Phosphorylation of Ornithine Decarboxylase at Both Serine and Threonine Residues in the ODC-Overproducing, Abelson Virus-Transformed RAW264 Cell Line'

[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 85â€” 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 4Â°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 37Â°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 â€œthatincells were harvestedinto bufferA [1 mMdithiothreitol,0.5%(v/v) Brij35, 25 mM ODC is phosphorylated at a single location (serine 303)â€•(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 4Â°C.Pansorbin(Calbiochem,Laiolla, CA) was addedat a dilution of 20 @.d/mlandincubated for 20 min at 4Â°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 4Â°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

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 110Â°C.Onemg/mi of phosphoserine, phosphothreonine, and expression from 3â€”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 100â€”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 60Â°Cfor10 mm, sprayed with 0.25% ninhydrin/acetone, and To determine whether the overexpression of ODC in RAW264 cells baked at 60Â°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 to S h, and then progres in RAW264 cell ODC activity when added alone. When all of the sively declined in parallel to the changes in enzyme activity. At 3 h, stimulants were combined, a synergistic induction of ODC occurred. the RAW264 cell ODC enzyme protein was 200 ng/mg cell protein, When these combined stimulants were added to serum-starved a level iOOâ€”i000-fold greater than that present in stimulated normal RAW264 cells, the cell tine overexpressed ODC activity in a regu tissues in which ODC constitutes 1â€”10ppmof the cellular protein (1). lated manner, as shown in Fig. IA. In serum-starved cells and after The ODC induced in the RAW264 cells at 3 h had a calculated 1 h of stimulation, no ODC activity was detected. After 2 h, enzyme specific activity of 20 @.tm/min/mgenzyme protein, consistent with the specific activity of ODC purified from these cells. Multisite Phosphorylation of ODC in the RAW264 Cell Line. A RAW264 cells were metabolically radiolabeled in the presence of [32P]orthophosphate for 30 mm prior to harvesting at different times after stimulation. Cell extracts were incubated with anti-ODC anti serum, and the immunoprecipitates were analyzed by SDS-PAGE and II: autoradiography. As shown in Fig. iC, no immunoprecipitable, ra @ 40 diolabeled ODC was present in RAW264 cells at zero-time when no enzyme activity was detectable. At 3 h, a radiolabeled band was detected which migrated coincident with ODC at Mr 52,000. The amount of radiolabeled ODC remained elevated to 5 h and then 2 4 S S @ 1@. declined. The incorporation of radiolabeled phosphate into ODC occurred throughout the time of ODC expression. S ODC was purified from stimulated RAW264 cells. The purified @7â€” enzyme was separated by two-dimensional gel electrophoresis and detected by silver staining. As shown in Fig. 2et, the purified enzyme @--.,.-- preparation contained two major and one minor isoelectric forms. To determine if all the isoelectric forms of ODC were reactive with the 30- anti-ODC antiserum, a companion gel was electrophoretically trans

01234507 S COC ferred to nitrocellulose and immunoblotted, as shown in Fig. 28. The at anti-ODC antiserum reacted with all three isoelectric forms of the C enzyme present in the purified preparation. To determine whether all of the isoelectric forms of ODC contained phosphate, RAW264 cells, @ 97- - which had been stimulated for 4 h, were metabolically radiolabeled @ 50- â€¢ for 30 min in the presence of [32P]orthophosphate and then immuno precipitated and analyzed by two dimensional electrophoresis. Fig. 46- 2C,animmunoblotofthatgel,showsthatthethreeisoelectricforms

