Histone kinase activity and histone phosphorylation from Pinus pinea cotyledons following x-irradiation

Environmentaland ExperimentalBotany, Vol. 22, No. 2, pp. 227 to 232, 1982

0098-8472/82/020227-06 $03.00/0

Printed in Great Britain

H I S T O N E KINASE A C T I V I T Y AND H I S T O N E P H O S P H O R Y L A T I O N F R O M PINUS PINEA COTYLEDONS FOLLOWING X-IRRADIATION R. M. ROY and J. M. SAMBORSKY

Department of Biological Sciences, Concordia University, 1455 de Maisonneuve Blvd., W., Montreal, Canada

( Received 26 September 1980; in revisedform 21 May 1981; infinalform 29 September 1981) Roy R. M. and SAMBORSKYJ. M. Histone kinase activity and histone phosphorylation in Pinus pinea cotyledons following X-irradiation. ENVIRONMENTALAND EXPERIMENTALBOTANY 22, 227-232, 1982.--Cotyledons of Pinus pinea have been irradiated with various dosages of 260 kVp X-ray up to 30 Gy at 13-14 days post-initiation of germination which is a period of active mitosis throughout the tissue. Although there was no apparent effect on electrophoretic mobility ofhistone isolated 16 hr post-irradiation, the net incorporation of inorganic phosphate was significantly depressed in varying degrees among histone subfractions. Histone kinase activity of irradiated cotyledons (30 Gy) was reduced 65% whereas histone kinase irradiated in vitro exhibited 20% loss of activity. Phosphorylation ofhistones isolated from irradiated cotyledons was reduced by 40%. Irradiation of isolated histones with the same dose had no effect on subsequent phosphorylation by nonirradiated kinase. The relationship between radiosensitivity ofhistone kinase and mitotic arrest is discussed. INTRODUCTION A L T H O U G H many studies have indicated that the

nucleus is the critical target in the cell, the mechanisms involved with radiation-induced inhibition of cell division remain obscure.
sites, elevated histone phosphatase activity and/or depressed histone kinase activity. The relationship between net phosphorylation of histones and mitotic arrest and subsequent recovery is of particular interest. The present study investigates radiation effects on the in vivo phosphorylation of histones in cotyledons of P. pinea during a period of active cell division. A comparison of histone kinase is described as well as the capacity of irradiated and non-irradiated kinase to phosphorylate in vitro both irradiated and non-irradiated histones.

MATERIALS

AND

M]ETHODS

Seeds ofPinus pinea (Arturo Ansaloni, Bologna, Italy) were surface sterilized with 1°/o javel water and germinated in sterile sand for 13 or 14 days at 21°C. Histories were extracted from soluble nucleohistone prepared from cotyledons and sep227

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R . M . ROY and J. M. SAMBORSKY

arated by polyacrylamide gel electrophoresis as previously describedJ 12) Histone kinase was partially purified from cotyledons by a modification of the method used by Nakaya et al. ~a3) and enzyme activity was assayed by the method of Yamamura et al. ~x4) Excised cotyledons were homogenized at high speed in a Waring blender for 90 sec in 0.05 M NaCI, 6 mM 2-mercaptoethanol in 0.05 M Tris-HC1 pH 7.8 and filtered through nylon mesh filters (375, 100, 25 and 10#m). The filtrate was centrifuged at 500g for 10 min and the supernatant mixed with Polyclar AT (Serva Biochemicals, Heidelberg, W. Germany) and recentrifuged at 500g for 10 min. Following centrifugation of the supernatant at 105,000g for 60 min, the 'crude extract' was combined with Dowex-I X2 (CI-) for 20 min and separated by filtration. Precipitation in 35-65% ammoniumsulfate was followed by overnight dialysis of the re-dissolved protein and subsequent desalting on Sephadex G-25 column (Pharmacia) at pH 8.5. The fraction with greatest kinase activity was further purified by elution from a DEAESephadex A-50 column with a linear gradient of NaC1 at pH 8.5. Enzyme activity was assayed by collecting acidprecipitated histone which had been labelled with 32phosphate derived from adenosine 5'-triphosphate 7 32p (New England Nuclear) on glass filters (AP-200, Millipore). The reaction mixture (0.25ml) contained 50#g/ml histone, 5#g/ml kinase extract, 2.5/~M y 32p-ATP (0.2/~Ci), 3.0/~M 2-mercaptoethanol, 5 m M magnesium acetate and 50 #M Tris-HCl pH 7.5. After incubation at 30°C for 5 min (optimal reaction time) the reaction was halted by addition of cold 5% TCA with 0.25% sodium tungstate (pH 2.0). The precipitate was washed on glass fibre filters with 5% TCA, 0.25% sodium tungstate, acetone, 95% ethanol and diethyl ether. Radioactivity was determined by liquid scintillation counting in 0.4% PPO and 0.01% P O P O P in toluene. Counts were corrected for quenching. Seedlings were removed from sand and placed between moist sheets of filter paper in unsealed plastic bags. Treated seedlings received 5, 10 or 30 Gy of 260kVp X-ray at 0.35 Gy/min from a Muller MG 300 X-ray machine with 1.0mm aluminum added filtration. In vivo phosphory-

