EXPERIMENTAL

CELL

RESEARCH

195,

255-262

(1991)

Nuclear Protein Kinases in Rat Liver: Evidence for Increased Histone HI Phosphorylating Activity during Liver Regeneration A.M. lstitufo

MARTELLI,* di Anatomia

CCARINI,~ l~mana $Department

~.MARMIROLI,$

Norm&, *I~niuersitci of Biochemistry,

M. MAZZONI,~P.J.BARKER,$

di Bologna e tFerrara, Italy; $lstituto Institute of Animal Physiology, Rabraham,

MATERIALS

INTRODUCTION Protein phosphorylation is a major bioregulatory event taking place throughout the cell in response to a wide variety of stimuli [11. The cell nucleus as well has long been recognized as a site of action of protein kinases, and many types of phosphotransferases have been described in the inner nuclear compartment [2-51. In rat liver cells, it has been found that protein phosphorylation changes after partial hepatectomy, and a correlation has been proposed between proliferative events and levels of whole cell protein kinase activity, particularly concerning CAMP-independent enzymes and protein kinase C [6, 71. This latter undergoes profound changes in activity and subcellular localization in regenerating compared to normal liver, and it has been thought to be involved in proliferative events [7]. Nuclear protein kinase C has been largely studied in rat liver, even though conflicting results have been reported concerning molecular size and regulation by Ca2+ and lipid cofactors [2, g-101. In this paper we report an analysis of the phosphorylation of normal and regenerating rat liver nuclei and characterize a protein kinase activ-

requests Normale,

di Citomorfologia Cambridge,

del IJnited

CNR, Kingdom

Bologna,

It&y;

and

ity overexpressed during the replicative phase at 22 h from partial hepatectomy.

Comparison of protein kinase activity in normal and regenerating rat liver nuclei indicates that exogenous histone Hl is hyperphosphorylated in 22-h regenerating nuclei. The protein kinase involved is not sensitive to protein kinase A inhibitor, is inhibited by staurosporine and by an anti-PKC polyclonal antibody, utilizes only ATP, and also phosphorylates the C-terminal fragment of histone Hl. These data suggest that protein kinase C is responsible for the observed effects, in agreement with the presence of this enzyme in normal and regenerating nuclei demonstrated by immunoblotting. cl 1991 Academic Press, Inc.

‘To whom correspondence and reprint dressed at: Istituto di Anatomia Umana Mortara, 66, 44100 Ferrara, Italy.

R.S.GILMOUR,§ANDS.CAPITANI~~'

