Biochimica et Biophysica Acta, 698 (1982) 211-213 211 Elsevier Biomedical Press

BBA Report

BBA 90012

PI-IOSPHORYLATION OF RIBOSOMAL PROTEINS DURING THE CELL CYCLE OF PHYSARUM POL YCEPHALUM

GILLES PARADIS, ANNE BARDEN and GI~RALD LEMIEUX

Department of Biochemistry, Faculty of Sciences, Laval University, Quebec G1K 7P4 (Canada)

(Received March 8th, 1982)

Key words: Ribosomal protein; Phosphorylation; Cell cycle," (P. polycephalum)

Physarum polycephalum has been used as a model system to study the phosphorylation of ribosomal proteins during the cell cycle. The results showed that the phosphate content of $3, the major ribosomal phos- phoprotein in this organism, was constant during all phases of the cell cycle. No additional ribosomal phosphoproteins were observed. These results differ significantly from those reported earlier by Rupp, R.G., Humphrey, R.M. and Shaeffer, J.R. (Biochim. Biophys. Acta (1976) 418, 81-92) and suggest that the use of thymidine or hydroxyurea to synchronize cell population may affect the phosphorylation of ribosomal proteins. The results are discussed in relation to protein synthesis and cAMP level during the cell cycle.

The phosphate content of the major ribosomal phosphoprotein can be altered by a large number of conditions. However, it is not yet clear what relationship exists between this phenomenon and the regulation of protein synthesis (for a recent review, see Ref. 1).

The phosphorylation of ribosomal proteins dur- ing the cell cycle has not yet been thoroughly studied. A few years ago, Rupp et al. [2] observed the specific phosphorylation of a unique ribosomal protein during the mitotic phase of the cell cycle in Chinese hamster ovary cells. Unfortunately, the ribosomal phosphoproteins were not identified. Furthermore, the use of hydroxyurea or thymidine block to synchronize the cells might have had an effect on the phosphorylation of the ribosomal proteins.

In order to study further this important cellular event in relation to ribosomal protein phosphory- lation, we have used Physarum polycephalum as a model system. This organism can be easily grown in the form of a giant cell containing up to 108 nuclei, all dividing with an exact natural syn- chrony [3]. The ribosomal proteins of P. poly-

0167-4781/82/0000-0000/$02.75 © 1982 Elsevier Biomedical Press

cephalum have been characterized [4,5] and five of these proteins are phosphorylated in vivo [6]. $3, the major phosphoprotein is equivalent to $6 in mammalian cells [7]. Since this protein is the only one to have a variable phosphate content [ 1,8], we have quantitatively studied the phosphorylation level of $3 during " the cell cycle.

Cultures and P. polycephalum, strain M3C , were grown in semi-defined medium, from which KH2PO 4 was omitted [3,9]. Macroplasmodia were grown on Millipore filters supported by glass beads in the same medium. The various phases of the cell cycle were determined as previously described [10].

The purified ribosomes [4,11] were denatured by the addition of 200 #1 sample buffer (0.0625 M Tris-HC1/1% fl-mercaptoethanol/2% SDS/10% sucrose, pH 6.8) and heating for 2 min at 100°C. The ribosomal proteins were separated by SDS- polyacrylamide slab gel electrophoresis in a 12- 16% linear acrylamide gradient [12]. After electro- phoresis, the gels were fixed and heated for 10 rain at 90°C in 10% trichloroacetic acid in order to hydrolyse the ribosomal RNA. The gels were then stained with Coomassie brilliant blue G-250 and

212

des ta ined in m e t h a n o l / a c e t i c a c i d / H 2 ° (10: 10: 80, v/v). The phosphorylated proteins were detected by autoradiography and cut out of the gel. The fixed dye was extracted by an overnight incubation of the gel pieces in 1 ml of 50% methanol. The absorbance of the supernatant was determined at 595 nm. The gel pieces were oxidized with 0.5 ml of H 2 0 2 a t 50°C for 12 h in tightly capped vials. After cooling, 10 ml Aquasol were added and the radioactivity was determined by liquid scintillation counting (LS350, Beckman).

We have recently shown that ribosomal protein $3 can be separated from all other ribosomal proteins by one-dimensional SDS-polyacrylamide gel electrophoresis [5]. This method, which is more convenient and requires a lower amount of protein than the standard two-dimensional gel system [ 13], enabled us to study the phosphorylation of $3 by using a single macroplasmodia for each determina- tion.

The autoradiogram of a gel is presented in Fig. 1. It can be seen that the phosphate content of the ribosomal phosphoproteins was constant dur- ing all phases of the cell cycle. Moreover, no additional phosphoproteins were observed at any stage. Since $3 is the only ribosomal protein with a

Fig. 1. Analysis of ribosomal protein phosphorylation during the cell cycle. Macroplasmodia were grown in the presence of 32po 4 (100 /~Ci/ml). The ribosomes were purified at various phases of the cell cycle and the ribosomal proteins were sep- arated by SDS-polyacrylamide gel electrophoresis in 12-16% linear polyacrylamide gradient. The gels were fixed and then heated for 10 min in 10% trichloroacetic acid. The gels were finally stained and subjected to autoradiography. The molecu- lar weight of the phosphoproteins, and their identity were determined as previously described [6].