lation of histones in cotyledons with 32phosphate was achieved by incubating in 250ml water containing 125#Ci Na Hz32po4 (carrier-free) with light and aeration for 16 hr immediately following irradiation. Histones were isolated and 32phosphate activity determined as described above. Phosphatase activity and histone degradation in sonicated nuclear preparations in isolation medium were checked using 32phosphate calf thymus H1 histone (Sigma) as substrate and assayed by precipitation in perchloric acid (PCA) and silicotungstic acid followed by extraction of inorganic phosphate from the supernatant as molybdate complex into butyl acetate followed by liquid scintillation counting. ~15) Proteolytic activity was assessed by measuring 32phosphate activity in the PCA-silicotungstate soluble fraction. Both extracted histone and partially purified kinase obtained by elution from DEAE-sephadex with NaC1 from non-irradiated cotyledons were irradiated as an aqueous solution on crushed ice. Kinase assays as described above were carried out using both irradiated histone and/or irradiated kinase. Radiation dose was measured by ferrous ammonium sulfate dosimetry as described by Law.(16) Mitotic activity of cotyledons irradiated at 13 days post-initiation of germination was determined from Feulgen squash preparations as described previously(1~) at various fixation times to 48 hr post-irradiation. Approximately 10,000 cells were examined for 4 individual seedlings for each dose and fixation interval.

RESULTS

The cells of cotyledons o f Pinuspinea are actively dividing at 13-14 days post-initiation of germination at 21°C; the mitotic index is approx. 4% (Fig. 1) and begins to decline by the 15th day of germination. After irradiation (5, 10, 30 Gy) the mitotic index approaches zero within 4 to 6 hr and then exhibits a response which depends upon dose. A recovery period is observed for the 5 and 10 Gy groups; however, the mitotic index during the 14-30 hr period is reduced in the cotyledons receiving 10 Gy. After dosage to 30 Gy there is no

HISTONE KINASE ACTIVITY AND HISTONE PHOSPHORYLATION

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rio. 1. Mitotic index of P. pinea cotyledons at various times following irradiation. (A 0, /k 5, ~ 10, • 30 Gy.) recovery of mitotic activity during the 48-hr sampling period. Seedlings incubated with 32phosphat e for 16 hr after irradiation incorporate variable amounts of label into their 8 histone subfractions (Table 1). There was no detectable alteration of electrophoretic mobility resulting from radiation exposure; however, both total activity and specific activity ofhistone fractions declined drastically (P < 0.01, 2-way analysis of variance) even at the lowest dosage. Specific activity of both H1 and core

histones exhibited varying degrees of phosphorylation and all were reduced following irradiation. There is a slight increase in this effect for most fractions at 10 Gy compared to the 5 G y dose. There was no evidence of increased histone phosphatase or histone hydrolase activity in sonicated nuclear preparations of irradiated cotyledons using 3zP-labelled calf thymus histone (H1) as substrate. Histone kinase was purified approx. 270-fold (8.5% yield) and had a p H o p t i m u m of 7.8. T h e enzyme was markedly Mg2+-dependent and cAMP-independent. Enzyme activity required the presence of mercaptoethanol and was inhibited by both monoiodoacetate and pchloromercuribenzoate. Temperature o p t i m u m was observed at 30°C. T h e enzyme half-life was found to be approx. 3 months at - 2 0 ° C and approx. 48 hr at 0°C. All assays were carried out at optimal conditions of time, substrate concentration, co-factor concentration and temperature. Both histone and histone kinase have been isolated at 16 hr post irradiation of cotyledons and were used in various in vitro enzyme assays to evaluate the effect of irradiation on histone kinase activity and on the susceptibility of histone to phosphorylation. The results of these experiments are presented in Fig. 2. The susceptibility of histone isolated from irradiated cotyledons to in vitro phosphorylation by kinase is reduced by approx. 40% at the lowest

Table 1. Incorporation of 32phosphate into histone subfractions of irradiated (5, I0 Gy) and non-irradiated cotyledons 0fPinus pinea Histone fraction I (HI) 2 (H1) 3 (H1) 4 (H1) 5 (H3) 6 + 7 (H2A) (H2B) 8 (H4) TOTAL

229

0 Gy Protein (/~g)

Specific activity (cpm/#g)

5 Gy Protein (#g)

0.5+0.03 5.3+0.80 5.6+0.65 1.2+0.10 0.3+0.01 11.2+1.07 4.4+0.02 28.5

506+39 58+7.4 51+7.2 213+19 344_+28 56+4.1 86-+9.1 77

0.2+0.01 1.9+0.21 4.1+0.37 1.6+0.14 3.6+0.42 11.9-+1.02 6.8+0.52 30.1

Specific activity (cpm/#g)

10 Gy Protein (#g)

Specific activity (cpm/#g)

384_+32* 0.6+0.02 213+13" 38+1.9" 3.1_+0.74 14+1.7" 30+1.9" 4.0+0.61 4.8+0.21" 43+4.1" 0.4+0.12 60_+8.2 21 _+ 1.8" 1.3+0.06 17_+1.0 7.8_+0.6* 12.2+0.90 3.4+0.48* 6.2+0.5* 8.4-+0.70 1.3_+0.07" 15 30.0 10.0

Count rates represent average of four separate experiments. Standard deviations are indicated. Significant differences (P < 0.05) between 0 and 5 or 10 Gy based on 2-way analysis of variance and Mann-Whitney test. * Significantly different (P < 0.05, t-test) comparing corresponding fractions of 0 and 5 Gy or 5 and 10 Gy.