should be adVia Fossato di

AND METHODS

Source of materials. [r-““PIATP (5000 Ci/mmol) was obtained from Amersham, IJK and [y-““P]GTP (6000 Ci/mmol) from New England Nuclear, West Germany. Histone Hl (fraction III-S), protein kinase inhibitor (I’KAi), 1,2-dioleoyl-mc-glycerol (D(i), bovine brain phosphatidylserine (PS), CAMP-dependent protein kinase (PKA), calmodulin (CM), casein, soybean trypsin inhibitor, trypsin, N-bra mosuccinimide (NBS), leupeptin, phenylmethylsulfonyl fluoride (PMSF), L-trans-epoxysuccinic acid (E 64), dithiothreitol (DTT), and phenyl-Sepharose were from Sigma (St. Louis, MO). Calpain inhibitor I and II, staurosporine (ST), and aprotinin were purchased from Boehringer-Mannheim, West Germany, and DEAE cellulose from Whatman, Maidstone, IJK. Electrophoresis reagents were from BioRad Laboratories (Richmond, CA), and X-OMAT S films from Kodak, France. All other reagents were of analytical grade. Partial hcpatectomy. Male Wistar rats (150-200 g body wt) were operated on according to Higgins and Anderson [ 111. Approximat,ely two-thirds of the liver was surgically removed, and sham-operated rats were used as controls for normal livers. The rats were fed ad libitum and then sacrificed at different time intervals ranging from 3 to 26 h. Livers were removed and used for isolation of nuclei. Isolation of rat liuw nuclei. The livers, minced in small pieces and blotted on filter paper to remove blood, were homogenized in 5 vol of 0.25 M sucrose, 5 mM MgCl,, 0.5 mM PMSF, 0.5 mM DTT, 10 mA4 Tris-HCl, pH 7.4, in a glass-Teflon homogenizer. The liver homogenate was filtered through four layers of cheesecloth and sedimented at 7OOg for 15 min at 2°C. The crude nuclear pellet was resuspended in 2.4 M sucrose, 5 mA4 MgCl,, 0.5 mM PMSF, 0.5 mM DTT, 10 mM Tris-HCl, pH 7.4, and sedimented at 50,OOOg for 1 h at 2°C. The clear nuclear pellet was washed with homogenization buffer by sedimentation at 7OOg and stored at ~25°C until use. Membrane-depleted nuclei were obtained by adjusting the sucrose density buffers to 0.2% Triton X-100. Nuclear extractions were carried out in the presence of 50 @g/ml leupeptin, 50 pg/ml soybean trypsin inhibitor, 15 pg/ml calpain inhibitor I, 7 pg/ml calpain inhibitor II, 1 Kg/ml E 64, and 2 pg/ml aprotinin as protease inhibitors. For ultrastructural analysis, nuclei were fixed in 2.5% glutaraldehyde in 0.1 M phosphate buffer (pH 7.2) for 30 min, postfixed in 1% osmium tetroxide, and embedded in plastic resin. Thin sections were stained with uranyl acetate and lead citrate and observed with a Philips CM 10 electron microscope. Assays of cytoplasmic enzyme markers were performed as previously described [ 21. Preparation of HI-NBS. Histone III-S was digested with N-bromosuccinimide as described 112, 131. The peptide mixture was fractionated by HPLC and each fraction was assayed by polyacrylamide gel electrophoresis. The fractions showing the required molecular

255 All

Copyright G 1991 rights of reproduction

0014-482:/91 _c w.00 by Academic Press, Inc. in any form reserved.

