Translocation of ornithine decarboxylase to the surface membrane during cell activation and transformation
1999; Springer Nature; Volume: 18; Issue: 5 Linguagem: Inglês
10.1093/emboj/18.5.1214
ISSN1460-2075
Autores Tópico(s)Amino Acid Enzymes and Metabolism
ResumoArticle1 March 1999free access Translocation of ornithine decarboxylase to the surface membrane during cell activation and transformation Marja Heiskala Corresponding Author Marja Heiskala Department of Pathology, Haartman Institute, and Helsinki University Hospital, University of Helsinki, Haartmaninkatu 3, FIN-00014 Helsinki, Finland The R.W.Johnson Pharmaceutical Research Institute, 3535 General Atomics Court, Suite 100, San Diego, CA, 92121 USA Search for more papers by this author Jian Zhang Jian Zhang Department of Pathology, Haartman Institute, and Helsinki University Hospital, University of Helsinki, Haartmaninkatu 3, FIN-00014 Helsinki, Finland Search for more papers by this author Shin-ichi Hayashi Shin-ichi Hayashi Department of Biochemistry II, The Jikei University School of Medicine, Minato-ku, Tokyo, 105 Japan Search for more papers by this author Erkki Hölttä Erkki Hölttä Department of Pathology, Haartman Institute, and Helsinki University Hospital, University of Helsinki, Haartmaninkatu 3, FIN-00014 Helsinki, Finland Search for more papers by this author Leif C. Andersson Leif C. Andersson Department of Pathology, Haartman Institute, and Helsinki University Hospital, University of Helsinki, Haartmaninkatu 3, FIN-00014 Helsinki, Finland Institute for Oncology and Pathology, Karolinska Institute, S-171 76 Stockholm, Sweden Search for more papers by this author Marja Heiskala Corresponding Author Marja Heiskala Department of Pathology, Haartman Institute, and Helsinki University Hospital, University of Helsinki, Haartmaninkatu 3, FIN-00014 Helsinki, Finland The R.W.Johnson Pharmaceutical Research Institute, 3535 General Atomics Court, Suite 100, San Diego, CA, 92121 USA Search for more papers by this author Jian Zhang Jian Zhang Department of Pathology, Haartman Institute, and Helsinki University Hospital, University of Helsinki, Haartmaninkatu 3, FIN-00014 Helsinki, Finland Search for more papers by this author Shin-ichi Hayashi Shin-ichi Hayashi Department of Biochemistry II, The Jikei University School of Medicine, Minato-ku, Tokyo, 105 Japan Search for more papers by this author Erkki Hölttä Erkki Hölttä Department of Pathology, Haartman Institute, and Helsinki University Hospital, University of Helsinki, Haartmaninkatu 3, FIN-00014 Helsinki, Finland Search for more papers by this author Leif C. Andersson Leif C. Andersson Department of Pathology, Haartman Institute, and Helsinki University Hospital, University of Helsinki, Haartmaninkatu 3, FIN-00014 Helsinki, Finland Institute for Oncology and Pathology, Karolinska Institute, S-171 76 Stockholm, Sweden Search for more papers by this author Author Information Marja Heiskala 1,2, Jian Zhang1, Shin-ichi Hayashi3, Erkki Hölttä1 and Leif C. Andersson1,4 1Department of Pathology, Haartman Institute, and Helsinki University Hospital, University of Helsinki, Haartmaninkatu 3, FIN-00014 Helsinki, Finland 2The R.W.Johnson Pharmaceutical Research Institute, 3535 General Atomics Court, Suite 100, San Diego, CA, 92121 USA 3Department of Biochemistry II, The Jikei University School of Medicine, Minato-ku, Tokyo, 105 Japan 4Institute for Oncology and Pathology, Karolinska Institute, S-171 76 Stockholm, Sweden *Corresponding author. E-mail: [email protected] The EMBO Journal (1999)18:1214-1222https://doi.org/10.1093/emboj/18.5.1214 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Ornithine decarboxylase (ODC) is highly up-regulated in proliferating and transforming cells. Here we show that upon induction, an initial cytosolic increase of ODC is followed by translocation of a fraction of the enzyme to the surface membrane. ODC membrane translocation is mediated by a p47phox membrane-targeting motif-related sequence, as indicated by reduced ODC activity in the membrane fraction of cells treated with a competing, ODC-derived (amino acids 165–172) peptide, RLSVKFGA, which is homologous to the p47phox membrane-targeting sequence. p47phox membrane translocation