KINASE

ACTIVITIES PROTEINS

IN THE NON-HISTONE OF RESTING BHK21

CHROMOSOMAL

AND PROLIFERATING

Cl3 CELLS

C. BLAT, M. MARTY DE MORALES and L. HAREL Institut de Recherches Scienti’ques SW le Cancer, F-94 800 Villejug, France

SUMMARY When BHK21 Cl3 cells in saturation density are stimulated to proliferate by a change of medium, a synchronized DNA synthesis occurs with a maximum at about 15 h. After stimulation the nonhistone chromosomal proteins (NHC proteins) phosphorylation in vivo increases and reaches a maximum in late G 1 phase. We show that the endogenous phosphorylation in vitro of NHC proteins is 4-fold larger when the NHC proteins are extracted from cells in late G 1 phase than from resting cells. The electrophoretic patterns of FP] distribution show that the [“‘PI incorporation enhancement in NHC proteins from stimulated cells is more significant for the slowly migrating proteins. The NHC protein kinase activity was fractionated on polyacrylamide gel in 10 fractions, in resting as in stimulated cells. Only some of these kinase activities were increased after cell stimulation. The enhancement of phosphorylation may be due in part to an increase in the number of phosphorylation sites and in part to an increase in kinase activities. The use of phosvitin as exogenous substrate allow the conclusion that the activity of some kinase is enhanced after stimulation. Furthermore, by fractionation on polyacrylamide gel five phosvitin kinase activities were found in low molecular weight NHC protein fractions, in control as in stimulated cells. After cell stimulation, two phosvitin kinase activities only are specifically increased. The results show the multiplicity of kinase activities and support the hypothesis that these various kinase activities correspond to specific differentiated functions.

of BHK21 Cl3 cells enter the stationary phase, the metabolism is decreased and RNA and DNA synthesis becomes minimal. A change of medium stimulates the cells to proliferate and a synchronized enhancement of DNA synthesis is observed, exhibiting a maximum about 15 h later [ 11.We have previously shown [l] that soon after stimulation the non-histone chromosomal protein (NHC protein) phosphorylation in vivo increases and reaches a maximum in late G 1, before the onset of DNA synthesis. The NHC protein phosphorylation remains elevated during early S and decreases in late S. When cultures

ExptlCell

Res 98 (1976)

It has been shown that chromatin isolated from stimulated cells is more active in in vitro RNA synthesis than chromatin from control cells [2-31. Kamiyama et al. [4] and Shea & Kleinsmith [5] have suggested that phosphorylation of NHC proteins might regulate RNA synthesis in chromatin. We have therefore compared the phosphoprotein kinase activity of the NHC proteins in stimulated cells with that found in resting cells. MATERIALS

AND METHODS

Baby hamster kidney cells (BHK21 C13) obtained from Dr Stocker’s laboratory [6, 71 were routinely

105

Kinase activities in the NHCproteins

ing electrophoresis the gels were cut into 2 mm slices. Each slice was eluted overnight at 4°C with 0.3 ml of 50 mM Tris buffer (pH 7.4) containing 1 mM MgCl, and the protein kinase activities were determined in aliquots of the eluant. 0.1 ml of eluant was incubated for 5 min at 37°C in 0.25 ml (final volume). The incubation mixture contained 2.5 pmoles Tris-buffer, pH 7.4, 1.25 pmoles MgQ and 0.5 pmoles y[32P]ATP (8 &i/ pmole). The reactions were stopped by addition of TCA (30% final concentration) added of 200 pg of ATP. The mixtures were filtered on Mihioore filters, the precipitates washed twice with 20%- TCA. The radioactivities were measured bv Cerenkov effect. For phosvitin kinase activity 5d pg phosvitin was added to 0.1 ml of eluant.

Protein kinase activity.

1 2

5

xl

time (min); ordinate: (cpm [32P]/mg proteins)x 10e4. -, stimulated cells; - * -, control cells. Kinetics of [“PI incorporation into NHC proteins of BHK21 Cl3 cells.

