Biochimica et Biophysica Acta,
1052 (1990) 345-347
345
Elsevier
B B A Report
BBAMCR 10256
Phosphorylation of ornithine decarboxylase in intact erythroleukemia cells Flavio Flamigni
1, S a n d r a
M a r m i r o l i 2, F l a v i o M e g g i o a n d L o r e n z o A. P i n n a 3
3, C a r l o
Guarnieri 1
Dipartimento di Biochimica, Universit& di Bologna, e Istituto di Citomorfologia, CNR, c / o lstituto Rizzoli, Bologna and ~ Dipartimento di Chimica Biologica, Unioersit6 di Padova, Padova (Italy)
(Received 10 October 1989) (Revised manuscript received29 December1989)
Key words: Ornithinedecarboxylase;Protein phosphorylation; (Erythroleukemiacell)
32p-labeled ornithine decarboxylase was isolated by immunoprecipitation from murine erythroleukemia cells incubated in a medium containing [32p]ortophosphoric acid. Analysis of immunoprecipitate by SDS-polyacrylamide gel electrophoresis and autoradiography revealed a radiolabeled band, which corresponded to the position of mouse ornithine decarboxylase, phosphorylated in vitro by casein kinase.2. A preparation of casein kinase-2 purified from nuclei of erythroleukemia cells could also phosphorylate mouse ornithine decarboxylase.
Ornithine decarboxylase (ODC), the key enzyme of the polyamine biosynthetic pathway, is characterized by a striking instability with a half-life that in mammalians can be less than 20 min [1]. Since the rate at which ODC turns over in cells appears to be subjected to physiological control [1-3], it is generally accepted that the coordinated controls of synthesis and degradation of this enzyme may represent a short term regulatory device. On the other hand, the occurrence of post-translational modifications is also suggested by a variety of observations. In particular ODC is readily phosphorylated in vitro by casein kinase-2 (CK-2) [4-9]. We have initially shown that rat heart ODC can be phosphorylated by rat liver CK-2 [4] and subsequently this finding has been confirmed and extended by using enzymes from different sources [5,8,9]. However, despite the in vitro phosphorylation of ODC by CK-2 is well established, evidence for phosphorylation of this enzyme in vivo has actually been indirect [5,8] and incorporation of phosphate into mammalian ODC within intact cells has not yet been incontrovertibly documented: in a recent report [10], phosphate was found to be specifi-
Abbreviations: ODC, ornithine decarboxylase;CK-2, casein kinase-2; MEL, murine erythroleukemia; PAGE, polyacrylamidegel electrophoresis. Correspondence: L.A. Pinna, Dipartimento di Chimica Biologica,Via Trieste 75, 35131 Padova, Italy.
cally but not covalently associated with ODC isolated from HTC cells. This report describes the isolation of 32p-radiolabeled ODC from Friend murine erythroleukemia (MEL) cells incubated in a medium containing 32pi. Since ODC is usually present in very low amounts in mammalian cells, MEL cells were diluted in fresh medium containing 10% serum to induce ODC and a-methyl ornithine was also added to the medium. This compound is a competitive inhibitor of ODC and can partially stabilize the enzyme, thus leading to an increase in its concentration [11]. In fact, 16 h after cell dilution, ODC activity was 2.5-fold higher in a-methyl omithine treated cells with respect to control MEL cells. Phosphorylation of ODC in intact MEL cells is documented in Fig. 1. MEL cells were incubated with 32pi for 1, 2 or 3 h. Then cells were collected and ODC was extracted and immunoprecipitated as described in the legend of Fig. 1 (A). The immunoprecipitates were subjected to P A G E / S D S and autoradiographed. A single protein radioactive band was evident and corresponded to the position of authentic ODC phosphorylated in vitro by CK-2. In parallel experiments it was shown that over 95% of ODC activity was immunoprecipitated under these conditions. Treatment of 32pi incubated MEL cells with pre-immune rabbit serum neither precipitated ODC activity nor gave rise to the 54 kDa radiolabeled band (not shown). Radiolabeling of ODC only slightly increased between 1 to 3 h incuba-
0167-4889/90/$03.50 © 1990 ElsevierSciencePublishers B.V. (BiomedicalDivision)
346
@
©
®
® kDa
kDa
--93---68--
x
--ODC--
oDc
--
93
- -
68
- -
45
- -
25
- -
--45---d.
