Appearance of a Nuclear Histone H1 Kinase at the Start of DNA Synthesis of Regenerating Rat Liver(Biochemistry)

7 Pages·1992·2.7 MB·English

by  Kouichi Asami

ZOOLOGICAL SCIENCE 9: 1001-1007 (1992) © 1992 Zoological SocietyofJapan
Appearance of a Nuclear Histone HI Kinase at the Start of
DNA
Synthesis of Regenerating Rat Liver
KOUICHI ASAMI
'Division ofBiology, National Institute ofRadiological Sciences,
Chiba-shi 263, Japan
—
ABSTRACT Phosphorylation ofhistone HI and elevation ofnuclear protein kinase activity occurat
the start of DNA synthesis during regeneration of rat liver. X ray irradiation prior to partial
hepatectomydepressed bothelevationofthe nuclearprotein kinaseactivityand DNA synthesis. Thus,
the protein kinase seemed to play a role in the startofDNA synthesis, but the propertyofthe enzyme
wasnotwellcharacterized. The nuclearprotein kinaseactivitywasmeasuredwithcaseinorhistone HI
as substrate. A protein kinase activity specific for histone HI was found in the extract from the
regeneratinglivernuclei, but notthatfromthe irradiatedandpartiallyhepatectomized livernuclei. On
theotherhand,caseinkinaseactivitywasfoundintheextractfromthe regeneratingliverirrespectiveof
prior X ray irradiation. Gel filtration chromatography ofthe nuclear extract revealed the presence of
thehistone HI kinaseandtwocasein kinasesintheregeneratingliver. Histone HI kinasewasobserved
in the regenerating liver, but not in the non-hepatectomized nor the irradiated and hepatectomized
liver. Two casein kinaseswere found in the non-hepatectomized liverand partial hepatectomy with or
without prior X irradiation induced a little increase in the activites. The present results indicate that
appearance ofthe nuclear histone HI kinase activity is required forphosphorylation ofhistone HI and
DNA
the start of synthesis in the regenerating liver.
INTRODUCTION the same period [2]. However, causal re-
latiopnships among these events are not clear. A
Hepatocyte in the adult liver does not prolifer- dose ofXraysenough to inhibit the onsetofDNA
ate in a normal physiological state. But when a synthesis inhibited phosphorylation and synthesis
part of the liver is removed, the rest of the liver ofhistone HI, while it failed to inhibit phosphory-
starts proliferating and when the liver mass has lation andsynthesisofHMGproteins [2, 3]. Thus,
gained the former size the cells return to the phosphorylation ofhistone HI seemed to be close-
quiescent state. Proliferation of hepatocytes are ly related tothe onsetofDNA synthesis. Changes
stimulated by several growth factors, such as in nuclear protein kinase activity were measured,
A
hepatocyte growth factor or hepatopoietin [1]. therefore, during the prereplicative phase of re-
DNA
Before the onset of synthesis the stimulated generation in the liver of partially irradiated rat
cells show many responses such as increase in [4], The protein kinase activity increased at the
Na+- H+ exchange, polyamine metabolism, and onset ofDNA synthesis and the rise ofthe activity
amino acid transport [2]. Phosphorylation of his- was inhibited by X ray irradiation delivered just
HMG
tones and proteins which belong to nuclear before partial hepatectomy [4]. Consequently this
non-histoneproteins, andsynthesisofhistonesand protein kinase seemed to be responsible for phos-
HMG
(high mobility group) proteins also occur phorylation ofhistone HI, although its nature was
DNA
before and during synthesis, and the en- unknown.
zymesofnucleotide motabolism are synthesized at Rat liver nuclei contain several kinds ofprotein
kinases such as histone HI kinase [5, 6], casein
ARPrececcseeeipnvtteeddadMJduarleyyss18:,5,119D99e92p2artment of Biology, Sapporo lkiinna-sdeespe[n7d,en8]t, pprrootteeiinnkikniansaeseC[[190]], Caan/dcalcmAoMdPu--
Medical College. Sapporo 060, Japan. dependent protein kinase [4, 11]. Our previous
1002 K. Asami
results suggested that growth-associated histone The hepatectomized rats were killed at 24h after
kinase of Langan, a protein kinase specific for partial hepatectomy, when DNA synthesis started
histone HI found in growing cells [5], was re- [3] and the protein kinase activity increased [4].
