at 50 °C for 30 min in 62.5 mm Tris-HCl, pH 6.7, 2% SDS containing 0.1 m 2-mercaptoethanol. PAM-1 was visualized using a rabbit polyclonal antibody (JH629) to Exon A (rPAM-1-(394–498)); this antibody recognizes both PHM and PAL produced from PAM-1 (11Yun H.Y. Milgram S.L. Keutmann H.T. Eipper B.A. J. Biol. Chem. 1995; 270: 30075-30083Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar). Rabbit polyclonal antisera to phospho-Ser949 (JH 2541) (12Steveson T.C. Zhao G.C. Keutmann H.T. Mains R.E. Eipper B.A. J. Biol. Chem. 2001; 276: 40326-40337Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar) and phospho-Ser937 (JH1922) (13Steveson T.C. Keutmann H.T. Mains R.E. Eipper B.A. J. Biol. Chem. 1999; 274: 21128-21138Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar) were used to detect PAM-CD phosphorylated at these residues. A polyclonal antibody (C-stop) to the final 12 residues of PAM was raised by immunizing rabbits with the Cys-extended peptide (Cys-Y965SAPLPKPAPSS976) conjugated to keyhole limpet hemocyanin (Covance); following ammonium sulfate precipitation and dialysis, C-stop antibody was affinity-purified using AffiGel 10 (Bio-Rad) resin conjugated to peptide antigen. Actin was immunoblotted with a monoclonal antibody (JLA20) from the Developmental Studies Hybridoma Bank (University of Iowa). Polyclonal antibody to histone H3 (Cell Signaling Technology, Inc.) was used to identify nuclear fractions. Immunoblots of two-dimensional gels were analyzed using ImageJ software (National Institutes of Health). Where indicated, membranes were cut above the 25 kDa marker; the two pieces were incubated separately with the same stock of C-stop and second antibody to eliminate any possibility of competition for limited amounts of antibody and to allow the use of different exposure times. Membrane extracts prepared from AtT-20 cell lines overexpressing PAM-1 or Myc-TMD-CD were immunoprecipitated with Exon A or C-stop polyclonal antibody using Protein A beads (200 μg of membrane protein). Bound proteins were eluted by boiling in Laemmli sample buffer and separated on 4–20% Tris-glycine gels (Invitrogen). Gels were stained with Coomassie Brilliant Blue R250 or GelCode Blue (Pierce), and the band of interest was excised for mass spectrometric analysis. Gel pieces were processed for in situ enzymatic digestion with endoproteinase Lys-C or chymotrypsin (24Stone K.L. Williams K.R. Curr. Protocols Protein Sci. 2004; : 11.3Google Scholar), followed by separation into flow-through and titanium dioxide-bound fractions. TiO2 Top Tips (Glygen Corp.) were washed with 40 μl of 100% acetonitrile, followed by 0.2 m sodium phosphate, pH 7.0, and then by 0.5% trifluoroacetic acid, 50% acetonitrile. The acidified digest was spun onto the TopTip (1000 rpm for 1 min and then 3000 rpm for 2 min) prior to a 0.5% trifluoroacetic acid, 50% acetonitrile (40 μl) wash to generate the flow-through fraction. Phosphopeptides were eluted using 3 × 30 μl of 28% NH4OH. Both fractions were dried prior to dissolving in 3 μl of 70% formic acid and dilution with 7 μl of 0.1% trifluoroacetic acid. Liquid chromatography-MS/MS analysis (5 μl injected) was performed on a Thermo Scientific LTQ-Orbitrap XL equipped with a Waters nanoAcquity (25Stone K.L. Crawford M. McMurray W. Williams N. Williams K.R. Methods Mol. Biol. 2007; 386: 57-77PubMed Google Scholar). MS/MS and MS3 spectra were obtained utilizing neutral loss (32.7 triply, 49 doubly, and 98 singly charged). Spectra were analyzed using the Mascot algorithm (26Hirosawa M. Hoshida M. Ishikawa M. Toya T. Comput. Appl. Biosci. 