:
d,
__/3
--25--
--13.7-1
2
3
4
5
6
7
8
9
Fig. 1. (A) Phosphorylation of ODC in intact MEL cells incubated with 32pi for 3 h (lane 1), 2 h (lane 2) or 1 h (lane 3). ODC was induced by dilution of stationary-phase MEL ceils in RPMI 1640 medium plus 10% serum [13] in the presence of 5 mM a-methyl ornithine. After 13, 14 or 15 h, batches of 3.107 cells were collected, washed and incubated in 3 ml of phosphate-free medium containing serum, a-methyl ornithine and 1.2 mCi of carrier-free [32p]H3PO4 at 37°C for 3, 2 or 1 h, respectively. Then cells were washed with phosphate-buffered saline and lysed as previously described [13] in the presence of 100 mM NaF. The cell extracts were treated with rabbit antibody to mouse kidney ODC, previously shown to precipitate very selectively [35S]methioninelabeled ODC from L1210 cells [3], and insoluble protein A. After extensive washing, the immunoprecipitates were analyzed by PAGE/SDS and autoradiography [4]. (B) Phosphorylation of mouse kidney ODC by CK-2 in vitro. ODC was highly purified from kidney of testosterone-treated mice [14] and phosphorylated in the presence of polylysine (0.1 mg/ml) by incubation with [-/-32p]ATP and CK-2 purified from rat liver cytosol (4). Lane 4: ODC plus CK-2; lane 5:CK-2 alone. (C) Phosphorylation of mouse kidney ODC by CK-2 partially purified from nuclei of MEL cells. The phosphorylation reaction was carried out in the absence (lane 6) or in the presence of polylysine (lane 7). Nuclei were isolated from MEL cells [15] and CK-2 was extracted with 1 M NaC1, dialyzed and partially purified by phosphocellulose chromatography [16]. (D) Coomassie patterns of mouse kidney ODC (lane 8) and rat liver CK-2 (lane 9). ODC, a and fl denote the position of authentic ODC from mouse kidney and of the a- and/3-subunits of CK-2. Both a- and fl-subunits are radiolabeled (lane 5) since the ODC phosphorylation medium includes polylysine which has been shown to shift autophosphorylation of CK-2 from the fl- to the a-subunits [4,17]. The protein markers used were glycogen phosphorylase (subunit 93 kDa), bovine serum albumin (68 kDa), ovalbumin (45 kDa), trypsinogen (25 kDa) and ribonuclease (13.7 kDa).