230

R . M . ROY and J. M. SAMBORSKY apparent that in vitro irradiation of isolated histone with dose up to 30 Gy had no effect on histone phosphorylation; however, a slight but progressive loss of kinase activity (21%) occurred from 5 to 30 Gy.

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FIO. 2. In vitro phosphorylation ofhistones isolated from non-irradiated and irradiated (5, 10, 30 Gy) cotyledons by histone kinase from irradiated and non-irradiated cotyledons. (O 0, O 5, • I0, [] 30 Gy represent dose to histone.)

dosage (5 Gy) with little further inhibition up to 30 Gy. Partially purified kinase from irradiated cotyledons also responded to radiation dose with a large decrease up to 30 Gy (approx. 70% loss of activity). Combining both enzyme and substrate from irradiated cotyledons resulted in a further decrease in phosphorylation of histones. The effect of in vitro irradiation of either isolated histone and/or histone kinase on phosphate incorporation into histone is presented in Fig. 3. It is I

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Phosphorylation of nuclear proteins has been associated with mitotic activity and D N A replication. (18't9'1°'21) These reports for mammalian cell systems indicate that radiation inhibition of phosphorylation may be a significant event in mitotic arrest. (6-9) The histone kinase described in this paper is cAMP-independent, presumably similar to 'growth' kinase associated with cell proliferation.(1°'22) There appears to be some correlation between the drastic decline of both histone phosphorylation and arrest of mitotic activity following irradiation. The mitotic index of cotyledons receiving 5 and 10 Gy are virtually identical to each other over the 16-hr phosphorylation incubation period but significant (P < 0.01 t-test) differences between the 5 and l0 Gy groups with respect to phosphorylation are observed. This suggests that there is inherent radiosensitivity to the process of histone phosphorylation which does not entirely result from inhibition of mitosis and of histone synthesis. Furthermore, the mitotic activity subsequent to the 16-hr phosphorylation period does not recover in the 10 Gy group to the same extent as that of the 5 Gy group; this may be a result of the greater degree of inhibition of phosphorylation following the l0 Gy dose. On the basis of this observation, it is more likely that reduced levels of histone phosphorylation may be a contributing factor to mitotic arrest than mitotic arrest causing reduced histone phosphorylation. It is not clear to what extent inhibition ofhistone phosphorylation immediately following irradiation contributes to mitotic arrest; however, rapid onset of inhibition of histone phosphorylation has been previously reported in isolated nuclei of Pinus pinea. (it) In synchronously dividing plasmodia ofPhysarum polycephalum exposed to dosages of ionizing radiation inducing G 2 mitotic delay (1-2 hr), in vivo net phosphorylation was significantly depressed in both HI and core (H3/4, H2a/2b) histones, t9) The severe depression of

HISTONE KINASE A C T I V I T Y AND HISTONE PHOSPHORYLATION 32phosphate activity in histones of irradiated cotyledons following in vivo phosphorylation m a y be due to one or more of the following: radiation d a m a g e to histones or chromatin altering accessability of phosphorylation sites, reduced histone kinase activity, increased histone phosphatase activity, or increased phosphate or triphosphate nucleotide pools. However, phosphatase assays using 32phosphate-labelled H1 histone as substrate and crude nuclear sonicates as an enzyme source do not suggest increased phosphatase activity following 30 Gy dose. In vitro assays using both isolated kinase and histone from irradiated and non-irradiated cotyledons suggest there are factors associated with both substrate and enzyme which contribute to the overall depression ofhistone phosphorylation. Altered histone synthesis or turnover rates m a y result in fewer available sites for phosphorylation. With respect to the kinase effect, it is possible that enzyme synthesis has been altered or that the enzyme which is dependent upon reduced sulfhydryl groups for its function is particularly susceptible to oxidizing free radical attack. In vitro irradiation of isolated histone has no apparent effect on its susceptibility to phosphorylation by kinase. This suggests there is little direct d a m a g e to histones and that altered turnover in irradiated cells probably contributes in part to the depression of histone phosphorylation in vivo as well as depressed phosphorylation ofhistone from irradiated cotyledons for in vitro assays. There is, however, some evidence for direct d a m a g e to kinase irradiated in vitro. This tends to support the view that depressed histone phosphorylation in vivo is at least partially due to radiation d a m a g e to histone kinase directly. T h e importance ofhistone kinase radiosensitivity in the overall p h e n o m e n o n of radiation inhibition of cell division requires further investigation. Acknowledgement-This work has been supported by operating grants from National Science and Engineering Council of Canada. REFERENCES

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