Fig. 1. Abscissa:

cultivated at 37°C in BHK medium [7] supplemented with 10% tryptose phosphate broth and 10% foetal calf serum. The cells were stimulated to proliferate, as was previously described [I]. The nuclei were prepared by the technique previously described [l] and the NHC proteins were isolated by Wang’s method [8] as modified by Loeb [9]. Isolated NHC proteins (90 fig) were incubated at 37°C in 1 ml (final volume). The reaction mixture consisted of 40 pmoles Tris-buffer pH 7.4, 5 pmoles MgCl,, 1 pmole dithiothreitol, 0.5 pmole -#*P]ATP (8 @Zi/~mole). Reaction was stopped with TCA (30% final concentration) containing ATP (5 mM). The precipitate was collected on Millipore filters and washed twice with 20 % TCA. The filters were transferred into vials. The radioactivity was measured by the Cerenkov effect in a Beckman Scintillator Counter.

Phosphorylation

of NHC proteins.

electrophoresis. Analytical gels (8 cm in length and 5.5 mm in diameter) contained 10% acrylamide and 0.2 % N, N’-methylene-his-acrylamide, dissolved in the gel buffer, pH 8.7, described by Uriel [lo]. We modified this by the addition of 2 mM Na, EDTA, 5 mM mercaptoethanol, 4 M urea and 40 mM NaF. Polymerization was accomplished by the addition of ammonium persulfate (75 mg/lOO ml) and N,N, N’N’-tetramethylethylenediamine (0.075 ml/100 ml). Electrophoresis was performed in the cold (+4”C) at 4 mA/tube until the bromophenol blue (used as a front indicator) reached the bottom (+) of the gel. Gels were frozen by contact with a block of CO9 and sliced into 1 mm sections. To each slice was added I ml of soluene solubilizer (Packard). The [“*PI radioactivity was measured in the presence of 10 ml of liquid scintillator (PPO and POPOP in toluene) in a Beckman scintillator counter.

Analytical

of protein kinnses. The protein kinases were separated by polyacrylamide gel electrophoresis as described by Dastugue et al. [ll]. Samples of 200 pg of NHC proteins were applied to each gel. Foilow-

Separation

RESULTS Endogenous phosphorylation of NHC proteins (a) Kinetic of [3zP] incorporation in NHC proteins extracted from stimulated and non-stimulated cells. BHK21 Cl3 cells

maintained at saturation density for 24 h were stimulated to proliferate by a change of medium. Control cells were left in old medium. The NHC proteins were extracted from both control and stimulated cells after 15 h. We have previously observed that DNA synthesis is at its maximum at this time [ 11. The NHC proteins were incubated for 1 to 10 min in the presence of JJ[~~P]ATP in the medium described in Materials and Table 1. Phosphorylation proteins from stimulated control cells

in vitro of NHC (11 and 15 h) and

All experimental details are as described in Materials and Methods. Incubation time 5 min Time after change of medium (hours) 11 15

cpm [“*PI per 100pg NHC proteins % increase Expt ConStimuS$XlOO no. trol lated 1 2 I 2 3

5 350 4 850 4 740 4 650 5 200

20800 20 200 12 900 17 100 16 000

290 317 173 268 210

Exptl Cell Res 98 (I 976)

1

I r)

3l

1 93

1

w

I

I a

30

xl

Fig.

2. Abscissa: gel fractions; ordinate: cpm [“‘PI. [32P] labelling patterns of NHC phosphoproteins from control and stimulated cells. NHC nroteins (500 wg) were incubated for 5 min at 37°C in-the reackon mixture described in Materials and Methods in the presence of 0.5 Fmole Y[~~P]ATP (16 &i/pmole). The reaction was stopped by cooling the tube in ice and addition of 4 M urea, IO mM ATP, 40 mM NaF. The

mixture was dialysed for I7 h at 4°C against the gel buffer. NHC proteins (100 big) were fractionated bv electrophoresis as described in-Materials and Methods. The radioactivity of [“‘PI incorporated in NHC proteins is plotted against migration distance in the gel. NHC proteins from (A) control cells (I I h); (B) stimulated cells (I I h-late G I phase); (C) control cells (IS h); (D) stimulated cells (IS h S phase).