tion of M E L cells with 32pi. It should be noted, however, that i n c u b a t i o n of the cells was carried out at a very high density (107 c e l l s / m l ) . U n d e r these conditions, O D C d e g r a d a t i o n prevails over synthesis as indicated by a progressive decline in enzyme activity. I n particular, O D C activity i n M E L cells after 1, 2 a n d 3 h of i n c u b a t i o n was 22.3, 17.8 a n d 15.4 n m o l / h per mg, respectively. CK-2 appears the most likely c a n d i d a t e for such p h o s p h o r y l a t i o n i n intact M E L ceils: C K - 2 is the only p r o t e i n kinase which has b e e n shown to p h o s p h o r y l a t e m a m m a l i a n O D C in vitro; it exhibits very low K m values for O D C [7] a n d the p o t e n t i a l p h o s p h o r y l a t i o n site, which is conserved in the m a m m a l i a n species, overlaps a so called P E S T region [6,7], typical of rapidly degraded proteins. I n d i r e c t evidence that part of O D C occurs in various tissues as a form p h o s p h o r y l a t e d b y C K - 2 has also b e e n provided [5,8]. Regrettably the a m o u n t s of 32p-ODC isolated from M E L cells were too
small to allow 32p-peptide m a p p i n g , which could have assessed the actual i n v o l v e m e n t of C K - 2 as p h o s p h o r y lating agent. H o w e v e r M E L cells c o n t a i n a remarkable, h e p a r i n sensitive, casein kinase activity, which can be characterized a n d identified as CK-2. Interestingly, C K - 2 extracted a n d purified from nuclei of M E L cells is able to p h o s p h o r y l a t e O D C (Fig. 1C). The reaction, s t i m u l a t e d b y polylysine, occurs at a rate c o m p a r a b l e to that catalyzed b y cytosolic CK-2. This f i n d i n g rules out the possibility, suggested b y a previous study [12], that only the cytosolic form of C K - 2 could p h o s p h o r y l a t e ODC. I n conclusion, the present data provide clear-cut evidence that O D C actually undergoes p h o s p h o r y l a t i o n within i n t a c t M E L cells. This work was s u p p o r t e d b y grants from the I t a l i a n M i n i s t e r o della P u b b l i c a Istruzione. The authors are grateful to Dr. L. Persson for a generous gift of O D C antibody.
347
References 1 Tabor, C.W. and Tabor, H. (1984) Annu. Rev. Biochem. 53, 749-790. 2 Murakami, Y. and Hayashi, S. (1985) Biochem. J. 226, 893-896. 3 Persson, L., Holm, I. and Heby, O. (1988) J. Biol. Chem. 263, 3528-3533. 4 Meggio, F., Flamigni, F., Caldarera, C.M., Guarnieri, C. and Pinna, L.A. (1984) Biochem. Biophys. Res. Commun. 122, 9971004. 5 Donato, N.J., Ware, C.F. and Byus, C.V. (1986) Biochim. Biophys. Acta 884, 370-382. 6 Meggio, F., Flamigni, F., Guarnieri, C. and Pinna, L.A. (1987) Biochim. Biophys. Acta 929, 114-116. 7 Flamigni, F., Meggio, F., Marmiroli, S., Guarnieri, C., Pinna, L.A. and Caldarera, C.M. (1988) in Progress in Polyamine Research (Zappia, V. and Pegg, A.E., eds), pp. 45-53, Plenum Press, New York. 8 Peng, T. and Richards, J.F. (1988) Biochem. Biophys. Res. Commun. 153, 135-141.
9 Lougovoi, C.P. and Kyriakidis, D.A. (1989) Biochem. Biophys. Acta 996, 70-75. 10 Mitchell, J.L.A., Hicks, M.F., Chert, H.J. and Hoff, J.A. (1988) in Progress in Polyamine Research (Zappia, V. and Pegg, A.E., eds.), pp. 55-70, Plenum Press, New York. 11 McCann, P.P., Tardiff, C., Duchesne, M.C. and Mamont, P.S. (1977) Biochem. Biophys. Res. Commun. 76, 893-899. 12 Seely, J.E., Stetler, D.A., Jacob, S.T. and Peg,g, A.E. (1984) Biochem. Biophys. Res. Commun. 120, 219-225. 13 Flamigni, F., Marmiroli, S., Caldarera, C.M. and Guarnieri, C. (1989) Cell Biochem. Funct. 7, 213-217. 14 Seely, J.E., Poso, H. and Pegg, A.E. (1982) Biochemistry 21, 3394-3399. 15 Cocco, L., Gilmour, R.S., Ognibene, A., Letcher, A.J., Manzoli, F.A. and Irvine, R.F. (1987) Biochem. J. 248, 765-770. 16 Meggio, F., Donella Deana, A. and Pinna, L.A. (1981) J. Biol. Chem. 256, 11958-11961. 17 Meggio, F., Brunati, A.M. and Pinna, L.A., (1983) FEBS Lett. 160, 203-208.