is known to be dependent on the phosphorylation of the targeting motif. Analogously, overexpressed ODC.S167A, a mutant ODC lacking the putative phosphorylation site Ser67, is unable to move to the surface membrane. Cells blocked with the RLSVKFGA peptide showed defective transformation, indicating that the motif-mediated translocation of ODC is prerequisite to its biological function. Constitutive targeting of ODC to the membrane using a plasmid encoding the chimeric protein, wild-type ODC with C-terminal linkage to the farnesylation motif of K-ras, caused impaired cytokinesis with an accumulation of polykaryotic cells. Impaired cytokinesis confirms that ODC is involved in mitotic cytoskeletal rearrangement events and pinpoints the importance of relevant membrane targeting to its physiological function. Introduction Ornithine decarboxylase (ODC) regulates the rate of polyamine synthesis by catalyzing the generation of putrescine and CO2 from ornithine. ODC activity and polyamines are connected intimately to cell proliferation and transformation (Tabor and Tabor, 1984; Pegg, 1986, 1988; Heby and Persson, 1990). Quiescent cells display low ODC activity, whereas mitogenic triggering induces a rapid up-regulation of ODC activity. Moreover, the activity of ODC varies during the cell cycle in normal cells. The activity starts to rise in mid-G1 and peaks in G2 before the onset of mitosis (Fredlund et al., 1995). Transformed cells, on the other hand, display constitutively elevated ODC activities (Pegg, 1988; Auvinen et al., 1992). We reported previously that the elevated ODC activity found in malignant cells is not merely a consequence of cell transformation, but that ODC activity and/or polyamines are involved in the mechanisms that lead to the transformed phenotype. When rat 2R cells infected with a temperature-sensitive (ts) mutant of v-src were treated with the selective ODC inhibitor, difluoromethylornithine (DFMO), and shifted to the permissive temperature in the absence of exogenous polyamines, cell transformation did not occur. Putrescine restored cell transformation at the permissive temperature. Furthermore, overexpression of normal human ODC in NIH 3T3 cells induced malignant transformation, including the ability to grow as colonies in semi-solid medium and to form rapidly progressing tumors in nude mice (Auvinen et al., 1992, 1997; Moshier et al., 1993; Clifford et al., 1995). We have found that ODC regulates the level of tyrosine phosphorylation of the Src substrate p130cas (Auvinen et al., 1995), a phosphoprotein involved in the formation of signaling complexes at the cytoplasmic side of the plasma membrane in focal adhesion sites (Parsons, 1996). Regulation of phosphorylation of p130cas, together with the suggested association of ODC with transmembrane calcium and/or calmodulin signaling (Ginty and Seidel, 1989) and with activation-mediated membrane translocation of protein kinase C (PKC) (Groblewski et al., 1992), point to the plausibility of ODC also exerting activity at the plasma membrane. However, ODC has been regarded as a cytoplasmic protein (Pegg, 1986). Direct visualization of the subcellular location of ODC has been difficult due to its low concentration and short half-life. To determine its localization, we combined double-sandwich immunostaining of ODC and confocal microscopy with measurements of ODC activity in subcellular fractions. Using this combined approach, we observed that a fraction of ODC translocates from the cytosol to the surface membrane during cell activation and transformation. We also identified a p47phox-related peptide motif, containing a putative serine phosphorylation site that is involved in the physiological membrane translocation of ODC. To study the consequences of constitutive binding of ODC to the plasma membrane, we transfected cells with a chimeric ODC plasmid that contained a 3′ sequence encoding the 17 C-terminal amino acids of K-ras, including the membrane-targeting farnesylation motif. Results Localization of ODC in resting and stimulated Rat-1 tsRSVLA29 cells by immunostaining and confocal microscopy Rat-1 tsRSVLA29 cells were grown on glass coverslips at the non-permissive temperature (39.5°C) for 2 days and were then