256

MARTELLI

weight were prohed with PKA, PKC, and related activators or inhihitars under the same conditions described for detection of kinase activity in nuclei. I’reparution of t>KC and PKM. Protein kinase C was obtained from rat brain essentially as described by Kikkawa et al. [ 141, omitting the final chromatographic step. The partly purified enzyme showed a specific activity of 24 nmol ATP/min/mg protein. PKM was obtained by partial proteolysis of PKC in the presence of 50 @g/ml trypsin for 3 min at 30°C [15,16]. The digestion was stopped with 3 mg/ml soybean trypsin inhibitor and by chilling in ice. The preparation of PKM was characterized on the basis of Ca” and PS independency and by immunoblotting. I’rc~paration of “‘1’.labeled h&one HI. Histone Hl was phosphorylated with PKC purified from rat brain. The incubation mixture, containing 10 mg histone, 40 pg PKC, 20 mMTris-HC1, pH 7.5,5 mM MgCI,, 0.5 mA4 DTT, 0.25 mM CaCl,, 0.1 mg PS, 4 fig DG, 0.13 mM [y-““PJATP (sp act 461 Ci/mol) in a final volume of 1 ml, was incuhated for 3 h at 30°C. The reaction was terminated with 25% TCA, and the phosphorylated histone was recovered as described by Meisler and Langan [l’i]. A.ssa,~ of protein phosphatuse actic?ity. Phosphohistone phosphatase activity was assayed by measuring the release of “2P1 from phosphorylated histone Hl, either under the conditions employed for the assays of nuclear kinase activity or as described by Zwiller et al. [ 181, in which case the incubation mixture contained, in a final volume of 0.1 ml, 1 mA4 MnCl,, 0.5 mM DTT, 1 mM EDTA, 50 mM Tris+HCl, pH 7.5, 0.15 mg “21’-laheled histone (sp act 2000 cpm/wg), and 200 pg protein as nuclei. The ‘=P, released was determined as the phosphomolyhdate complex [19]. I’r-otcin phosphorylation and clssa.y of hinaw nctiuit\,. Nuclei (100 pg protem) were incubated at 30°C for 10 min in a reaction mixture containing in a final volume of 50 ~1, 5 mM MgCl,, 3 mM DTT, 100 PM vanadate, 50 mA4 Tris-HCI, pH 7.4, 13 PM ATP, and 1 FCi of [“‘P]ATP. When indicated, 250 PM CaCl,, 100 pg/ml phosphatidylserine, 4 pg/ml 1.2 dioleoyl-rat-glycerol, 1 mM EGTA, 20 pg/ml calmodulin, 10 PM CAMP, 500 pg/ml PKA inhibitor, 10 nM staurosporine were used. To assess the nucleoside triphosphate requirement, GTP substituted for ATP at the same concentration and specific activity. For phosphorylation of exogenous substrates, 25 pg of casein, histone Hl, or Hl-NBS was included in the assay. For characterization of kinase specificity of Hl and Hl-NBS, 25 pg of suhstrate protein was inruhated with 3 pg of PKC, 10 pmol units of PKA X catalytic subunit, under the same conditions described above for nuclei phosphorylation. The reactions were stopped with appropriate volumes of’4~ sample buffer (0.25 M Tris-HCl, pH 6.8, 8% SDS, 40% glycerol, 20% 0.mercaptoethanol, 0.0050; hromophenol blue), boiled 5 min, and electrophoresed on 12.5 or 15?& acrylamide-0.1% SDS gels according to Laemmli [20]. The gels were stained with Coomassie R-250, destained, dried, and autoradiographed with Kodak X-OMATS S films. Protein determination was performed according to Lowry (211. Pwparation of anti-PKC’ pol&onal antibodies. The antibodies were prepared by injecting rabbits with the synthetic peptide CVVNPQFVHPILQSAV derived from the C-terminal sequence of protein kinase C as described by Parker et al. [22]. The peptide was conjugated to a purified protein derivative oftuberculin and administered as described by Lachmann et al. [23]. Serum was stored at 70°C until use. Nuclei were incuIncubation of w&i with polyclonul antibodies. hated with either preimmune or immune serum at a 1:lO dilution in the presence of .jO pg/ml leupeptin, 50 pg/ml soybean trypsin inhihitor. 20 pg/ml aprotinin, 1 mA4 PMSF. After 1 h at 4”C, the phosphotransferase activity was assayed with exogenous histone Hl as described above. Immunoblofting. Samples were separated onaO.l% SDS, 8% polyacrylamide gel. Proteins were then transferred to nitrocellulose in

ET

AL.

0.192 M glycine, 0.025 M Tris-HCI pH 8.3, 20% methanol at 40 volts (constant) for 24 h, at 4°C. Nitrocellulose strips were saturated for 1 h at 37°C in Dulhecco’s phosphate-butfered saline (PBS), pH 7.4, containing 3% bovine serum albumin (BSA). They were then reacted for 6 hat room temperature in PBS, 0.1% BSA containing a 1:50 dilution of the serum. After four washes in PBS, 0.1% Tween 20, they were incubated for 2 h at room temperature in PBS, 0.1% BSA, 0.1% Tween 20 containing a 1500 dilution of an alkaline phosphatase-conjugated anti-rahhit IgG (Sigma). Strips were washed as above in PBS-Tween 20 and positive binding was detected in 100 mM Tris-HC1, pH 9.5, 4 mM MgCl,, containing 0.05 mg/ml 5.hromo-4.chloro-3-indolylphosphate and 0.1 mg/ml p-nitrohlue tetrazolium as substrates.