Methods. The results (fig. 1) show that [32P]incorporation in NHC proteins is rapid and reaches a plateau 5 or 10 min after incubation. The rate of [32P]incorporation is greater in NHC proteins obtained from S phase cells than from cells in the G 0 phase. The maximum radioactivity incorporated in NHC proteins from stimulated cells is 4fold larger than in NHC proteins from control cells. In other experiments NHC proteins were

extracted from: (N) non-stimulated cells; (6) late G 1 phase cells 11 h after stimulation; and (c) S phase cells at 15 h poststimulation (the G 1 and S phase states were determined by in vivo labelling with [3H]thymidine). The proteins were incubated as before in the presence of Y[~*P]ATP for 5 min. Table 1 shows that [““PI incorporation in NHC proteins from stimulated cells is greater than in NHC proteins from control cells. Maximum [“‘PI incor-

Exptl Cell Res 98 (1976)

Kinase activities in the NHCproteins

107

men&. Some variations in the labelling pattern were seen between G 1 and S phase, specifically the high molecular weight proteins are more phosphorylated in late G 1 than in S phase cells. (c) Fractionation of protein kinases by polyacrylamide gel electrophoresis. We

have investigated whether the kinase activity associated with the NHC proteins represented the presence of multiple enzymes I r 20 30 as it has been found in rat and beef liver 1 5 10 NHC proteins [ 11, 181. The NHC proteins Fig. 3. Abscissa: gel fractions; ordinate: cpm [=P]. - - -, Stimulated cells (late G 1 phase); O-O, control cells. were fractionated by polyacrylamide gel Fractionation of protein kinases by polyacrylamide electrophoresis as described in Materials gel electrophoresis. After electrophoresis (see Maand Methods and each eluted fraction was terials and Methods) slices were eluted. Aliquot of each eluant was incubated as described in Materials tested for endogenous kinase activity in the and Methods. The radioactivity of [“‘PI incorporated in NHC proteins was plotted against migration disabsence of added substrate. We have detance in the gel. tected at least 10 fractions of kinase activity in NHC proteins from control cells as from poration was observed for cells in late G 1 stimulated cells (fig. 3). After stimulation the [“*PI incorporation into protein fractions phase where the NHC protein phosphorylais enhanced, but the per cent increase is not tion is enhanced by 300%, on average. the same for the different fractions. As in (b) Distribution of [32P] in NHC proteins separated by gel electrophoresis. Since the the preceding experiments, the radioactivNHC proteins are heterogenous [ 12-171, ity of the heaviest fractions is the most inwe investigated whether the observed phos- creased. The increased [32P]incorporation in NHC phorylation is equal for all the protein proteins after cell stimulation may reflect species or if the increase is selective. NHC proteins from cells in GO, in late G I and in an augmentation of phosphorylable sites or S phase were phosphorylated in vitro in an increase in kinase activity. To verify the presence of Y[~~P]ATP, and then sepa- that kinase activity is enhanced in stimulated cells we have compared the phosrated by electrophoresis in polyacrylamide gels. The patterns of [32P] distribution (fig. phorylation of phosvitin (an exogenous 2) show the same number of bands in con- substrate) by NHC proteins from cells in trol and stimulated cells. We observed an GO and G 1 phase. increase in the phosphorylation of total Phosvitin kinase activity in resting NHC proteins when the cells were stimuand stimulated cells lated, but the per cent augmentation of phosphorylation was not the same for the (a) Phosphor?,lation ofphosvitin. Phosvitin different phosphoproteins. In stimulated was incubated in the presence of total NHC cells in late G 1 as in S phase the phosphoryproteins extracted from resting or stimulation increase is more significant for the lated cells. Table 2 shows that phosvitin slowly migrating proteins. The same dif- can be phosphorylated by NHC protein ferences have been found in all experi- kinases from BHK2 1 C 13 cells. The [32P]inExprl Cell Res 9.4 (1976)