@ 30- of ODC that were present in the purified enzyme preparation were also present in and immunoprecipitable from RAW264 cell extracts. @ 0 1 3 5 7 9 Fig. 2D is the autoradiograph of the blot shown in Fig. 2C, and the at inset, 2E, is an alignment of the sections of the blot and autoradio Fig. 1. Temporalpatternof alterationsinODC activity,enzymeproteinandphosphor ylatedenzymeproteinafter stimulationof RAW264cells.Cellswere serum-starved graph where ODC was detected. No radioactivity was associated with overnight and stimulated at zero-time; cellular extracts were prepared and analyzed as the major basic isoelectric form of ODC, whereas the major and minor described in â€œMaterialsandMethods.â€•A,cells were harvested and assayed for ODC acidic isoelectric forms of the enzyme were radioactive. Multiple activity.TheODCactivityateachtimepointisexpressedasa percentageofthemaximal activity achieved at 3 h of 4 nmol/min/mg cell protein. B, cells were harvested at zero-time isoelectric forms of ODC have been observed previously by two and hourly intervals after stimulation, separated by SDS-PAGE, transferred to nitrocel dimensional electrophoresis, protein staining of enzyme preparations lulose, and immunoblotted to determine ODC protein content. A purified ODC standard was also loaded on the gel; the lower molecular weight immunoreactive bands were ODC purified from kidney (iS) and epidermal papillomas (16), higli-reso degradationproductsgeneratedduringstorageof the purifiedenzyme.C, cells were lution slab gel isoelectricfocusing analysis, and immunoblotting of metabolically radiolabeled with [32Pjorthophosphate for 30 mm prior to preparation of stimulated RAW264 whole cell extracts (17). This is the first direct cell extracts for immunoprecipitation of ODC protein. Immunoprecipitates were separated by SDS-PAGE, and the radioactive protein immunoprecipitated by the anti-ODC anti demonstration that ODC can be present intracellularly in both unphos serum was detected by autoradiography. phorylated and phosphorylated states. 3968

SERINEANDTHREONINEPHOSPHORYLATIONOFODC

A C within the range of normal tissue expression (19). ODC expression in . this system is also solely dependent on the activation of the c-Myc

@ @_@4 chimeric protein and does not require activation by extracellular I@. I regulatorystimulants,asdoestheoverexpressionofODCinRAW264 @@ cells. ODC exists within the RAW264 cell in an unphosphorylatedform and at least 2 phosphorylated forms. This report demonstrates for the first time that ODC can be phosphorylated on both serine and threo nine residues in situ. It has recently been reported that ODC is phosphorylated intracellularly exclusively on serine residues by ca B Ct. sein kinase II (4, 5). There are several potential explanations for the different results reported here: (a) Abelson virus-transformed, murine macrophage-derived RAW264 cells were used in the present report, whereas murine myeloma 653-1 (4) or EXOD-1 (5) cells were used in the previous studies. Different cell lines and tissues may inherently display unique phosphorylation patterns of ODC; (b) cells may ac quire unique ODC phosphorylation patterns after transformation with different oncogenes; (c) in the previous reports, the cells were grown in the presence of FBS alone, whereas in this report, cells were also stimulated with cAMP, IBMX, and LPS. These stimulants may lead to Fig.2.Two-dimensionalelectrophoreticanalysisofpurifiedRAW264cellODCandof ODC immunoprecipitatedfrom stimulated cells metabolicallyradiolabeledwith activation of phosphorylation pathways for which ODC is a substrate [3@PJorthophosphate. A, silver-stained gel of purified ODC. B, immunoblot of gel of which are not activated by FBS; and (d) in the previous reports, acid purifiedODC.C, immunoblotofgelof ODCimmunoprecipitatedfrom[32P]orthophos hydrolysis of metabolically radiolabeled ODC was conducted for just phate metabolically radiolabeled RAW264 cells. D, autoradiograph ofthe gel shown in C. E, alignment of ODC containing areasof the immunoblot and the autoradiographof the a single time, 2 h. Thus, the selection of only a single, relatively short gel shownin (C) and(D). hydrolysis time could obscure the detection of radiolabeled phospho threonine (18). To determine whether the in situ incorporation of 32Pinto ODC was the result of phosphorylation of amino acids, cells were stimulated, metabolically radiolabeled with [32Pjorthophosphate at 3 h, immuno precipitated, separated by SDS-PAGE, and electrophoretically trans A. ferred to Immobion-P. Radiolabeled ODC bands were excised and subjected to acid hydrolysis; then the phosphoamino acids were separated by two-dimensional high voltage electrophoresis. Fig. 3A shows the separation achieved between the ninhydrin-stained internal phosphoserine, phosphothreonine, and phosphotyrosine standards. Fig. 3B shows the Beta-Scan image of the radiolabeled phosphoamino acids present after 2 h of acid hydrolysis of the ODC, a time optimal x for the detection of phosphoserine (18). Phosphoserine was the major radiolabeled amino acid detected, and phosphothreonine was also 5, present. Fig. 3C shows the Beta-Scan image of a similar plate on .s which the radiolabeled ODC phosphoamino acids were analyzed â€¢1 after i8 h of acid hydrolysis, a time optimal for the detection of phosphothreonine (i8). Only radiolabeled phosphothreonine was V detected, and it was present in a greater amount than after 2 h of acid hydrolysis. These results are consistent with the fact that the stability to acid hydrolysis of both the peptide bond and the 0 phosphodiester bond is significantly greater for phosphothreonine