3000

200C

g

1 0 0 0

I " I i

m

I

I

M2 M3 Time(h )

Fig. 2. Phosphorylation level of $3 during the cell cycle. Macro- plasmodia were labeled in vivo with inorganic 32po 4 (100 /~Ci/ml). The various phases of the cell cycle were determined by phase contrast microscopy [9]. After the purification of ribosomes, the proteins were separated by SDS-polyacrylamide gel electrophoresis in 12-16% acrylarnide gradient according to Laemmli [12]. The phosphate content in $3 was determined by liquid scintillation counting and related to the amount of $3, which was estimated from the dye content of the band.

variable phosphate content [8,14], we have de- termined quantitatively the radioactivity associ- ated with this band and related the values ob- tained to the amount of dye present. The results are shown in Fig. 2. It can be concluded that effectively the phosphate content of $3 was con- stant through all stages of the cell cycle.

The results are relatively different from those publislied by Rupp et al. [2], on the phosphoryla- tion of ribosomal proteins during the cell cycle of Chinese hamster ovary cells. These authors ob- served the presence of five ribosomal phos- phoproteins, having a molecular weight of 17500, 23000, 30000, 38000 and 45000, respectively. The protein with a molecular weight of 45000 was specifically phosphorylated at the mitotic phase of the cell cycle. The protein with a molecular weight of 38000 contained the higher proportion of radio- activity. This information suggests that this latter protein is probably $6. If this is the case, the nature of the phosphoprotein with a molecular weight of 45000 remains to be elucidated. The phosphorylation of this protein may be a conse- quence of the use of thymidine or hydroxyurea for cell synchronization. In this regard, the results presented in this work can be interpreted unam-

biguous ly since P. polycephalum is na tu ra l ly syn- ch ronous th roughou t its cell cycle. However , it c anno t be exc luded that the p h o s p h o r y l a t i o n of a specific r i bosoma l p ro t e in dur ing the cell cycle is a p h e n o m e n o n specific to the higher eukaryo t i c cells.

I t has been observed in m a n y systems that a r a p i d cel lular p ro l i f e ra t ion is genera l ly associa ted with a high p h o s p h a t e conten t of $6, or its equiva- lent [1]. However , the ra te of p ro te in synthesis is r educed by 75% dur ing the mi to t ic phase of the cell cycle [15]. The results p resen ted in this work showed tha t this r educ t ion is no t a c c o m p a n i e d by a concomi t an t decrease of the phospha t e con ten t of $3. Similar results, showing the absence of a re la t ion with g lob in synthesis and the p h o s p h a t e con ten t of $6 have been ob ta ined in re t iculocytes [16,17].

The phospha t e con ten t of $6 in m a m m a l i a n cells is also affected by the c A M P level [1,7]. However , in t he cell cycle of P. polycephalum this r e la t ionsh ip d id no t have to be taken into account since there is no var ia t ion of c A M P concen t ra t ion du r ing the cell cycle [18].

References

1 Leader, D.P. (1980) in Recently discovered systems of enzyme regulation by reversible phosphorylation (Cohen, P., ed.), pp. 203-223, Elsevier North-Holland, Amsterdam

213

2 Rupp, R.G., Humphrey, R.M. and Shaeffer, J.R. (1976) Biochim. Biophys. Acta 418, 81-92

3 Daniel, J.W. and Baldwin, H.M. (1964) in Methods in Cell Physiology (Prescott, D.M., ed.), Vol. 1, pp. 9-41. Academic Press, NY

4 Lemieux, G., B61anger, G., Nicole, L. and Bellemare, G. 91979) Biochim. Biophys. Acta 578, 351-364

5 B61anger, G. and lemieux, G. (1982) Can. J. Biochem., in the press

6 B61anger, G., bellemare, G. and lemieux, G. (1979) Bio- chem. Biophys. Res. Commun. 86, 862-868

7 Wool, I.G. (1979) Annu. Rev. Biochem. 48, 719-754 8 B61anger, G., Godin, C. and Lemieux, G. (1981) Eur. J.

Biochem. 120, 143-148 9 Bersier, D. and Braun, R. (1974) Biochim. Biophys. Acta

340, 463-471 10 Guttes, E. and Guttes, S. (1964) in Methods in Cell Physiol-

ogy (Prescott, D.M., ed.), Vol. 1, pp. 43-54, Academic Press, NY

11 Brewer, E.N. (1972) Biochim. Biophys. Acta 277, 639-645 12 Laemmli, U.K. (1970) Nature 227, 680-682 13 Kaltschmidt, E. and Wittmann, H.G. (1970) Anal. Biochem.

36, 401-412 14 Courtois, G., Paradis, G., Barden, A. and Lemieu:~, G.

(1982) Biochim. Biophys. Acta 696, 87-93 15 Hodge, L.D., Robbins, E. and Scharff, M.D. (1969) J. Cell

Biol. 40, 497-507 16 Floyd, G.A. and Traugh, J.A. (1980) Eur. J. Biochem. 106,

269-277 17 Floyd, G.A. and Traugh, J.A. (1980) Eur. J. Biochem. 117,

257-262 18 Garrison, P.M. and Barnes, L.D. (1980) Biochim. Biophys.

Acta 633, 114-121