Polyamines modulate phosphorylation and acetylation of non-histone chromosomal proteins of the cerebral cortex of rats of various ages

Arch. GerontoL Geriatr., 1 (1982) 339-348

339

Elsevier Biomedical Press

Polyamines modulate phosphorylation and acetylation of non-histone chromosomal proteins of the cerebral cortex of rats of various ages Ratna Bose and M.S. Kanungo

1

Biochemistry Laboratory, Department of Zoology, Banaras Hindu University, Varanasi 221 005, India

(Received 5 July 1982; accepted in revised form 29 September 1982)

Summaw In vitro phosphorylation and acetylation of NHC (non-histone chromosomal) proteins and their modulation by spermine and spermidine were studied using slices of cerebral cortex of female albino rats of various ages, The total DNA, histone and.NHC proteins do not change significantly with age. Phosphorylation and acetylation of total and individual NHC proteins decreases with increasing age of the rat. Spermine and spermidine stimulate phosphorylation and acetylation of specific NHC proteins in immature rats. This effect decreases with increasing age. It is suggested that such modulatory effects of polyamines may cause alterations in the expression of specific genes during aging. aging; acetylation; NHC proteins; phosphorylation; spermine; spermidine

Introduction N o n - h i s t o n e c h r o m o s o m a l ( N H C ) proteins which are present in the c h r o m a t i n are believed to p l a y a key role in regulating the p a t t e r n of gene expression in higher organisms (Barrett et al., 1974; Elgin a n d W e i n t r a u b , 1975). In c o n t r a s t to histones, the N H C p r o t e i n s are acidic, larger in number, highly heterogeneous, a n d tissue- a n d species-specific (Paul a n d G i l m o u r , 1975). T h e y u n d e r g o covalent m o d i f i c a t i o n s such as p h o s p h o r y l a t i o n a n d acetylation p o s t - t r a n s l a t i o n a l l y which m a y affect their b i n d i n g with D N A (Paul and G i l m o u r , 1975) a n d m a y be required for the regulation o f gene activity.

To whom reprint requests should be addressed. Address for correspondence." Dr. Ratna Bose, Department of Immunology, Faculty of Medicine, University

of Manitoba, 770 Bannatyne Avenue, Winnipeg, Manitoba, Canada R3E OW3. 0167-4943/82/0000-0000/$02.75

© 1982 Elsevier Biomedical Press

340 Phosphorylation of N H C proteins has been implicated in the expression of genes (Jansing et al., 1977; Park et al., 1977) and is reported to increase transcription, whereas, dephosphorylation is reported to decrease it (Offerbacher and Klein, 1975). An increase in acetylation of N H C proteins has been reported during high genetic activity (Jungmann and Schweppe, 1972; Suria and Liew, 1974). Polyamines have been implicated in cell multiplication and growth (Fillingame, 1975; Janne et al., 1978). Our earlier study showed a sharp decrease in spermine and spermidine concentration in brain of rats with increasing age (Das and Kanungo, 1982). Spermine and spermidine showed stimulatory effects on phosphorylation and acetylation of specific histones in immature rats. This effect was observed to decrease in adult and old rats (Das and Kanungo, 1979). Hence, we have undertaken the following in vitro approach to understand the role of polyamines on phosphorylation and acetylation of N H C proteins of the brain of rats as a function of age.

Materials and methods

Animals Albino rats of Wistar strain were used. They were maintained, bred and kept in a rat colony at 24 + 2°C with artifical illumination for 12 h followed by a dark period for 12 h.

Chemicals The biochemicals were procured from Sigma Chemical Co., U.S.A. Orthophosphoric acid (carrier-free) 32p and sodium [U-t4C]acetate (sp. act. 40-60 m C i / m m o l ) were purchased from Bhabha Atomic Research Centre, Trombay, Bombay, India.

Preparation of tissue for incubation The rats were killed by cervical dislocation. Cerebral cortex was immediately taken out, washed in cold saline and then cut into slices of approximately 0.4 mm thickness with a clean blade (Takagaki et al., 1959). Approximately 1.0 g of the sliced tissue was used for the in vitro studies.