1052 (1990) 345-347
345
Elsevier
B B A Report
BBAMCR 10256
Phosphorylation of ornithine decarboxylase in intact erythroleukemia cells Flavio Flamigni
1, S a n d r a
M a r m i r o l i 2, F l a v i o M e g g i o a n d L o r e n z o A. P i n n a 3
3, C a r l o
Guarnieri 1
Dipartimento di Biochimica, Universit& di Bologna, e Istituto di Citomorfologia, CNR, c / o lstituto Rizzoli, Bologna and ~ Dipartimento di Chimica Biologica, Unioersit6 di Padova, Padova (Italy)
(Received 10 October 1989) (Revised manuscript received29 December1989)
Key words: Ornithinedecarboxylase;Protein phosphorylation; (Erythroleukemiacell)
32p-labeled ornithine decarboxylase was isolated by immunoprecipitation from murine erythroleukemia cells incubated in a medium containing [32p]ortophosphoric acid. Analysis of immunoprecipitate by SDS-polyacrylamide gel electrophoresis and autoradiography revealed a radiolabeled band, which corresponded to the position of mouse ornithine decarboxylase, phosphorylated in vitro by casein kinase.2. A preparation of casein kinase-2 purified from nuclei of erythroleukemia cells could also phosphorylate mouse ornithine decarboxylase.
Ornithine decarboxylase (ODC), the key enzyme of the polyamine biosynthetic pathway, is characterized by a striking instability with a half-life that in mammalians can be less than 20 min [1]. Since the rate at which ODC turns over in cells appears to be subjected to physiological control [1-3], it is generally accepted that the coordinated controls of synthesis and degradation of this enzyme may represent a short term regulatory device. On the other hand, the occurrence of post-translational modifications is also suggested by a variety of observations. In particular ODC is readily phosphorylated in vitro by casein kinase-2 (CK-2) [4-9]. We have initially shown that rat heart ODC can be phosphorylated by rat liver CK-2 [4] and subsequently this finding has been confirmed and extended by using enzymes from different sources [5,8,9]. However, despite the in vitro phosphorylation of ODC by CK-2 is well established, evidence for phosphorylation of this enzyme in vivo has actually been indirect [5,8] and incorporation of phosphate into mammalian ODC within intact cells has not yet been incontrovertibly documented: in a recent report [10], phosphate was found to be specifi-
Abbreviations: ODC, ornithine decarboxylase;CK-2, casein kinase-2; MEL, murine erythroleukemia; PAGE, polyacrylamidegel electrophoresis. Correspondence: L.A. Pinna, Dipartimento di Chimica Biologica,Via Trieste 75, 35131 Padova, Italy.
cally but not covalently associated with ODC isolated from HTC cells. This report describes the isolation of 32p-radiolabeled ODC from Friend murine erythroleukemia (MEL) cells incubated in a medium containing 32pi. Since ODC is usually present in very low amounts in mammalian cells, MEL cells were diluted in fresh medium containing 10% serum to induce ODC and a-methyl ornithine was also added to the medium. This compound is a competitive inhibitor of ODC and can partially stabilize the enzyme, thus leading to an increase in its concentration [11]. In fact, 16 h after cell dilution, ODC activity was 2.5-fold higher in a-methyl omithine treated cells with respect to control MEL cells. Phosphorylation of ODC in intact MEL cells is documented in Fig. 1. MEL cells were incubated with 32pi for 1, 2 or 3 h. Then cells were collected and ODC was extracted and immunoprecipitated as described in the legend of Fig. 1 (A). The immunoprecipitates were subjected to P A G E / S D S and autoradiographed. A single protein radioactive band was evident and corresponded to the position of authentic ODC phosphorylated in vitro by CK-2. In parallel experiments it was shown that over 95% of ODC activity was immunoprecipitated under these conditions. Treatment of 32pi incubated MEL cells with pre-immune rabbit serum neither precipitated ODC activity nor gave rise to the 54 kDa radiolabeled band (not shown). Radiolabeling of ODC only slightly increased between 1 to 3 h incuba-
0167-4889/90/$03.50 © 1990 ElsevierSciencePublishers B.V. (BiomedicalDivision)
346
@
©
®
® kDa
kDa
--93---68--
x
--ODC--
oDc
--
93
- -
68
- -
45
- -
25
- -
--45---d.