induced to up-regulate ODC activity by treatment with the following stimuli: (i) 100 nM 12-O-tetradecanoyl-phorbol-13-acetate (TPA); (ii) hypotonic (50% volume H2O) medium; (iii) dialyzed human AB serum (15%) after 13 h of serum starvation; or (iv) transfer to permissive temperature (35°C) to induce ts-v-src-driven transformation. At selected time points, the cells were fixed, permeabilized and stained for ODC using double-fluorescein isothiocyanate (FITC) labeling. Cells subjected to the first three stimuli displayed a transient membrane staining (Figure 1) that was not observed in the unstimulated control cells. In Rat-1 tsRSVLA29 cells transformed at the permissive temperature (Figure 1), prolonged membrane staining with anti-ODC antibodies was seen. Figure 1.Fluorescent immunostaining of ODC in quiescent Rat-1 tsRSVLA29 cells (A) and in cells activated by 100 nM TPA for 2 (B), 4 (C) and 24 h (D) is shown. As a control, similarly treated cells were stained using monoclonal anti-CD3 (OKT3) as the primary antibody (E–H). ODC immunostaining in quiescent cells is shown (I), as well as cells exposed to the permissive temperature for 1 (J), 4 (K) and 24 h (L). Arrowheads indicate membrane staining. Bar, 25 μm. Download figure Download PowerPoint To substantiate further the membrane translocation of ODC observed by immunofluorescence, we measured ODC activity in the membrane and soluble fractions of activated and control Rat-1 tsRSVLA29 cells. In cells treated with 100 nM TPA for 2, 4 and 24 h, the increase of ODC activity in the soluble fraction peaked at 2 h, then decreased, reaching the control level at 24 h. For these cells, the membrane fraction showed the highest levels of activity at 4 h (Figure 2A). The kinetics of ODC activity were somewhat slower in the soluble fractions of hypotonia-treated cells, where the peak activity in both soluble and membrane fractions was reached at 4 h (Figure 2B). Similar kinetics of ODC activity were encountered in Rat-1 tsRSVLA29 cells subjected to serum after 13 h of starvation (Figure 2C). When the cells were shifted from 39.5°C to the permissive temperature (35°C), activation of ts-v-src induced ODC activity in the soluble fraction within 2–3 h. In the membrane, ODC activity was rising to 24 h concomitantly with the cells acquiring a transformed phenotype (Figures 2D and 1L). In activated cells, a fraction comprising 10–20% of the total elevated ODC activity displayed membrane translocation. Figure 2.The relative ODC activity/milligram of protein in Rat-1 tsRSVLA29 cell soluble (open bars) and membrane fractions (filled bars), expressed as mean ± SD of the percentage of the activity of the soluble fraction of quiescent control cells indicated by 100% (3–7 experiments/group). The mode of activation was TPA (A), hypotonia (B), 15% serum (C) and permissive temperature (D). Download figure Download PowerPoint The sequence surrounding Ser167, a membrane-targeting motif in ODC The cytosolic proteins p47phox and p67phox are components of the NADPH-dependent leukocyte respiratory burst oxidase. These proteins become phosphorylated on serine residues and translocate to the membrane in activated polymorphonuclear neutrophils (Clark et al., 1990). Treatment of leukocytes with TPA redistributes a proportion of p47phox to the membrane (Tisch et al., 1995). Nauseef et al. (1993) found that a putative serine phosphorylation domain corresponding to residues 323–332 of p47phox (AYRRNSVRFL) is crucial for the membrane translocation of p47phox. It is noticeable that Ser167 of ODC is surrounded by the sequence AVCRLSVKFG, which is homologous to the membrane-targeting motif of p47phox. In order to test the impact of this motif on ODC function, Rat-1 tsRSVLA29 cells were cultured at the non-permissive temperature for 3 h with either (i) 0.2–2 μg/ml of the synthetic peptide RLSVKFGA linked via a proline to an antennapedia (RQIKIWFQNRRMKWKK) peptide to facilitate transmembrane transport (Hall et al., 1996), or (ii) 0.2–2 μg/ml of an antennapedia-linked control peptide (ELFHQLACRECHVPL). After 3 h, cells were transferred to the permissive temperature. Incubation with the relevant peptide greatly delayed ts-v-src-driven transformation as compared with untreated