Labelling of chromosomal proteins The sliced tissue was incubated in 4.0 ml of Krebs-Ringer bicarbonate buffer (pH 7.4) with shaking at 37°C in Warburg flasks without central well. A mixture of 95% 02 and 5% CO 2 (v/v) was bubbled through the buffer. In vitro phosphorylation (Kadohama and Anderson, 1977) and acetylation (Sung et al., 1977) were studied by adding spermine (10 -5 M) or spermidine (6 × 10 -5 M) to the experimental flasks. Cycloheximide (2 × 10 -4 M) was added to each flask to inhibit protein synthesis. The flasks were incubated at 37°C in a water bath and shaken for 30 min. Then 0.1 mCi of 32p-orthophosphate and 0.1 mCi sodium [u-laC]acetate were added to the flasks set up for phosphorylation and acetylation, respectively. The buffer used for phosphorylation was free of phosphate. The incubation was continued for 60 min.

341

Isolation and characterization of chromosomal proteins The purification of chromosomal proteins was carried out at 0 + 2°C unless otherwise mentioned. At the end of the incubation period, the slices were taken out and washed thrice in cold Krebs-Ringer bicarbonate buffer. For the purification of chromatin (Bonner et al., 1968), a 5% (v/v) homogenate of the slices was made in saline-EDTA (0.075 M NaC1 and 0.024 M EDTA, pH 8.0). The crude nuclear pellet was collected by centrifugation at 1500 x g in an IEC PR-6 model refrigerated centrifuge for 30 min. After washing in saline-EDTA, the nuclei were lysed by homogenization in 0.05 M TRIS (pH 8.0), containing 1.0 mM NaHSO 3. The crude chromatin was collected by centrifugation at 10000 x g and washed twice in 0.05 M TRIS (pH 8.0), containing 1.0 mM NaHSO 3. It was then purified by centrifugation in 1.7 M sucrose (prepared in 0.01 M TRIS, pH 8.0) at 30000 x g for 3 h using a VAC 601 ultracentrifuge. The purified chromatin was then resuspended in 0.01 M TRIS (pH 8.0), and dialysed against the same buffer overnight. The dialysed suspension was then passed through a 26-gauge hypodermic needle thrice and stirred for 30 min, and then centrifuged at 10000 x g for 30 min. The purity of the chromatin was then checked by calculating the protein to DNA ratio (approximately 2.4) and observing the absorption spectrum of the supernatant obtained. This was then used for the isolation of proteins. N H C proteins were extracted from the dehistonised chromatin (Elgin and Bonner, 1970). The concentration of chromatin was adjusted to 0.5 mg D N A / m l with 0.01 M TRIS (pH 8.0). One-fourth volume of 2 N H2SO 4 was added dropwise to the vigorously stirring chromatin. The solution was then allowed to flocculate for 30 min on ice. Then the precipitate was collected by centrifugation at 17 000 x g for 20 min. The pellet containing N H C proteins bound to DNA was washed with cold 0.4 N H zSO4. The DNA was removed (Clark and Felsenfeld, 1971) and the N H C proteins were immediately brought to room temperature where all the subsequent steps were carried out. The N H C proteins-containing pellet was suspended in 1% SDS-I% mercaptoethanol-0.01 M TRIS (pH 7.5) and was dialysed overnight against 0.01 M sodium phosphate buffer (pH 7.0), containing 0.1% SDS, 0.1% mercaptoethanol and 10% glycerol, its protein content and radioactivity were determined. It was then analysed on 10% SDS-TRIS-glycine polyacrylamide gels (Laemmli, 1970). The gel was scanned at 500 nm. Two mm thick slices were cut by a Bio-Rad model 190 electrophoresis gel slicer. The slices were digested overnight at 37°C in 0.2 ml of 30% H202 (v/v). The radioactivity in each slice was counted in an LS-100 C Beckman scintillation counter. DNA (Schneider, 1957) and protein (Lowry et al., 1951) were estimated.