RESULTS

A crucial point concerning the use of isolated nuclei is the purity of nuclear preparations, and this was preliminarily assessed by means of morphological and biochemical criteria. The ultrastructural data indicate that the method employed yielded nuclei free of cytoplasmic contamination and, in the case of detergent-treated nuclei, depleted of the nuclear membrane (Fig. 1). As previously described [ 241, negligible amounts of plasma membrane and cytoplasmic enzyme markers were found (not shown). Under basal conditions, protein phosphorylation, as analyzed on denaturing gels, did not show significant differences between normal and regenerating nuclei, except for some bands including core histones and two proteins of approximate MW of 21 and 25 kDa, which were more phosphorylated in regenerating nuclei. The nuclear kinase activities were not modulated by Ca2+, PS, DG, CAMP, or CM (Fig. 2), and the same results were obtained regardless of the presence or the absence of the nuclear membrane (data not shown). Since the endogenous system did not reveal significant differences related to the partial hepatectomy, exogenous substrates were added to the assay. Casein was phosphorylated to the same extent in the two types of nuclei, while histone Hl was strongly hyperphosphorylated in regenerating nuclei. While this effect was not dependent on Ca2+, PS, DG, EGTA, CAMP, or CM (Fig. 3), nor was it affected by PKA inhibitor, it was totally inhibited by the PKC inhibitor staurosporine at concentrations as low as 10 nM (Fig. 4). The dependency of Hl hyperphosphorylation on nucleoside triphosphate was assayed by comparing ATP and GTP as phosphate donors. The phosphorylation in nuclei was totally dependent on ATP; no label was incorporated in the presence of GTP (Fig. 5). Since the response to staurosporine and the nucleoside triphosphate requirement suggested a possible involvement of PKC, a more specific substrate was used, namely, the N-bromosuccinimide cleavage fragment of

FIG.

absence

Electron micrograph (C, D) of Triton X-100.

1.

analysis of normal (A, C) and regenerating (B, D) rat liver nuclei isolated in either the presence (A, B) or the The nuclear membrane is completely removed when detergent is included in the sucrose density huffers.

258

MARTELLI

Mrx

N

IO-~

200

21

-

R

I

5-

---C

Ps Ca

DG

CAMP

cs

CM

FIG. 2. SDS gel electrophoretic analysis of protein tion in nuclei from normal (N) and 22-h regenerating Nuclei were isolated in the presence of Triton X-100 with the indicated factors as described under Materials

phosphoryla(R) rat liver. and incubated and Methods.

histone Hl, referred to as Hl-NBS, which corresponds to the C-terminal fragment and is specifically phosphorylated by PKC [12, 131 and not by PKA (Fig. 6). Hl-NBS was more phosphorylated in regenerating nuclei and showed the same insensitivity to cofactors together with the response to staurosporine (Fig. 7). In addition, exogenous histone hyperphosphorylation was restricted to the nuclei isolated from 22-h regenerating

Mrx 200

NRNRNRNR

1o-3 -

116.2 97.4

-

66.2

-

46

-

21.6

-

ET

AL.

liver, since it was absent in nuclei obtained from other regeneration times ranging from 3 to 26 h (Fig. 8). The turnover of phosphate groups during the assay was evaluated by employing 32P-labeled Hl under the incubation conditions for either protein kinases or phosphatases [ 181. The release of 32P was negligible under the standard incubation conditions and very low in the assay devised to optimize protein phosphatase activity. In any case, no differences were found between control and regenerating nuclei (Table 1). The inhibitory effect of staurosporine strongly suggested an involvement of PKC in the observed hyperphosphorylation of exogenous histone Hl and Hl-NBS, even though the phenomenon was independent of the presence of PKC activators. We reasoned that this could be due to the presence in our nuclear preparations of a proteolytically cleaved catalytic fragment of PKC (i.e., PKM), which reportedly does not require the presence of PS, DG, and Ca2+ for activity [ 15,161. Therefore, we probed our nuclear preparations for the presence of PKC and/or PKM by using immunoblotting techniques. We employed a rabbit polyclonal antiserum which had been raised against a synthetic peptide as described under Materials and Methods. The antiserum recognized a single band (80-82 kDa) when tested against partially purified protein kinase C from rat brain (Fig. 9). No reaction was detectable when the rabbit preimmune serum was used. The serum was also able to detect PKM which was prepared from PKC by limited trypsin digestion. The molecular weight of PKM was around 45-50 kDa, in good agreement with the reported molecular weight of the catalytic subunit of PKC