versus phosphoserine (18). C. Cell lines which overexpress ODC have been derived by a variety S of strategies, the most common being mutagenesis followed by se â€¢i lection by growth in the presence of inhibitors of ODC activity (1). In these cases, elevated levels of ODC are often constitutively expressed, V and growth factor addition stimulates a 5â€”10-foldincrease in enzyme activity. In contrast, the RAW264 cell line was derived by oncogene transformation. This cell line overexpresses ODC at levels compara x ble to the cell lines described above, but enzyme expression is tightly regulated in that it is temporally discrete in a manner similar to normal cell lines and tissues and it is absolutely dependent upon the addition Fig. 3. Two-dimensional hiaja voltage electrophoretic analysis of RAW264 cell ODC of extracellular growth factors. Expression of an active c-Myc chi radiolabeled amino acids@ODC was immunoprecipitated from stimulated RAW264 cells meric protein has been shown to induce ODC mRNA expression and metabolicallyradiolabeledbyincubationinthepresenceof[32Pjonhophosphate.Amino acid analysis was accomplished as described in â€œMaterialsand Methodsâ€•.A, ninhydrin elevate ODC activity in BALB/c 3D and Rat iA fibroblasts; how stained amino acid standards. ODC-radiolabeled phosphoamino acids after 2 h (B) or ever, the induced ODC activity is not overproduced but increases only 18h (C) of acidhydrolysis. 3969