Results

A sharp decrease in the concentration of all the three chromatin constituents was observed from 2 to 15 wk of age. A very slight decrease in N H C proteins and no change in histone and DNA occurred from 15 to 84 wk of age (Table I). Table II shows that phosphorylation and acetylation of total N H C proteins

342 TABLE I Contents of chromosomal proteins and D N A ( m g / g wet wt.) of the cerebral cortex of female rats of different ages. Age (wk)

Histone

DNA

Non-histone

Mean 5: SD

P

Mean _+ SD

P

Mean _+ SD

P

2 15

2.85 + 0.10 2.13+0.07

< 0.001

2.37_ 0.16 1.70+0.08

< 0.01

1.87 + 0.20 1.37+0.15

< 0.05

( - 25%) 2.09_+0.25

NS

< 0.05

( - 27%) 1.24_+0.26

NS

84

( - 28%) 1.44_+0.14 (-15%)

Each value represents the mean _+ SD of triplicate determinations done on samples from 4 to 5 rats. P values less than 5% were taken as significant; NS = not significant; SD = standard deviation. This applies for both of the tables.

decrease

with increasing

acetylation

of NHC

age. Both

the polyamines

proteins greatly in immature

stimulate

phosphorylation

r a t s . T h e e f f e c t is r e d u c e d

and

in adult

r a t s a n d is n e g l i g i b l e i n t h e o l d o n e s . The

densitometric

scanning

of NHC

proteins

(Fig. l) resolved

in SDS-poly-

T A B L E II Effects of spermine (10 -5 M) and spermidine (6 × 10 -3 M) on phosphorylation and acetylation of N H C proteins of cerebral cortex of female rats of different ages. Age

Experimental condition

Phosphorylation Mean

2 wk

Slice Slice+ Spm. Slice + Spd.

15 wk

84 wk

7.24 10.71 ( + 48%) 9.10

+

Acetylation

SD

P

0.65 1.16

<0.01

0.58

NS

Slice Slice + Spm.

3.95 4.41

0.34 0.46

NS

Slice + Spd.

3.52

0.37

NS

Slice Slice + Spin. Slice+ Spd.

1.96 1.72 1.67

0.35 0.35 0.06

NS NS

Mean

+

SD

P

1.22 2.14 ( + 75%) 2.25 ( + 84%)

0.32 0.11

< 0.001

0.21

< 0.001

0.46 0.55 ( + 20%) 0.67 ( + 46%)

0.01 0.04

< 0.02

0.01

< 0.001

0.25 0.18 0.14 (-44%)

0.07 0.03 0.01

NS < 0.05

Specific activity of phosphorylation and acetylation of N H C proteins are given as c p m / / t g protein. Each point represents the mean + SD values of triplicate determinations done on samples from 4 to 5 rats. Spm. = spermine; Spd. = spermidine.

343

2-WK

15-WK

"l~

84WK

3 ' ~ o.2

xS

2

I. ....":[,..;~ " ! ~"

",

°" ;E

....: ' ~ " ' ' ~ ' " " ' ~ J o

~g Or-

.-.

oo

g

.

i

.~..,\

,,,o.

•

~

.

. . . . --..

..,,,x,.."

!i

: ...... J ~ " . ....

IX

0A

0.4

q o.2

.2

o 0

0 I

0

I

I

3

6

I

I

1

I

9 0 3 6 Distance of m i g r a t i o n

I

9 (cm)

I

0

I

I

t

3

6

9

Fig. 1. Densitometric scans and phosphorylation and acetylation of N H C proteins of the cerebral cortex of female rats of different ages.

acrylamide gel at 500 nm does not show any significant difference among the three ages, whereas there is considerable variation in the degree of phosphorylation and acetylation of individual NHC proteins with age (Figs. 1-3). While considering the degree of phosphorylation (Fig. 2), the fractions 11, 12, 23 and 24 of adult rats show much higher phosphorylation than those of immature and old rats. The high Control

Spermine

Sper'midine

4-- 2 week

~0 2 -

u

,,..,.

12 16 19

8

17

23

12

8

17 23

15 week

o

c

r~

19 24

84 week

N

t~ 2

13

26

12

28

5

23

28

Fig. 2. Effects of polyamines on phosphorylation of individual N H C proteins of the cerebral cortex of female rats of different ages.