108

Blat, Marty de Morales and Hare1

Table 2. Phosvitin phosphorylation by NHC proteins of stimulated (II h) and control cells NHC proteins (90 pg) were incubated in the absence or presence of phosvitin (200 pg) for 5 min at 37°C in conditions described in Materials and Methods. Phosvitin phosphorylation was measured by the difference between the [s*P] incorporation in NHC proteins+phosvitin and the [‘*PI incorporation in NHC proteins alone C, control cells S, stimulated cells cpm per 100pg NHC proteins NHC proteins

NHC proteins +phosvitin

Expt no.

C

S

C

S

C

S

: 3

55000 500 2 620

I8 I7 300 500 8 540

87 900 410 6 270

22 21 850 740 I3 600

32 400 410 3 650

4 550 240 5 060

corporation in phosvitin is better when the NHC proteins are extracted from late G I phase cells; it is then enhanced by 50%, on average. These results clearly show that some kinase activity of NHC proteins is increased when the cells are stimulated to proliferate. (b) Fractionation of phosvitin kinase activity. We have fractionated the NHC proteins from resting and stimulated cells by polyacrylamide gel electrophoresis as above and compared the phosvitin kinase activities in each fraction. Each fraction was incubated in the absence or presence of 50 pg phosvitin, as described in Materials and Methods. Phosvitin kinase activity was obtained from the difference between the [3zP] incorporation in NHC proteins+phosvitin and the [3pP] incorporation in NHC proteins alone. Fig. 4 shows that 5 phosvitin kinase activities may be resolved by electrophoresis of control cell proteins. We observed that the high molecular weight fractions did not contain any phosvitin kinase activity. The same peaks are found when NHC proteins are extracted from stimulated cells, but the 5 kinase activities are not enhanced in the same manner. Only two of the phosvitin kinase activities appear to Exptl Cell Res 98 (I 976)

cpm [“‘PI incorporated in phosvitin

% increase of cpm [“‘PI incorporated in phosvitin +oo

33.8 76.0 38.6

be specifically modified, one increasing 3fold, the other 2-fold. These results, which can be observed repeatedly, demonstrate that some kinase activities selectively increase after cell stimulation to proliferate. DISCUSSION Our results show that endogenous phosphorylation in vitro of NHC proteins is enhanced when the cells are stimulated by a

'

(+l

1-j

gel fractions; ordinate: [s2P] cpm incorporated in phosvitin. Fractionation of phosvitin kinases from NHC proteins of control and stimulated (I I h) cells. The [“*PI radioactivity incorporated in phosvitin is plotted against migration distance in gel. All experimental details are described in Materials and Methods. Phosvitin kinases of NHC proteins from -, control cells; - - -. stimulated cells.

Fig. 4. Abscissa:

Kinase activities in the NHCproteins change of medium. When cells are in late G 1 phase, the increase is more pronounced than in S phase. These data agree with those obtained by us previously, showing increased in vivo phosphorylation of NHC proteins following cell stimulation [ 11.They are also in agreement with those recently reported by several authors [3, 191who observed an increase in phosphorylation in vitro of NHC proteins, following hormonal stimulation. The enhancement of phosphorylation in vitro of NHC proteins may reflect an augmentation of the phosphoprotein kinase activity of the NHC proteins or an augmentation after stimulation of the number of phosphorylable sites per mg of NHC proteins. The increase in phosphorylation of high molecular weight proteins might be due, in fact, to the accumulation of these proteins after cell stimulation. By labelling NHC proteins from resting cells with [3H]aspartic acid and NHC proteins from stimulated cells with [14C]aspartic acid and coelectrophoresis of the two fractions we have observed an enhancement of the synthesis of high molecular weight NHC proteins in late G 1 phase (this result confirms the data of Baserga & Rovera [20]). However, as the percentage increase in phosphorylation (unpublished results) is larger than the percentage increase in synthesis, it seems that synthesis of these proteins cannot explain the large increase in phosphorylation. Experiments using phosvitin as exogenous substrate show clearly that at least some kinase activity is enhanced after cell stimulation. Ishida & Ahmed [21] have also observed an augmentation of the chromatinassociated phosvitin kinase activity in the isoproterenol-stimulated rat submandibular gland. Our experiments demonstrate once more