sponsible for the rise ofthe protein kinase activity
in the nuclei, although participation of other Preparation ofnuclear extract
kinase could not be excluded completely [4]. Isolation of nuclei, essentially according to the
Therefore, we measured histone HI kinase and procedure of Blobel and Potter, and preparation
casein kinase activities in parallel. The present of the nuclear extract were described previously
M
results indicate that nuclear kinase specific for [4]. Briefly, the nuclei were extracted with 0.4
histone HI is responsible for the rise of protein NaCl in buffer A, which consisted of 20mM
kinase activity at the onset of DNA synthesis. TrisHCl pH7.5, 1 mM EDTA pH 7.5, 0.1 mM
PMSF, 5 mM benzamidine, 2 mM glycerol-2-
mM
phosphate and 10 2-mercaptothanol. After
MATERIALS AND METHODS
treatment with BioRex 70, the extract was mixed
with potassium phosphate pH 7.5 and cAMP to
Animals and materials give 0.1 M and \ /jM respectively in order to
Male Wistar strain rats, supplied from our separate cAMP-dependent protein kinase [5, 6].
breedingcolony, were usedbetween 8and 10week The protein precipitated with ammonium sulfate
oldofage. [y-32P]ATPwaspurchasedfrom Amer- between 17.5% and 35% saturation was collected.
sham, Japan. PMSF (phenylmethylsulfonyl The precipitate from one rat (ca. 5 g liver) was
fluoride), a-casein (Sigma), and Combithek (pro- dissolved in 0.5 ml buffer A containing 0.4 M
tein standard set: Boeringer) were used. Histone NaCl. The extracts thus obtained were dialyzed
HI was extracted from rat liver according to the overnight against about 50volume of buffer A
proceduresofSanders andJohns [12], andthe acid containing 10% glycerol. For resolution with
soluble fraction was passed through a column of chromatography the nuclei from 3 rats were com-
BioRex 70 (Bio-Rad Lab., USA) [13]. The frac- bined before extraction and the extractswere used
tion containing histone HI was collected, freeze- for gel filtration chromatography immediately af-
M
dried, anddissolved in water. Fractionscontaining ter extraction. Although 0.4 NaCl did not
mainly histone Hl were not used. Histone HI extract all the kinase activity, the portion of the
()
solution, which showed a single band with polyac- extracted activity was not changed much during
rylamide gel electrophoresis, was divided in small regeneration [4]. The protein kinase activities
—
aliquots and stored at 20°C until use. were hence compared on the nuclear extracts with
M
0.4 NaCl in the present experiments.
X
ray irradiation andpartial hepatectomy
X rayirradiation andpartial hepatectomyofrats Column chromatography ofthe extract
were carried out as described previously [3, 4]. FPLC system (Pharmacia) was used. When
During anesthesia with pentobarbital, rats were Mono O column was used to separate the extract,
irradiated to their upper abdomen including the dialysis of the extract before chromatography
liver with 4.8 Gy of X rays. The condition of caused aggregation of the proteins and almost all
irradiation was 200 kVp and 20mA and X rays proteins remained on the top of the Mono Q
were filtered through 0.5 mm Cu and 0.5 mm Al. column. Therefore, the extract was applied to the
Following irradiation, the median and the left column of Superose 12 immediately after extrac-
lateral lobe of the liver were removed. The tion. The column was equilibrated in 0.4 M NaCl
non-irradiated rats were also hepatectomized containing buffer A. and ca. 2 mg of the nuclear
under anesthesia with pentobarbital. The right extracts were applied. Fractions of 1 ml were
lateral and caudate lobes were used in the experi- collected, and the enzyme activity and protein
ments of non-hcpatectomized rats, since these concentration were determined. Protein was de-
lobeswere used in thecase ofhepatectomized rats. termined according to the procedureofBensadoun
HI Kinase in Regenerating Liver Nuclei 1003
and Weinstein [14]. Tabu: 1. Protein Kinasesinthe NuclearExtract of
Regenerating Liver
Assay ofprotein kinase activity Activity(nmol/10min/mgprotein)
A similar procedure with the previous experi- Substrates
Non-irradiated X-irradiated
ments was used with rat liver histone HI and
a-casein as substrate [4]. Briefly, the reaction histone HI 3.66+0.93 0.63+0.57 P<0.05
mixture contained in 0.05 ml; 50mM TrisHCl pH casein 4.75+1.60 3.65+0.80 NS
7.5, 10 mM MgCl2, histone HI (0.5 mg/ml) or (endogenous) 7.14+1.66 5.71+ 1.57 NS
a-casein (0.5 mg/ml), 0.5 mM ATP (containing 1 The nuclearextract was prepared from the rat at 24
juCi [y-32P]ATP), ImM EDTA pH7.5, 1 mM h after partial hepatectomy. Mean of 3 indepen-
dithiothreitol, and 20mM glycerol-2-phosphate. dent experiments with SEM.