:
d,
__/3
--25--
--13.7-1
2
3
4
5
6
7
8
9
Fig. 1. (A) Phosphorylation of ODC in intact MEL cells incubated with 32pi for 3 h (lane 1), 2 h (lane 2) or 1 h (lane 3). ODC was induced by dilution of stationary-phase MEL ceils in RPMI 1640 medium plus 10% serum [13] in the presence of 5 mM a-methyl ornithine. After 13, 14 or 15 h, batches of 3.107 cells were collected, washed and incubated in 3 ml of phosphate-free medium containing serum, a-methyl ornithine and 1.2 mCi of carrier-free [32p]H3PO4 at 37°C for 3, 2 or 1 h, respectively. Then cells were washed with phosphate-buffered saline and lysed as previously described [13] in the presence of 100 mM NaF. The cell extracts were treated with rabbit antibody to mouse kidney ODC, previously shown to precipitate very selectively [35S]methioninelabeled ODC from L1210 cells [3], and insoluble protein A. After extensive washing, the immunoprecipitates were analyzed by PAGE/SDS and autoradiography [4]. (B) Phosphorylation of mouse kidney ODC by CK-2 in vitro. ODC was highly purified from kidney of testosterone-treated mice [14] and phosphorylated in the presence of polylysine (0.1 mg/ml) by incubation with [-/-32p]ATP and CK-2 purified from rat liver cytosol (4). Lane 4: ODC plus CK-2; lane 5:CK-2 alone. (C) Phosphorylation of mouse kidney ODC by CK-2 partially purified from nuclei of MEL cells. The phosphorylation reaction was carried out in the absence (lane 6) or in the presence of polylysine (lane 7). Nuclei were isolated from MEL cells [15] and CK-2 was extracted with 1 M NaC1, dialyzed and partially purified by phosphocellulose chromatography [16]. (D) Coomassie patterns of mouse kidney ODC (lane 8) and rat liver CK-2 (lane 9). ODC, a and fl denote the position of authentic ODC from mouse kidney and of the a- and/3-subunits of CK-2. Both a- and fl-subunits are radiolabeled (lane 5) since the ODC phosphorylation medium includes polylysine which has been shown to shift autophosphorylation of CK-2 from the fl- to the a-subunits [4,17]. The protein markers used were glycogen phosphorylase (subunit 93 kDa), bovine serum albumin (68 kDa), ovalbumin (45 kDa), trypsinogen (25 kDa) and ribonuclease (13.7 kDa).