N

R

Ca

CM

N

R

----C

FIG. 3. Phosphorylation casein and histone HI were

PS Ca

W

EGTA

CAMP

of exogenous substrates in normal (N) used, and the indicated modulators were

and 22-h regenerating included as described

c

(R) rat liver nuclei. Twenty-five under Materials and Methods.

micrograms

of

Hl

Mrx

it3

200

PHOSPHORYLATION

IN

REGENERATING

LIVER

NRNRNR

Ca ps DG

-

Mrx

116.297.4-

259

NUCLEI

IO-~

Ca PS DG

: T A

E 7 A

66.2-

--P c FIG. 4. Effect of PKA inhibitor (10 nM) on histone Hl phosphorylation ating (R) rat liver nuclei.

PKAi

ST

(500 pg/ml) and staurosporine by normal (N) and regener-

--PKC

[25,26]. In control and 22-h regenerating rat liver nuclei the polyclonal antibody recognized a band with the same electrophoretic mobility asthat seen in partly purified PKC preparations which was present in similar amounts in the two types of nuclei. None of our preparations of nuclei contained detectable PKM. When regenerating nuclei were incubated with the antibody, the phosphotransferase activity was deeply

Hl

FIG. 6.

Probing of histone for PKC and PKA. Incubation assay of protein phosphorylation

reduced (Fig. lo), further ment of PKC.

PKA

PKC

PKA

HI-NBS

Hl and Hl-NBS substrate specificity conditions were as described for the in isolated nuclei.

substantiating

the involve-

DISCUSSION Mr

x10

NRNR

3

200

-

l$$

-

66.2

-

45

-

31

-

-Hl

21.5-

ATP-

GTP

FIG. 5. Dependency on nucleoside triphosphate of exogenous Hl phosphorylation by normal (N) and regenerating (R) rat liver nuclei. Concentration of ATP and GTP was 13 FM, and 1 PCi of both isotopes was used.

Although liver regeneration has been extensively studied, the molecular events involved in the cell proliferation are largely unknown. The possible role of CAMP has been proposed, and fluctuations of CAMP-dependent protein kinase activity have been reported during the prereplicative period [6]. PKC as well has been suggested to take part in liver regeneration, since the soluble fraction of the enzyme showed a biphasic decrease of activity prior to the initiation of DNA synthesis [7]. Our data indicate that the phosphorylation of exogenous histone Hl and Hl-NBS is strongly enhanced in nuclei obtained from 22-h regenerating rat liver as compared with controls and other regeneration times. The phosphotransferase activity involved in this phenomenon is insensitive to several cofactors and inhibitors which are specific for some types of protein kinases that are present within the nucleus. However, this activity is completely suppressed by staurosporine, a well-known inhibitor of PKC [27, 281, and largely inhibited by the anti-PKC antibody. In agreement with the recent results of other investigators [B, 91, our immunoblotting data revealed that only the BO-to 82-kDa form of PKC is

260 Mrr

MARTELLI

tom3

ET

AL.

TABLE

NRNRNRNR

200

-

116.2 97.4 66.2

=

45

-

1

Phosphohistone PhosphataseActivity in Control and 22-h Regenerating Nuclei

-

Assay conditions Control nuclei a b -Ii-NBS

321,

‘“P-labeled HI input

released

302,395

k 27,402

306,093

i- 39,741

290,847 294,410

t-i- 32,570 514,342

1868 4403

Phosphatase labile radioactivity

5 157 3 186

0.62

1951 205 4368 i+ 203

0.67

(%)

1.44

Regenerating nuclei it

1.49

Note. Dephosphorylation was assayed under the standard incubation conditions used for kinase activity (a) or as described under Materials and Methods for optimizing phosphatase activity (b). Data are expressed as cpm of “P and represent mean values of three independent experiments * SD. PS ca

C

FIG. 7. Phosphorylation ating (R) nuclei. Activators under Materials and Methods.