control cells or cells treated with the nonsense peptide (Figure 3). In other experiments, the RLSVKFGA peptide without the antennapedia was used. These experiments required higher concentrations of the peptide (100–400 μg) to produce identical results, probably a result of less efficient cellular uptake. Using the same concentrations of the scrambled control peptide (VARGFLESK) had no effect on the level or compartmentalization of ODC activity. Measuring ODC activities from the peptide-treated Rat-1 tsRSVLA29 cells consistently revealed a dose-dependent suppression of the permissive temperature-induced increase in the membrane-associated ODC activity. This suppression did not result from non-specific toxicity since the cells remained responsive as indicated by an undisturbed increase in the overall activity observed in response to the permissive temperature (Figure 4). Figure 3.Rat-1 tsRSVLA29 cells were cultured at the non-permissive temperature (39.5°C) for 2 days to make them quiescent. They were treated subsequently for 3 h either with the solvent [PBS; (A)], with 1 μg/ml of the RLSVKFGA (ODC Ser167-surrounding octapeptide, homologous to phox47 membrane-targeting motif)–antennapedia peptide chimera (B) or with 1 μg/ml of the control peptide–antennapedia chimera (C), whereafter they were transferred to the permissive temperature (35°C) to induce v-src-driven transformation. The cells were photographed 5 h later. Download figure Download PowerPoint Figure 4.Rat-1 tsRSVLA29 cells were treated with the solvent [PBS; (A) and (B)], with 400 μg/ml of control peptide VARGFLSK (C) or with 100 and 400 μg/ml of ODC Ser167-surrounding peptide RLSVKFGA [(D) and (E), respectively]. After a 3 h pre-treatment with PBS or peptides at the non-permissive temperature (39.5°C), the control cells (A) were maintained at 39.5°C, whereas the other cultures (B–E) were transferred to the permissive temperature (35.0°C) for 5 h before harvesting. The relative ODC activities are expressed as percentage of control (soluble cell fraction of the untreated cells at non-permissive temperature, counts/mg of protein). Open bars indicate ODC activity in the soluble fraction, and filled bars indicate the membrane fraction. (One representative experiment out of three is shown.) Download figure Download PowerPoint Ser167 is required for membrane translocation of ODC cDNAs encoding wild-type (wt) ODC and mutated ODC lacking the putative phosphorylation site of the phox47-related membrane-targeting motif RLSVKFGA (with a substitution of Ser167 by alanine) were amplified by PCR and cloned into the XbaI site of the pCHA expression vector (pCHA-wtODC and pCHA-ODC.S167A). The hemagglutinin (HA)-epitope tag of the pCHA vector enables endogenous ODC to be distinguished from transfected ODC. Neither the HA tag nor the S167A mutation invalidated the catalytic activity of ODC (data not shown). Upon transfection of Rat-1 tsRSVLA29 cells, overexpression of both wtODC-HA and ODC.S167A-HA increased total ODC activity. The proportion of increased ODC activity induced by the permissive temperature was lower in the membrane fraction of cells expressing ODC.S167A-HA than in cells expressing wtODC-HA. This finding was consistent in four independent experiments (Table I). Table 1. Distribution of ODC activity/mg protein in Rat-1 tsRSVLA29 cells after 12 h at the permissive temperature Experiment 1 Experiment 2 m c m cc Wt 4.2 6.1 5.9 11.3 EV ND ND 7.7 14.7 ODC 19.8 38.7 23.0 54.1 ODC.S167A 5.5 56.2 11.8 91.1 Wt, untransfected control Rat-1 tsRSVLA29 cells; EV, cells transfected with the empty vector; ODC, cells overexpressing wtODC; ODC.S176A, cells overexpressing mutant ODC; m, membrane fraction; c, cytosolic fraction; ND, not done. In order to follow the localization of overexpressed HA-tagged ODC visually, the wtODC-HA and ODC.S167A-HA fragments were cloned into the HindIII site of the pLTRpoly vector for higher expression levels. Resting cells transfected with pLTRpoly-wtODC or with pLTRpoly-ODC.S167A displayed mainly a diffuse cytosolic staining; after transfer to the permissive temperature, only cells expressing wtODC-HA showed clear, membrane-associated staining (Figure 5). Figure 5.Rat-1 tsRSVLA29 cells overexpressing