344

Control

Spermidine

Spermine

3

N" b x E

5 9 15 week

14

5

14

22

4

9

4

9

4

9

15 19

28

6

9

28

6 9

28

6 9

13

19

26

'-7.

g .o_

9

19

25

13

22

19

25

84 week

5

9

19

26

17

25

Fig. 3. Effects of p o l y a m i n e s o n acetylation of i n d i v i d u a l N H C proteins of the cerebral cortex of female rats of different ages.

phosphorylation of fractions 18 and 19 of immature rats is not seen in the adult and the old. Furthermore, the degree of phosphorylation of fractions 13 and 26 decreases with increasing age. Our studies (Fig. 2) show that polyamines have variable effects on phosphorylation of individual N H C protein fractions. Spermine is seen to stimulate the fractions 12, 16, 19 and 20 in immature rats, whereas, spermidine stimulates 7-10, 17 and 22-24 fractions. These stimulatory effects are lower in the adults and are not seen in the old rats. The studies on the incorporation of [14C]acetate into the individual N H C proteins show significant changes in the acetylation of only a few proteins as a function of age, for example, fractions 5, 9, 14, 22 and 25 (Figs. 1 and 3). Polyamines selectively stimulate acetylation of a few N H C proteins (Fig. 3). Spermine stimulates fractions 4, 5, 9, 13, 15, 19, 20, 22 and 28, whereas spermidine stimulates fractions 6, 9, 13, 16, 17, 19 and 25-27. This effect decreases and varies slightly with age.

Discussion

Aging is an universal biological phenomenon. Changes in the chemical nature of chromatin may be responsible for the structural and functional changes seen during aging. The decrease in the concentrations of chromatin constituents during 2 to 15 wk (Table I) expressed as per gram wet weight basis may be attributed to the significant increase in the brain weight as a result of increase in cell volume during the growing period.

345 Phosphorylation of NHC proteins is reported to result in conformational changes in chromatin (Gershey and Kleinsmith, 1969), augment the template activity of DNA-histone complexes and cause stimulation of RNA synthesis (Ord and Stocken, 1968). The decrease in the phosphorylation of NHC proteins with increasing age (Table II) observed by us may, therefore, be correlated with the decrease in the template activity with increasing age. Polyamines are cations. Therefore, they may act directly at the chromatin level by binding with DNA phosphates (Tabor, 1962). Hence, they may alter the structure of chromatin and thereby alter its template activity. Our studies show that polyamines, spermine and spermidine, stimulate phosphorylation of NHC proteins greatly in immature rats and this effect decreases with increasing age (Table II). This decrease may be due to increasing condensation of chromatin with increasing age as stated by earlier workers (Gurley et al., 1977). Alternatively, lesser numbers of phosphate acceptors (serine and threonine) may be available for the action of nuclear protein kinases in older ages. As the effect of spermine on the phosphorylation of total NHC proteins is greater than that of spermidine (Table II), it shows that the end product of the polyamine biosynthetic pathway, spermine, plays a greater role at the genetic level. An increase in acetylation of NHC proteins has been reported during high genetic activity (Jungmann and Schweppe, 1972; Suria and Liew, 1974). The sharp decrease in acetylation of NHC proteins with increase in age (Table II) indicates, therefore, a decrease in template activity of the brain chromatin. Since acetylation of histones increases prior to an increase in RNA synthesis (Allfrey et al., 1964; Ruiz-Carrillo et al., 1975), the stimulation of acetylation of NHC proteins by polyamines in immature and adult rats suggests their involvement in the developing phase (Moruzzi et al., 1968; 1975). From the densitometric scanning of NHC proteins resolved in SDS-polyacrylamide gel, there appears to be a loss of specific NHC proteins as a function of age. As NHC proteins are believed to be involved in gene expression (Kleinsmith et al., 1970), these specific NHC proteins may be necessary for the expression of certain genes during development and reproduction. The age-dependent differential phosphorylation of individual NHC proteins observed by us (Figs. 1 and 2) may be due to differences in the activities of specific protein kinases and phosphatases (Jungmann and Krainas, 1977). Also, conformational changes in chromatin that may decrease the accessibility of serine and threonine residues to protein kinases may alter the phosphorylation pattern. Polyamines may alter the structure of chromatin and also its template activity by their direct action at the chromatin level (Tabor, 1962). The effect of polyamines on differential phosphorylation of individual NHC proteins (Fig. 2) indicates that polyamines have a specific role at the genetic level, and the age dependent variations in their levels may alter the expression of specific genes. Acetylation of lysine residues of chromosomal proteins is reported to alter the structure and function of chromatin and stimulate DNA-dependent RNA synthesis (Huang and Bonner, 1962). The general decrease in the acetylation of NHC proteins (Figs. 1 and 3) may be due to conformational changes in chromatin that may