tion of M E L cells with 32pi. It should be noted, however, that i n c u b a t i o n of the cells was carried out at a very high density (107 c e l l s / m l ) . U n d e r these conditions, O D C d e g r a d a t i o n prevails over synthesis as indicated by a progressive decline in enzyme activity. I n particular, O D C activity i n M E L cells after 1, 2 a n d 3 h of i n c u b a t i o n was 22.3, 17.8 a n d 15.4 n m o l / h per mg, respectively. CK-2 appears the most likely c a n d i d a t e for such p h o s p h o r y l a t i o n i n intact M E L ceils: C K - 2 is the only p r o t e i n kinase which has b e e n shown to p h o s p h o r y l a t e m a m m a l i a n O D C in vitro; it exhibits very low K m values for O D C [7] a n d the p o t e n t i a l p h o s p h o r y l a t i o n site, which is conserved in the m a m m a l i a n species, overlaps a so called P E S T region [6,7], typical of rapidly degraded proteins. I n d i r e c t evidence that part of O D C occurs in various tissues as a form p h o s p h o r y l a t e d b y C K - 2 has also b e e n provided [5,8]. Regrettably the a m o u n t s of 32p-ODC isolated from M E L cells were too
small to allow 32p-peptide m a p p i n g , which could have assessed the actual i n v o l v e m e n t of C K - 2 as p h o s p h o r y lating agent. H o w e v e r M E L cells c o n t a i n a remarkable, h e p a r i n sensitive, casein kinase activity, which can be characterized a n d identified as CK-2. Interestingly, C K - 2 extracted a n d purified from nuclei of M E L cells is able to p h o s p h o r y l a t e O D C (Fig. 1C). The reaction, s t i m u l a t e d b y polylysine, occurs at a rate c o m p a r a b l e to that catalyzed b y cytosolic CK-2. This f i n d i n g rules out the possibility, suggested b y a previous study [12], that only the cytosolic form of C K - 2 could p h o s p h o r y l a t e ODC. I n conclusion, the present data provide clear-cut evidence that O D C actually undergoes p h o s p h o r y l a t i o n within i n t a c t M E L cells. This work was s u p p o r t e d b y grants from the I t a l i a n M i n i s t e r o della P u b b l i c a Istruzione. The authors are grateful to Dr. L. Persson for a generous gift of O D C antibody.
347
References 1 Tabor, C.W. and Tabor, H. (1984) Annu. Rev. Biochem. 53, 749-790. 2 Murakami, Y. and Hayashi, S. (1985) Biochem. J. 226, 893-896. 3 Persson, L., Holm, I. and Heby, O. (1988) J. Biol. Chem. 263, 3528-3533. 4 Meggio, F., Flamigni, F., Caldarera, C.M., Guarnieri, C. and Pinna, L.A. (1984) Biochem. Biophys. Res. Commun. 122, 9971004. 5 Donato, N.J., Ware, C.F. and Byus, C.V. (1986) Biochim. Biophys. Acta 884, 370-382. 6 Meggio, F., Flamigni, F., Guarnieri, C. and Pinna, L.A. (1987) Biochim. Biophys. Acta 929, 114-116. 7 Flamigni, F., Meggio, F., Marmiroli, S., Guarnieri, C., Pinna, L.A. and Caldarera, C.M. (1988) in Progress in Polyamine Research (Zappia, V. and Pegg, A.E., eds), pp. 45-53, Plenum Press, New York. 8 Peng, T. and Richards, J.F. (1988) Biochem. Biophys. Res. Commun. 153, 135-141.
9 Lougovoi, C.P. and Kyriakidis, D.A. (1989) Biochem. Biophys. Acta 996, 70-75. 10 Mitchell, J.L.A., Hicks, M.F., Chert, H.J. and Hoff, J.A. (1988) in Progress in Polyamine Research (Zappia, V. and Pegg, A.E., eds.), pp. 55-70, Plenum Press, New York. 11 McCann, P.P., Tardiff, C., Duchesne, M.C. and Mamont, P.S. (1977) Biochem. Biophys. Res. Commun. 76, 893-899. 12 Seely, J.E., Stetler, D.A., Jacob, S.T. and Peg,g, A.E. (1984) Biochem. Biophys. Res. Commun. 120, 219-225. 13 Flamigni, F., Marmiroli, S., Caldarera, C.M. and Guarnieri, C. (1989) Cell Biochem. Funct. 7, 213-217. 14 Seely, J.E., Poso, H. and Pegg, A.E. (1982) Biochemistry 21, 3394-3399. 15 Cocco, L., Gilmour, R.S., Ognibene, A., Letcher, A.J., Manzoli, F.A. and Irvine, R.F. (1987) Biochem. J. 248, 765-770. 16 Meggio, F., Donella Deana, A. and Pinna, L.A. (1981) J. Biol. Chem. 256, 11958-11961. 17 Meggio, F., Brunati, A.M. and Pinna, L.A., (1983) FEBS Lett. 160, 203-208.