DG

EGTA

ST

of Hl-NBS in normal (N) and inhibitors were used

and regeneras described

present in rat liver nuclei, thus ruling out the possibility that isolated nuclei would contain a cofactor-insensitive form of PKC (PKM). A possible explanation for the lack of effect observed when PKC cofactors are added in vitro is that rat liver nuclei already contain the molecules which are necessary for PKC to be active. This would not be unprecedented because it has been re-

Mr

hrs after hepatectomy 22 3 16

xlo-3 200

cently reported that erythroid nuclei contain a PKC activity which does not require the addition of exogenous cofactors [29]. The authors suggested that this could be due to the presence of the activators in the nucleus. Indeed Ca2+ ions are known to be present inside the nucleus [30,31] and mobilized by IP, for activating nuclear PKC [32]. Our laboratory has repeatedly documented the existence of phosphatidylserine and diacylglycerol in isolated nuclei [33,34], suggesting a potential role for these lipid cofactors in a different regulation of PKC in normal and regenerating nuclei, which contain similar amounts of the enzyme. Accordingly, the diacylglycerol levels found in regenerating nuclei are higher than in normal nuclei (unpublished observations). In addition, Mr x~O-~

26

-

205-

1gp7:; 1 66.2

-

11697.s

31

-

as.-

66--

Hl

45-

21.5-

A

FIG. 8. Time course analysis ating liver nuclei obtained from after partial hepatectomy.

of Hl phosphorylation by regenerrat sacrificed at 3, 16, 22, and 26 h

BCDEFGH

FIG. 9. Immunoblots showing the proteins recognized by the anti-PKC polyclonal antibody. Lanes A, C, E, G: immune serum; B, D, F, H: preimmune serum; A, B: partly purified rat brain PKC; C, D: PKM obtained by limited proteolysis of PKC; E, F: normal rat liver nuclei; G, H: 22-h regenerating rat liver nuclei.

Hl

R

R

PHOSPHORYLATION

N

IN

REGENERATING

R

*.a-HI

LIVER

261

NlJCLEI

tivity which hyperphosphorylates exogenous histone Hl and Hl-NBS. This activity is inhibited by the PKC inhibitor staurosporine, but is insensitive to PKC cofactors. Although in agreement with increasing evidence on the presence and functional role of PKC in the nuclei of many cell types [8-10,44-481, these results await further work to definitely assessthe nature and the regulation of this protein kinase more active in proliferating nuclei. We are indebted to Sandra Matteucci for preparation Lucia Cocco, Maurizio Previati, Anna Maria Billi, and tagnolo for helpful discussion; and to Maurizio Stroscio assistance. This research was supported by grants from Research Council (CNR 89.04123.04 and 89.02470.04) and 60%).

1

2

3

of PKC; to Valeria Berfor technical the National and MPI (40

REFERENCES

4

FIG. 10.

Inhibition by anti-PKC polyclonal antibody of exogenous Hl iperphosphorylation in regenerating rat liver nuclei. After preincubation with the antibody for 1 h at 4”C, the kinase activity was assayed as described under Materials and Methods. Lane 1: immune serum, regenerating (R) nuclei; 2: preimmune serum, regenerating (R) nuclei; 3,4: standard conditions in the absence of antibody, showing the hyperphosphorylation of histone Hl in regenerating (R) versus normal (N) nuclei.

1.

Blackshear, 2, 2957-2969.

2.

Capitani, S., Girard, I’. R., Mazzei, and Manzoli, F. A. (1987) Rio&em. 367 -375.

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