346

decrease the accessibility of lysyl residues for acetyl transferase may decrease with age and account for the decrease in acetylation. The differential effects of the two polyamines on acetylation of specific NHC proteins (Fig. 3) indicate that they may have specific effects on gene expression. Similar results were observed in our laboratory with epinephrine and oestradiol where both stimulated acetylation of N H C proteins in young, but not in adult and old rats (Thakur et al., 1978). On the other hand, calcium was shown to stimulate NHC proteins (Kanungo and Thakur, 1977, 1979). Our results suggest that differential phosphorylation and acetylation of NHC proteins may play a role in the template activity of chromatin. Furthermore, modulation of these modifications by polyamines may be responsible for the expression of specific genes and repression of others at different phases of the life span. This is consistent with the model for aging (Kanungo, 1975) according to which differential activation and repression of genes by various effectors may be responsible for differentiation, development and senescence of an organism.

Acknowledgements This research was supported by grants from the Nuffield Foundation, England, PL-480 (IN-ARS-24), Department of Science and Technology and University Grants Commission, India, to M . S . K . R . B . thanks the C.S.I.R., India, for a research fellowship.

References Allfrey, V.G., Faulkner, R. and Mirsky, A.E. (1964): Acetylation and methylation of histones and their possible role in the regulation of RNA synthesis. Proc. Natl. Acad. Sci. U.S.A., 51,786-794. Barrett, T., Maryanka, D., Hamlyn, P.H. and Gould, H.J. (1974): Nonhistone proteins control gene expression in reconstituted chromatin. Proc. Natl. Acad. Sci. U.S.A., 71, 5057-5061. Bonner, J., Chalkley, G.R., Dahmus, M., Frambroug, D., Fugimura, F., Haung, R.C., Huberman, J., Jenaen, R., Marushige, K., Ohlenbusch, H., Olivera, B. and Widnolm, J. (1968): Isolation and characterization of chromosomal nucleoproteins. Methods in Enzymol., 12(B), 3-65. Clark, R.J. and Felsenfeld, G. (1971): Structure of chromatin. Nature (London), 229, 101-106. Das, R. and Kanungo, M.S. (1979): Effects of polyamines on in vitro phosphorylation and acetylation of histones of cerebral cortex of rats of various ages. Biochem. Biophys. Res. Commun., 90, 708-714. Das, R. and Kanungo, M.S. (1982): Activity and modulation of ornithine decarboxylase and concentrations of polyamines in various tissues of rats as a function of age. Exp. Gerontol., in press. Elgin, S.C. and Bonner, J. (1970): Limited heterogeneity of the major nonhistone chromosomal proteins. Biochemistry, 9, 4440-4447. Elgin, S.C.R. and Weintraub, H. (1975): Chromosomal proteins and chromatin structure. Ann. Rev. Biochem., 44, 725-774. Fillingame, R.H., Jorstad, C.M. and Morris, D.R. (1975): Characterization of acetylated liver nuclear acidic proteins. Proc. Natl. Acad. Sci. U.S.A., 72, 4042-4045. Gershey, E.L. and Kleinsmith, L.J. (1969): Phosphorylation of nuclear proteins in avian erythrocytes. Biochim. Biophys. Acta., 194, 519-525. Gurley, L.R., Waiters, R.A., Hildebrand, C.E., Hohmann, P.G., Barham, S.S., Deaven and Tobey, R.A.