Since casein kinase H phosphorylates only serine residues on induced destabilization of ornithine decarboxylase (ODC) is mediated by accumula purified RAW264 cell ODC in vitro,4 as also reported for bacterial tion of antizyme in ODC-overproducing variant cells. J. Biol. Chem., 268: 9393â€”9399,1993. expression vector pET-8C-expressed ODC (4), it can be inferred from 6. Kahana,C., andNathans,D. Nucleotidesequenceof murineornithinedecarboxylase the present results that a different protein kinase must be phosphoryl mRNA. Proc. Nail. Acad. Sci. USA, 82: 1673â€”1677,1985. ating the ODC threonine residue(s) in intact RAW264 cells. The 7. Coff@mo,P.,and Chen, E. L Nucleotide sequence of the mouse ornithine decarbox ylase gene. Nucleic Acids Res, 16: 2731â€”2732,1988. phosphorylation of ODC throughout the entire time of enzyme cx 8. Meggio, F., Caldarera, C. M., Guarnier, C., and Pinna, L A. Phosphorylation of rat pression studied indicates that the protein kinases responsible belong heart ornithine decarboxylase by type-2 casein kinase. Biochem. Biophys. Res@ to signal transduction pathways activated throughout the time of cell Commun., 122: 997â€”1004,1984. 9. Tipnis, U. R., and Haddox, M. K. Phosphorylationof ornithinedecarboxylaseby proliferation, such as the cyclin regulated cdc-2 family of protein casein kinase II from RAW264 cells. Cell. Mol. BioL, 36: 275-289, 1990. kinases (20). Current investigation is directed towards identifying the 10. Raschke,W. C., Baird,S., Ralph,P., andNakoinz,L Functionalmacrophagecelllines protein kinases which phosphorylate ODC and elucidating the func transformed by Abelson leukemia virus. Cell, 15: 261â€”267,1978. 11. Taffet,S. M., andHaddox,M. K. Bacteriallipopolysaccharideinductionof ornithine tional significance of the multisite in situ phosphorylation of the decarboxylase in the macrophage-like cell line RAW264: requirement of an inducible enzyme. soluble factor.J. Cell. Physiol., 122: 215â€”220,1985. 12. Seely, J. E., and Pegg, A. E. Omithine decarboxylase (mouse kidney). Methods Acknowledgments Enzymol., 94: 158â€”161,1983. 13. O'Farrell,P. H. High resolutiontwo-dimensionalelectrophoresisof proteins.J. Biol. We thankDr. VictoriaP. Knutsonfor her helpful suggestions duringthe Chem., 250: 4007â€”4021,1975. 14. Greenfleld, A. R. L, Taffet, S. M., and Haddox, M. K. Immunocytochemical course of this study and Ellen Fenley for her preparation of this manuscript. localization of omithine decarboxylase in cultured murine cells. Cell Tissue Rca., 243: 33â€”40,1986. References 15. Persson, L, Seely, J. E., and Pegg, A. E. Investigationof structureand rate of synthesis of ornithine decarboxylase protein in mouse kidney. Biochemistry, 23: I. Pegg, A. E. Polyamine metabolism and its importance in neoplastic growth and as a 3777â€”3783,1984. target for chemotherapy. Cancer Res., 48: 759â€”774,1988. 16. O'Brien,T. G., Madara,T.,Pyle,J.k, andHolmes,M. Ornithinedecarboxylasefrom 2. Reich, D. J., worth, L., and Haddox, M. K. Phosphorylation of ornithine decarbox mouse epidermis and epidermal papillomas: differences in enzymatic properties and ylase in situ. J. Cell Biol., 101: 246a, 1985. 3. Flamigni, F., Marmiroli, S., Meggio, F., Guarnieri, C., and Pinna, L A. Phosphoryl structure. Proc. Nati. Acad. Sci. USA, 83: 9448â€”9452,1986. ation of ornithine decarboxylase in intact erythroleukemia cells. Biochim. Biophys. 17. Reddy, S. G., Cochran, B. J., Worth, L L, Knutson, V. P., and Haddox, M. K. Acts, 1052: 345-347, 1990. High-resolutionslabgelisoelectricfocusing:methodsforquantitativeelectrophoretic 4. Rosenberg-Hasson,Y., Strumpf,D., and Kahana,C. Mouseornithinedecarboxylase transfer and immunodetection as applied to the study of multiple isoelectric forms of is phosphorylated by casein kinase-il at a predominant single location (serine 303). ornithine decarboxylase. Anal. Biochem., 218: 149â€”156,1994. Eur. J. Biochem., 197: 419â€”424, 1991. 18. Duclos, B., Marcandier,S.,andCozzone,A. J. Chemicalpropertiesandseparationof 5. Kanamoto, R., Kameji, T., Iwashita, S., Igarashi, K., and Hayashi, S. Spermidine phosphoamino acids by thin-layer chromatography and/or electrophoresis. Methods Enzymol.,201B:10â€”21,1991. 19. Wagner,A. J., Meyers,C., Laimins,L A., andHay,N. c-Myc inducestheexpression 4S. R. Reddyand M. K. Haddox,RA@264cell ornithinedecarboxylaseisphospho and activity of ornithine decarboxylase. Cell Growth & Differ., 4: 879â€”883,1993. rylated in situ at multiple sites in addition to that phosphorylated by casein kinase II, 20. Wang, J. Y. J. Oncoprotein phosphorylation and cell cycle controL Biochim. Biophys. submitted for publication. Acts, 1114: 179â€”192,1992.

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Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1994 American Association for Cancer Research. 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 Mari K. Haddox

Cancer Res 1994;54:3967-3970.

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