ODC.S167A-HA (A) and wtODC-HA (B) were transferred to the permissive temperature for 12 h, fixed and immunostained for ODC-HA. Extensive membrane staining is only seen in cells expressing wtODC. Bar, 25 μm. Download figure Download PowerPoint Expression of wtODC and ODC.C360A containing the K-ras-2 membrane-targeting motif and the HA epitope To investigate the functional role of the membrane translocation of ODC that was observed in activated cells, we designed plasmid constructs to constitutively target the expressed protein to the plasma membrane. In order to test whether the decarboxylation activity of overexpressed ODC is required to induce the transformed phenotype or whether the mere overexpression and/or membrane targeting of the protein is sufficient, Cys360 was mutated to alanine, an alteration known to abolish >90% of the enzymatic activity of ODC (Coleman, 1993). NIH 3T3 cells were transfected with the following plasmids: pNHA, pCHA, pCHA-ODC, pNHA-ODC-K-ras-2 adhesion sequence, pCHA-ODC.C360A and pNHA-ODC.C360A-K-ras-2 adhesion sequence. After G418 selection, the transfected cells were stained for the HA epitope. Positive immunofluorescence was evident in the membranes of all the cells transfected with a plasmid encoding the ODC-K-ras adhesion sequence, whereas morphological changes only occurred when a construct encoding enzymatically active wtODC was used (Figure 6). In early passages, these cells showed a high frequency of polykaryosis (Figures 6 and 7) and could not be recovered for continuous cultures. The cells that remained in prolonged cultures displayed a round, flattened shape with multiple cellular protrusions (Figure 7). Cells transfected with the pNHA-ODC-K-ras-2 plasmid showed a 40-fold elevated ODC activity in the membrane fraction as compared with the control; the cells transfected with the pCHA-ODC plasmid had a 5-fold elevated enzyme activity in both the cytosolic and the membrane fractions (Figure 8). These elevated activities suggest that the high, persistent ODC activity in the plasma membrane was related causally to the morphological changes. Assays for ODC enzyme activity also revealed that the cells transfected with the pNHA-ODC.C360A plasmid did not display increased activity but rather marginally lower ODC activities than seen in control cells, indicating that ODC.C360A may have a dominant-negative impact (Figure 8). Figure 6.NIH 3T3 cells, transfected with empty pCHA (A) and pNHA vectors (D), pCHA-ODC (B), pNHA-ODC-K-ras-2 adhesion tag (C), pCHA-ODC.C360A (E) and pNHA-ODC.C360A-Ki-ras-2 adhesion tag (F), were immunostained using monoclonal anti-HA as the first antibody. Bar, 25 μm. Download figure Download PowerPoint Figure 7.NIH 3T3 cells transfected with pCHA-ODC.C360A (A), pCHA-ODC (B), pNHA-ODC.C360A-K-ras-2 adhesion tag, early passages (C), pNHA-ODC-K-ras-2 adhesion tag, early passages (D), pNHA-ODC.C360A-K-ras-2 adhesion tag, later passages (E) and pNHA-ODC-K-ras-2 adhesion tag, later passages (F). Download figure Download PowerPoint Figure 8.ODC activity in NIH 3T3 cells, transfected with an empty pNHA vector (A), pCHA-ODC (B), pNHA-ODC-Ki-ras-2 adhesion tag (C), pCHA-ODC.C360A (D) and pNHA-ODC.C360A-Ki-ras-2 adhesion tag (E), expressed as a percentage of control (the activity, counts/mg, of the soluble fraction of cells transfected with empty pNHA vector). Open bars indicate ODC activity in the soluble fraction, and filled bars indicate ODC activity in the membrane fraction. (One representative experiment out of three is shown.) Download figure Download PowerPoint These findings demonstrate that ODC-related alterations in cell morphology and growth behavior are dependent on intact ODC enzyme activity. These findings also show that for the physiological function of ODC, membrane targeting using the phosphorylation-regulated, p47phox-related motif is mandatory. Constitutive (irrelevant) membrane targeting of the active enzyme leads to defective cytokinesis. Discussion The cellular level of ODC is strictly feedback controlled. Elevated cellular concentrations of polyamines induce up-regulated expression of antizyme, an ODC-inhibitory protein, that binds ODC and induces its rapid degradation by the 26S proteasome complex independently