347 (1977): International Cell Biology, pp. 420-429. Editors: B.R. Brinkley and K.R. Poter. Rockefeller University Press, New York. Huang, R.C. and Bonner, J. (1962): Histone, a suppressor of chromosomal RNA synthesis. Proc. Natl. Acad. Sci. U.S.A., 48, 1216-1222. Janne, J., P6s6, H. and Raina, A. (1978): Polyamines in rapid growth and cancer. Biochim. Biophys. Acta., 473, 241-293. Jansing, R.L., Stein, J.L. and Stein, G.S. (1977): Activation of histone gene transcription by nonhistone chromosomal proteins in WI-38 human diploid fibroblasts. Proc. Natl. Acad. Sci. U.S.A., 74, 173-177. Jungmann, R.A. and Schweppe, J.S. (1972): Mechanism of action of gonadotropin. I. Evidence for gonadotropin-induced modifications of ovarian nuclear basic and acidic protein biosynthesis, phosphorylation and acetylation. J. Biol. Chem., 247, 5535-5542. Jungmann, R.A. and Krainas, E.G. (1977): Nuclear phosphoprotein kinases and the regulation of gene transcription. Int. J. Biochem., 8, 819-830. Kadohama, N. and Anderson, K.M. (1977): Acetylation and phosphorylation of nuclear proteins from growing or dividing rat ventral prostate. Can. J. Biochem., 55, 513-520. Kanungo, M.S. (1975): A model for aging. J. Theor. Biol., 53, 253-261. Kanungo, M.S. and Thakur, M.K. (1977): Phosphorylation of chromosomal proteins as a function of age and its modulation by calcium. Biochem. Biophys. Res. Commun., 79, 1031-1036. Kanungo, M.S. and Thakur, M.K. (1979): Phosphorylation and acetylation of nonhistone chromosomal proteins of the brain of rats of various ages and its modulation by calcium and estradiol. Biochem. Biophys. Res. Commun., 86, 14-19. Kleinsmith, L.J., Heidema, J. and Carrol, A. (1970): Specific binding of rat liver nuclear proteins to DNA. Nature (London), 226, 1025-1026. Laemmli, U.K. (1970): Cleavage of structural proteins during the assembly of the head of bacteriophage. Nature (London), 227, 680-685. Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. (1951): Protein measurement with the folin phenol reagent. J. Biol. Chem., 193, 265-275. Moruzzi, G., Barbiroli, B. and Galdarera, C.M. (1968): Polyamines and nucleic acid metabolism in chick embryo. Incorporation of labelled precursors into nucleic acids of subcellular fractions and polyribosomal patterns. Biochem. J., 107, 609-613. Moruzzi, G., Barbiroli, B., Moruzzi, M.S. and Tadolini, B. (1975): The effect of spermine on transcription of mammalian deoxyribonucleic acid - dependent ribonucleic acid polymerase. Biochem. J., 146, 697-703. Offerbacher, S. and Klein, E.S. (1975): Differential phosphorylation of rat liver nuclear non-histone proteins in vitro. Biochem. Biophys. Res. Commun. 66, 375-382. Ord, M.G. and Stocken, L.A. (1968): Variations in the phosphate content and thio-disulphide ratio of histones during the cell cycle. Studies with regenerating rat liver and sea urchins. Biochem. J., 107, 403-410. Park, W., Jansing, R., Stein, J. and Stein, G. (1977): Acetylation of histone gene transcription in quiescent WI-38 cells or mouse liver by a nonhistone chromosomal protein fraction from HeLa $3 cells. Biochemistry, 16, 3713-3721. Paul, J. and Gilmour, R.S. (1975): In: The structure and Function of Chromatin, Ciba Foundation, Symposium 28, pp. 181-198. Editors: D.W. Fitzsimons and G.E.M. Wolstenholme. Ruiz-Carrilo, A., Wangh, L.J. and Allfrey, V.G. (1975): Processing of newly synthesized histone molecules. Science, 190, 117-128. Schneider, W.C. (1957): Determination of nucleic acids in tissues by pentose analysis. Methods Enzymol., 3, 680-684. Sung, M.T., Harford, J., Bundman, M. and Vidalakas, G. (1977): Metabolism of histones in avian erythroid cells. Biochemistry, 16, 279-285. Suria, D. and Liew, C.C. (1974): Isolation of nuclear acidic proteins from rat tissues. Biochem. J., 137, 355-362. Tabor, H. (1962): The protective effect of spermine and other polyamines against heat denaturation of deoxyribonucleic acid. Biochemistry, 1,496-501. Takagaki, G., Hirano, S. and Nagata, Y. (1959): Some observations on the effect of D-glutamate on the

348 glucose metabolism and the accumulation of potassium ions in the brain cortex slices. J. Neurochem., 4, 124-134. Thakur, M.K., Das, R. and Kanungo, M.S. (1978): Modulation of acetylation of chromosomal proteins of the brain of rats of various ages by epinephrine and estradiol. Biochem. Biophys. Res. Commun., 81, 828-831.