1993; 9: 161-167PubMed Google Scholar) along with manual verification of the sequence. Cardiomyocyte and pituitary nuclei were purified from tissue taken from adult female Sprague-Dawley rats (27Boheler K.R. Chassagne C. Martin X. Wisnewsky C. Schwartz K. J. Biol. Chem. 1992; 267: 12979-12985Abstract Full Text PDF PubMed Google Scholar). Tissue rinsed with L-15 medium was suspended in 10–15 volumes of MA buffer (10 mm Tris-HCl, pH 7.4, 2 mm MgCl2, 0.25 m sucrose) with phenylmethylsulfonyl fluoride and protease inhibitors. Tissue was homogenized for 10–12 s with a Polytron homogenizer at setting 4 (homogenate), followed by centrifugation at 1000 × g for 10 min. The pellet was resuspended in 10 volumes of MA buffer, further homogenized with a Teflon homogenizer (6 strokes), and filtered through a nylon membrane (pore size 70 μm); the supernatant (S1) was retained for analysis. The filtrate was centrifuged at 1000 × g for 10 min, and the pellet was resuspended in MA buffer containing 0.5% Triton X-100; the supernatant (S2) was retained for analysis. After centrifugation as above, the pellet was resuspended in 800 μl of MB buffer (10 mm Tris-HCl, pH 7.4, 2 mm MgCl2, 2.2 m sucrose) with phenylmethylsulfonyl fluoride and protease inhibitor mixture. Resuspended nuclei (gradient input) were layered onto a 1.4-ml layer of 2.4 m sucrose in MB buffer and centrifuged for 2 h at 86,000 × gavg in a TLS55 rotor in a Beckman TL-100 ultracentrifuge. All but 200 μl of buffer covering the white pellet at the bottom of the tube was removed; the remainder was diluted with 4 volumes of MA buffer so that nuclei could be pelleted by centrifugation at 2000 × g for 20 min. The supernatant was discarded, and the white nuclear pellet was resuspended in 40 μl of MA buffer. Freshly isolated nuclei were fixed in 3.7% (w/v) formaldehyde in PBS (50 mm sodium phosphate, 150 mm NaCl, pH 7.4) for 30 min at 4 °C, and aliquots were placed onto polylysine-coated coverslips as described (28Yao I. Iida J. Nishimura W. Hata Y. J. Neurosci. 2002; 22: 5354-5364Crossref PubMed Google Scholar). After rinsing with PBS, nuclei were permeabilized with 0.075% Triton X-100 in PBS for 20 min and blocked with 2 mg/ml bovine serum albumin in PBS for 1 h at room temperature. Fixed nuclei were incubated with affinity-purified C-stop antibody (1:200) diluted in blocking buffer overnight. Specificity was confirmed by preblocking the antibody with antigenic peptide (10 μg/ml) for 1 h at room temperature prior to immunostaining. Following extensive rinsing with PBS, nuclei were incubated in Cy3-conjugated second antibody (Jackson ImmunoResearch, West Grove, PA) for 1 h at room temperature in the dark. TOPRO3 (1:500 in PBS for 30 min) was used to identify nuclei. Coverslips were mounted on slides using Prolong Gold anti-fade reagent (Molecular Probes). Confocal microscopy was performed in the Center for Cell Analysis and Modeling using a Zeiss LSM510 microscope (Thornwood, NY). The cytosolic domain of PAM, although not as well conserved as its catalytic cores, is more highly conserved than its signal sequence or transmembrane domain (29Eipper B.A. Stoffers D.A. Mains R.E. Annu. Rev. Neurosci. 1992; 15: 57-85Crossref PubMed Scopus (562) Google Scholar). Basic residues predominate near the transmembrane domain, with the K893KSR sequence meeting the criteria for a monopartite nuclear localization signal (30Lange A. Mills R.E. Lange C.J. Stewart M. Devine S.E. Corbett A.H. J. Biol. Chem. 2007; 282: 5101-5105Abstract Full Text Full Text PDF PubMed Scopus (861) Google Scholar); acidic residues are prevalent near the C terminus (Fig. 1A). Of the 15 Ser/Thr residues in rat PAM-CD, Disphos (Disorder-enhanced Phosphorylation Predictor) (available on the World Wide Web) identifies 11 as likely phosphorylation sites (probability score of >0.5) (Fig. 1A). The five sites previously identified by in vitro phosphorylation of recombinant PAM-CD using purified protein kinase C, protein kinase A, casein kinase 2, and P-CIP2 bring the total number of sites to be considered to 13 (11Yun H.Y. Milgram S.L. Keutmann H.T. Eipper B.A. J. Biol. Chem. 1995; 270: 30075-30083Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar, 31Caldwell B.D. Darlington D.N. Penzes P. Johnson R.C. Eipper B.A. Mains R.E. J. Biol. Chem. 1999; 274: 34646-34656Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). Mean hydrophobicity and mean net charge can be used to predict regions expected to have a natively unfolded structure (32Uversky V.N. Gillespie J.R. Fink A.L. Proteins. 