of ubiquitination (Tokunaga et al., 1994). This degradation leads to a fast turnover of ODC, which is, in fact, one of the most short-lived enzymes in mammalian cells, having a half-life of 10–20 min. Given these features, the cellular concentration of ODC is very low, estimated to comprise 0.0003–0.01% of total cellular proteins (Heby and Persson, 1990). Therefore, visualizing the subcellular distribution of ODC has been difficult. Previously, ODC was regarded mainly as a cytosolic protein (Pegg, 1986). To follow the distribution of ODC in resting and in activated and/or transformed cells, we combined confocal microscopy with the following five-step sandwich immunostaining treatment: (i) monoclonal anti-ODC or anti-HA antibodies; (ii) biotinylated anti-mouse antibodies; (iii) FITC/avidin; (iv) biotinylated anti-avidin; and (v) FITC/avidin. In response to cell activation, regardless of the mode (TPA, hypotonic medium or serum after 13 h of starvation), as well as in response to transformation (induced at permissive temperature by ts-v-src), we consistently found translocation of immunoreactive ODC to the plasma membrane. The overexpressed ODC product was monitored by the monoclonal antibody anti-HA 12CA5 against the HA epitope and was found to display the same membrane location as endogenous ODC upon stimulation. Using core-labeled [3H]ornithine as the substrate, ODC enzyme assays of cell fractions revealed an initial rise in soluble fraction activity followed by translocation of a proportion of the ODC activity to the membrane fraction. These findings demonstrate that ODC behaves analogously to several other signaling molecules (i.e. PKC, GTP-binding proteins of the Rho–Rac family, c-Raf and the SH2 domain-containing Shc) which, upon cell activation, are recruited transiently close to the plasma membrane (Bokoch et al., 1994; Lankester et al., 1994; Stokoe et al., 1994; Matowe and Ginsberg, 1995). When investigating the mechanism of ODC membrane translocation, we found that intact catalytic activity of ODC is not required, since the HA-tagged, C360A-mutated ODC displayed normal membrane association in activated cells (data not shown). Treating Rat-1 tsRSVLA29 cells with an antennapedia sequence-carried peptide, representing the eight amino acids around Ser167 of ODC and having homology to the previously described membrane-targeting motif of p47phox (Nauseef et al., 1993), dramatically delayed ts-v-src-induced transformation and suppressed the permissive temperature-induced increase in ODC activity in the membrane compartment. The concomitant rise in cytosolic ODC remained intact, indicating that the cells were responsive and suggesting that the motif surrounding Ser167 is important for ODC membrane translocation, which, in turn, is crucial for cell transformation. A direct demonstration of phosphorylation of Ser167 is technically difficult due to the low amount of ODC protein present, even after overexpression. Instead, to substantiate further the role of the p47phox-related motif, we mutated Ser167 to alanine. This single amino acid substitution abolished the membrane translocation of overexpressed ODC upon cell activation, indicating that phosphorylation of Ser167 is the prerequisite of ODC membrane translocation and confirming that the phox47-related Ser167-surrounding octamere is involved in ODC membrane association. While v-src-driven transformation induced an extended localization of ODC to the plasma membrane, cells activated with hypotonic medium, TPA or serum displayed a transient rise in membrane-associated ODC activity. To study the impact of constitutively membrane-associated ODC, we constructed plasmids to express chimeric ODC containing the C-terminal farnesylation motif of K-ras. We used vectors with the cytomegalovirus (CMV) promoter, which yields modest levels of protein in rodent cells. Upon transfection into NIH 3T3 cells, these plasmids still had a dramatic impact on cell morphology. At an early phase of G418 selection, multinucleated, large, flattened cells with extended cytoplasmic protrusions were seen. The generation of polykaryosis suggests that constitutive overexpression of ODC at the membrane interferes with cytokinesis. Defective cytokinesis