[CANCER RESEARCH 54, 3967-3970, August 1, 1994] Advances in Brief Phosphorylation of Ornithine Decarboxylase at Both Serine and Threonine Residues in the ODC-overproducing, Abelson Virus-transformed RAW264 Cell Line' Laura L Worth, Barbara J. Cochran, and Man K. Haddox@ Department ofPharmacology, The University of Texas Medical Schoo4 Houston, Texas 77225 Abstract ylated and phosphorylated forms. Furthermore, the phosphorylated ODC contains both phosphoserine and phosphothreonine residues, Expression of ornithine decarboxylase (ODC), the initial enzyme in demonstrating that the enzyme can be subject to intracellular multisite polyamlne biosynthesis, is essential for cell growth. The Abelson virus phosphorylation. transformed, murine macrophage-derived RAW264 cell line, overex presses ODC activity and enzyme protein at a level 100-1000-fold greater Materials and Methods than In normal cells. Expression of ODC was completely dependent on extracellular Stimulants and followed a temporally discrete pattern sinai Cell Culture and ODC Purification. RAW264cells (10) weremaintained lax to that in normal cells. ODC was present in RAW264 cells as two in culture in Dulbecco's modified Eagle's medium (GIBCO, Grand Island, m*jor and one minor isoelectric forms. Analysis of ODC isolated from NY) supplementedwith0.11 g/l sodiumpyruvate,3.7 g/l sodiumbicarbonate, [32Pjorthophosphate metabolically radiolabeled cells demonstrated that and0.584 g/l L-glutamine.Cellswere platedat 1 X 10@cells/mlof serum-free the basic isoelectric enzyme form was usphosphorylated, the two more medium,incubatedovernight,and stimulatedby the additionof 10%FBS, 1 addic forms were phosphorylated, and both phosphoserine and phospho mM cyclic AMP, 1 mM IBMX, and 1 @g/mlLPS. Cellular ODC activity was threonine residues were present In the phosphorylated ODC. Therefore, in assayed as described (11). ODC was purified to homogeneity by sequential the RAW264 cell line, ODC Is overexpressed and phosphorylated at DEAE-Sepharose CL-6B (PharmacialLKB, Uppsala, Sweden) and pyridox multiple sites on the enzyme molecule. amine5-phosphateaffinitychromatography(12). Electrophoresis and Immunoblotting, SDS-PAGE was performed using Introduction 8% polyacrylamide gels, and two-dimensional gel electrophoresis was per formedas described(13). Gels were stainedwith silver and visualizedby ODC (EC 4.1.1.17)@ is the initial, inducible enzyme in the poly autoradiographyor transferredto nitrocellulose(Schleicherand Schuell, amine biosynthetic pathway. The polyamines, small aliphatic nitro Keene, NH) or to Immobion-P (Millipore,Bedford,MA). Experimentscon genous bases, fulfill structural and regulatory roles in protein and ductedwith iodinated,purifiedODC documenteda uniformtransferof 85— nucleic acid biosynthesis and function (1). ODC has been shown to be 90%of the enzyme proteinfromthe gel to the membranes.Forimmunoblot phosphorylated in situ by immunoprecipitation of radiolabeled en ting, the nitrocellulose was incubated for 2 h at room temperature in TBS (150 mM NaCl-50 mM Tris-HC1, pH 7.5) containing 5% bovine serum albumin and zyme from 32P@metabolically radiolabeled murine Abelson-virus thenfor 16 h at 4°Cwitha 1:1000dilutionof rabbitanti-ODCantiserum(14), transformed RAW264 cells (2), Friend murine erythroleukemia cells washedwithTBS, andthenincubatedwitha 1:1000dilutionofgoat anti-rabbit (3), murine 653-i myeloma cells and monkey COS cells (4), and a IgG conjugated with horseradish peroxidase (Bio-Rad Laboratories, Hercules, variant mouse FM3A cell line, termed EXOD-i (5). No studies have CA) for 2 h at room temperature.Thebound antibodieswere visualized by examined whether all the cellular ODC exists in a phosphorylated incubation with 4-chloro-l-naphthol and 0.015% H2O2 in 20 mM Tris-HC1, pH state. 7.5, containing 0.5 MNaC1.SDS-PAGE and immunoblotting of 0.5 to 20 ng A consensus phosphorylation sequence for casein kinase II is of pure ODC protein generated a linear increase in laser densitometric-detected conserved in the ODC amino acid sequence around sense 303 (6, 7), color. The amount of ODC protein present in whole cell extracts was quanti and casein kinase II will phosphorylate ODC in vitro (8, 9) with a Km tated by linear regression analysis of the density of the bands detected as as a substrate similar to that reported for other substrates (9). Phos compared to pure ODC standards.Apparentmolecular weight values for proteins were determinedby interpolationon a plot of log M1 versus the phoserine was the only phosphorylated amino acid detectable in the mobiities of ,nethyl-'4C-labeledstandardproteins: phosphorylase B (M1 radiolabeled ODC isolated from the 653-i myeloma (4) and EXOD-i 97,000), bovine serum albumin (Mr 69,000), ovalbumin (M1 46,000), and