109

[l 1, 181 the great number of different species of kinases in the NHC proteins probably corresponding to specific differentiated functions. It is interesting to note that only two out of five phosvitin kinase activities are enhanced after cell stimulation. They are certainly of different species. The comparison between the distribution of endogenous protein kinases and phosvitin kinases after gel electrophoresis is also interesting. We observed that phosvitin is not a good substrate for high molecular weight protein kinases which are particularly enhanced after cell stimulation. This result explains the discrepancies between the large increase of endogenous phosphorylation after stimulation (300% on average) and the only 50% increase of phosvitin phosphorylation. We now intend to study the large molecular weight protein kinase activity and to define more precisely a good substrate for these enzymes. Most likely they are involved in the proliferation process, perhaps in the transcription of mRNA which increases after cell stimulation [22]. We thank Dr J. Kruh for helpful and stimulating suggestions and Mrs M. de Monti for expert technical assistance. This research was supported in part by the Commissariat a I’Energie Atomique.

REFERENCES 1. Marty de Morales, M, Blat, C & Harel, L, Exp cellres86(1974) 111. 2. Stein, G, Chaudhuri, S & Baserga, R, J biol them 247 (1972) 3918. 3. Brade, W P, Thomson, J A, Chiu, J F & Hnilica, L S, Exp cell res 84 (1974) 183. 4. Kamiyama, M, Dastugue, B, Defer, N & Kruh, J, Biochim biophys acta 277 (1972) 576. 5. Shea, M & Kleinsmith, L J, Biochem biophys res commun 50 (1973) 473. 6. MacPherson, I & Stocker, M, Virology 16 (1962) 147. 7. MacPherson, I, J natl cancer inst 30 (1963) 795. 8. Wang, T Y, J biol them 242 (1%7) 1220. 9. Loeb, J E, Compt rend acad sci 267 (1968) 2185. 10. Uriel, J, Bull sot chim biol48 (1966) %9. 11. Dastugue, B, Tichonicky, L & Kruh, J, Biochimie 56 (1974) 491. Exptl Cell Res %S (1976)

I10

Blat, Marty de Morules and Hare1

12. Loeb, J E & Creuzet, C. FEBS lett 5 (1969) 37. 13. Dastugue, B, Tichonicky, L, Penit-Soria. J & Kruh, J, Bull sot chim biol 52 (1970) 391. 14. Elgin, S C R & Bonner, J, Biochemistry 9 (1970) 4440. 15. Shaw, L & Huang, R C. Biochemistry 9 (1970) 4530. 16. Kostraba, N C & Wang, T Y. Inst j biochem I (1970) 327. 17. MacGillivray, A J, Carroll, D & Paul, J, FEBS lett 13 (1971) 204.

Exprl Cell Res 98 (1976)

18. Kish, V M & Kleinsmith, L J. J biol them 249 (1974) 750. 19. Ishida, H & Ahmed, K, Exp cell res 78 (1973) 3 I, 20. Rovera & Baserga, R, J cell physio177 (1971) 201. 21. Ishida, H & Ahmed, K, Exp cell res 84 (1974) 127. 22. Blat, C, Marty de Morales, M & Harel. L, Proc 9th FEBS meeting Budapest 1974 (ed G Gardos) vol. 34, p. 137. North Holland, Amsterdam (1975). Received July 23, 1975