may be caused by constitutively high ODC activity in the membrane and/or may be due to the non-physiological mode of targeting, such as use of the Kras farnesylation motif instead of the p47phox-related phosphorylation-regulated membrane targeting motif, i.e. targeting of ODC to the membrane lipid layer instead of to the membrane cytoskeleton (Woodman et al., 1991; El-Benna et al., 1994). Since reorganization of the membrane actin skeleton is the prerequisite for cytokinesis and acquisition of a transformed phenotype, it appears plausible that the membrane translocation of ODC in activated cells indicates an association with the membrane actin skeleton rather than with the lipid membrane. The ultimate molecular mechanism(s) by which ODC contributes to the regulation of cell transformation remains to be elucidated. The disassembly of the organized cytoskeleton that takes place during malignant transformation apparently implies 'misuse' of the genetic program that physiologically regulates initiation of mitosis. Thus, it is tempting to speculate that the steeply increased ODC activity found in G2 in normal cells and the high ODC activity found in transformed cells represent a similar molecular role for ODC in both events. Indeed, we recently found that the ODC activity in the cell membranes of synchronized Rat-2 fibroblasts is low in S-phase cells, whereas in the membranes of G2- and/or M-phase cells an increase of >10-fold increase is seen (M.Heiskala and L.C.Andersson, in preparation). Understanding the regulation of the obviously essential phosphorylation of the p47phox related membrane targeting motif is important for a comprehensive view of how ODC eventually triggers the cellular machinery, which leads to the reorganization of the cytoskeleton. Phox47 has been reported to be phosphorylated in numerous serines by PKC, PKA and p65pak/PAK2. These kinases probably also act on the 323–332 residue motif, which is crucial for phox67 and membrane binding (Knaus et al., 1995; De Leo et al., 1996; El-Benna et al., 1996). It will be of interest to identify the kinase(s) that phosphorylate ODC. Not only do these kinases play a central role in controlling ODC-mediated cellular events, but they are also potential targets for potential drugs for treating proliferative diseases. Materials and methods Cells and reagents The ts RSV-infected rat fibroblast cell line, Rat-1 tsRSVLA29, was obtained from Dr J.Wyke (Wyke et al., 1980). Before experiments, cells were cultured at the non-permissive temperature (39.5°C) for 48 h in RPMI-1640 medium supplemented with 10% fetal calf serum, glutamine and antibiotics. Cells were grown until 80–90% confluent. NIH 3T3 cells were cultured in Dulbecco's modified Eagle's medium supplemented with 5% newborn calf serum, glutamine and antibiotics. TPA was obtained from Sigma Chemical Co. (St Louis, MO). Plasmid constructions and transfections Human ODC cDNA (Hickok et al., 1987) was used as the template for PCR amplification, and the oligonucleotides 5′-GCTCTAGAAACAACTTTGGTAAT-3′ (5′ primer) and 3′-GCTCTAGAAAGCTTCACATTAATACTA-5′ (3′ primer) were used to amplify cDNA encoding ODC. The ODC cDNA fragment was flanked by XbaI sites at both ends and a HindIII site at the 3′ end proximal from the XbaI site. Following amplification, the PCR product was isolated and cloned into the pCHA vector. The pCHA (C-terminal tagging of the protein product) and pNHA (N-terminal tagging of the protein product) vectors are designed for expressing HA epitope-tagged proteins in mammalian cells. The plasmids are composite constructs derived from the 5.4 kb Rc/CMV expression vector (Invitrogen, San Diego, CA), which retains features such as the CMV promoter, a high-copy ColE1 origin, the SP6 and T7 promoters, the neomycin and ampicillin resistance genes and the M13 origin for mutagenesis (Pati, 1992). The insert was sequenced, and the plasmid was used for transfections. To express ODC containing a membrane-targeting motif, we prepared a cDNA fragment encoding a c-K-ras-derived, C-terminal membrane adhesion tag. The pUC 13/c-Ki-ras-2 (human, ATCC; McCoy et al., 1984) plasmid was used as a template, and the 5′ and 3′ primers,
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