cell lines (5). Expression vectors for wild-type ODC and for ODC carbonic anhydrase (M1 30,000; Amersham, Arlington Heights, IL). with a serine 303 to alanine mutation were transiently transfected into Metabolic Radiolabeling of Cells and Immunoprecipltation. Thirty min monkey COS cells, which were then metabolically radiolabeled with priorto harvestingstimulatedcells, the mediumwas replacedwith 1 ml of 32Pi and immunoprecipitated with anti-ODC antibody. Only the wild 37°Cphosphate-freesupplemented Dulbecco's modified Eagle's medium con type ODC was phosphorylated, and serine was the only radiolabeled taming 3 mCi of carrier-free [32P]orthophosphate (ICN, Irvine, CA). Cells amino acid detected, leading the authors to conclude “thatincells were harvestedinto bufferA [1 mMdithiothreitol,0.5%(v/v) Brij35, 25 mM ODC is phosphorylated at a single location (serine 303)―(4). In this NsF, 1 mM EDTA, 1 mg/mI bovine serum albumin, 150 m@sNaC1, and 50 mM Tris-HC1,pH 7.5]. Cell lysates were centrifuged at 10,000 X g for 5 mm, and report, we demonstrate that in the ODC-overproducing, Abelson the supernatantswere incubatedwith a 1:500 dilution of rabbitanti-ODC virus-transformed RAW264 cell line, ODC exists in both unphosphor antiserumfor 1 h at 4°C.Pansorbin(Calbiochem,Laiolla, CA) was addedat a dilution of 20 @.d/mlandincubated for 20 min at 4°C;theimmune complexes Received 5/27/94; accepted 6/16/94. wererecoveredby centrifugationat10,000 X g for5 min. Immunoprecipitates Thecostsof publicationofthisarticleweredefrayedinpartbythepaymentofpage were washed threetimes at 4°Cbyresuspensionin bufferA containing2 M charges@Thisarticle must therefore be hereby marked advertisement in accordance with 18 US.C. Section 1734 solely to indicatethis fact. NaG and 5% (v/v) Brij 35 and centrifuged at 10,000 X g for 5 mm; then they I This work was supported by Grant DK44331 from the NIH. were washed three additional times in buffer A. 2 To whom requests for reprints should be addressed, at Department of Pharmacology, Analysis of Radiolabeled Amino Acids. ODC immunoprecipitated from TheUniversityofTexasMedicalSchool,P.O.Box20708,Houston,TX77225. 32P1metabolically radiolabeled RAW264 cells was separated by SDS-PAGE. 3 The abbreviations used are: ODC, ornithine decarboxylase; FBS, fetal bovine serum; IBMX, isobutylmethylxanthine; LPS, lipopolysaccharide; SDS-PAGE, sodium dodecyl Proteins were electrophoretically transferred to an Immoblin-P membrane, sulfate-polyacrylamidegelelectrophoresis. which was immunoblotted to localize ODC as referenced to samples of pure 3967 Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1994 American Association for Cancer Research. SERINE AND ThREONINE PHOSPHORYLATIONOF ODC ODC.The radiolabeledODCbandwas excised, placedin 6 NHC1,andheated activity was detectable and continued to rise to a peak level of for 2 or 18 h at 110°C.Onemg/mi of phosphoserine, phosphothreonine, and expression from 3—5h.The enzyme activity then declined to 20% of phosphotyrosine were added to the hydrolysates before lyophilization. Ly the maximal level by 8 h. This temporal pattern of stimulated ODC ophilized samples were resuspended in 5 @lof 5.0% acetic acid-7.5% activity expression is typical of that seen in normal tissues and cell formic acid (pH 1.9) and spotted onto a thin-layer chromatography plate lines. However, the peak activity of ODC achieved at 3 h, 4 nmol/ which was run in the same buffer at 1000 V for 1 h. The thin-layer mm/mg cell protein, was 100—1000-foldgreater than that achieved in chromatographyplates were air dried, wetted with buffer containing 5.0% stimulated normal tissues (1). acetic acid-0.5% pyridine (jH 3.5), and run at 1100 V for 45 mm. Plates were dried at 60°Cfor10 mm, sprayed with 0.25% ninhydrin/acetone, and To determine whether the overexpression of ODC in RAW264 cells baked at 60°Cfor 10 mm to detect the phosphoamino acid standards and was the result of an overexpression of enzyme protein, whole cell scanned using a Betascope to detect the radiolabeled phosphoamino acids. extracts were harvested at hourly intervals after stimulation, separated by SDS-PAGE, and immunoblotted. As shown in Fig. 18, the poly Results and Discussion clonal anti-ODC antiserum was monospecific; no protein was de Regulation of Overexpression of ODC in the RAW264 Cell tected at zero-time in serum-starved cells, and only one protein was Line. The RAW264 cell line was derived from murine macrophages detected in stimulated cells which migrated coincident with authentic, by transformation with the Abelson virus (10). Each of the cell purified ODC at Mr 52,000. After cell stimulation, ODC protein was stimulants used, FBS, cAMP plus IBMX, or LPS, induced an increase first detected at 2 h, reached a peak from 3