2000; 41: 415-427Crossref PubMed Scopus (1771) Google Scholar). When applied to PAM, the catalytic cores (PHM and PAL), along with its single transmembrane domain, fell into the folded category (Fig. 1B). In contrast, both PAM-CD and the region between PHM and PAL (Exon A) fell into the natively unfolded category. IUPRED, a Web server that predicts intrinsically unstructured regions in proteins, also predicts PAM-CD to be intrinsically unstructured (33Dosztányi Z. Csizmok V. Tompa P. Simon I. Bioinformatics. 2005; 21: 3433-3434Crossref PubMed Scopus (1596) Google Scholar). Recombinant PAM-CD purified from bacterial lysates was used to test this prediction. Consistent with the protease sensitivity expected of natively unfolded proteins, MALDI-TOF analysis indicated that PAM-CD purified from bacteria expressing rat PAM-1-(898–976) terminated at Ser961. Antibody directed to the C terminus of PAM (-Ser976) confirmed the absence of this epitope, and this C-terminally truncated fragment of PAM-CD is here referred to as PAM-CD*. The secondary structure of purified PAM-CD* was analyzed by circular dichroism spectroscopy (Fig. 1C). The spectrum obtained, with its negative peak at 200 nm, indicated that PAM-CD* was largely a random coil at pH 7.4. NMR studies of purified 15N-labeled PAM-CD were consistent with this conclusion (data not shown). Antisera specific for the protein kinase C site in PAM-CD, Ser(P)937, and for the P-CIP2 or Casein Kinase 2 site, Ser(P)949, were used to compare PAM phosphorylation in AtT-20 corticotrope tumor cells and primary pituitary cells. In both systems, stimulated exocytosis evoked by treatment with the secretagogue phorbol myristate acetate (phorbol ester) was necessary before phosphorylation of Ser937 was detectable (Fig. 2, A and B). Although calyculin A alone was without effect, inclusion of this inhibitor of protein phosphatases 1 and 2A in phorbol ester-stimulated cultures increased levels of Ser(P)937 in PAM-1 AtT-20 cells and in primary cultures. In primary pituitary cells, Ser(P)937 was detected in PAM-1, PAM-2 (an alternatively spliced isoform that lacks exon A), and PALm (Fig. 2B, right). Although inclusion of a proteasome inhibitor (MG132) increased Ser(P)937 levels in AtT-20 cells, it did not do so in pituitary cells. When the same samples were examined using antibody specific for Ser(P)949, a site phosphorylated by P-CIP2 and by casein kinase 2, basal phosphorylation was apparent in both AtT-20 cells and primary pituitary cells (Fig. 2). Neither stimulation with phorbol ester nor the addition of calyculin A had any effect on the level of phosphorylation at this site. Although PAM-2 is more prevalent than PAM-1 in rat pituitary, it was barely detected by the Ser(P)949-specific antibody, revealing the occurrence of isoform-specific phosphorylation. This simple analysis of factors affecting phosphorylation at these two sites revealed rapid turnover and site-specific regulation, validating the use of stably transfected AtT-20 cells as a model for pituitary endocrine cells. To identify phosphorylation sites in PAM, membranes were prepared from AtT-20 cell lines stably expressing PAM-1 or its Myc-tagged transmembrane/cytosolic domain (Myc-TMD-CD). To facilitate i
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