The reaction was started with addition of the
enzyme. After incubation for 10 min at 37°C, the
reaction wasstoppedwith addition of0.25 ml 20% extract. Phosphorylation ofendogenous substrate
trichloroacetic acid. The precipitate was collected was also observed on all the groups measured
on a Millipore filterand washedwith 10% trichlor- (Table 1). Irradiation caused no substantial
oacetic acid. After drying in air, radioactivity of changes in the activity phosphorylating casein or
the filter was measured with a liquid scintillation endogenous substrate. These results indicated
counter. Since incubation without substante presence of histone HI kinase distinct from casein
caused substantial uptake of radioactivity, net up- kinase 1 and2and responsible forphosphorylation
take of radioactivity was calculated by subtracting ofhistone HI at the onset ofDNA synthesis in the
the values obtained without substrate. The zero regenerating liver nuclei.
time value was subtracted. To confirm the presence of a protein kinase
more specific to histone HI than casein in the
nuclei, the nuclear extracts were fractionated with
RESULTS
gel filtration chromatography using Superose 12
Normal liver nuclei contain a protein kinase or column. The nuclear extract dissolved in buffer A
M
kinases and partial hepatectomy causes partial containing0.4 NaCl was immediately applied to
DNA
increase in kinase activity at the time of Superose 12 column equilibrated in the same
synthesis [4]. More than one protein kinase could medium. The protein kinase activities of each
be measured simultaneously in the previous ex- fraction was measured in the presence of histone
periments, however, since histone mixture used as HI orcaseinorwithoutsubstrate addition (Fig. 1).
the substrate contained histone mixture as well as Elution profile of proteins did not change much
non-histone proteins (data not shown). In the among the non-hepatectomized, the hepatecto-
present experiments, therefore, protein kinase mized and the X-irradiated and hepatectomized
activitywasmeasuredwith moredefinedsubstrate; liver. The activity phosphorylating endogenous
rat liver histone HI and casein. Phosphorylation substrate(s) was found in the fraction eluted at the
due to endogenous substrate was subtracted from void volume and decreased in the later fractions.
the values with the substrates, and also shown in Endogenoussubstrate(s) wereprobably associated
the results separately (Table 1). The protein with its protein kinase in the nuclear extract and
kinase activity phosphorylating histone HI was pass through the column in the associated form.
observed in the extract from regenerating liver The activity phosphorylating casein was eluted in
nuclei, while in was not observed in the extract two peaks, i.e. fraction 10 and 15 as maximal
from the nuclei ofthe X ray-irradiated and partial- respectively, while that phosphorylating histone
ly hepatectomized rat (Table 1). On the other HI was maximal in fraction 12. The liver nuclei
hand, the protein kinase activity phosphorylating have been reported to contain two casein kinases
casein was observed in both the hepatectomized [7, 8]. The two peaks probably correspond to the
and the irradiated and hepatectomized nuclear nuclearcasein kinase 1 (fraction 15) and2(fraction
1004 Till K. Asami
— ii
100 iPROTiEIN 10) orNI and Nil [7, 8]. The casein kinase activity
A / '•-..... (mg) r0-5 after subtraction of the activity without substrate
addition were divided into two parts, i.e. the
-0 fractions 13-17 and the fractions 7-12, and they
were designated here as casein kinase 1 and 2
50 respectively. The integratedactivitiesare shown in
Table 2. Activities ofcasein kinases of hepatecto-
mized liver are a little higher than those of non-
hepatectomized liver, but X ray irradiation hardly
affected the activities.
II An activity phosphorylating histone HI was
c 100 I 1 1 1 i i eluted in the fractions of regenerating liver be-
E tween casein kinase 2 and 1. This indicates that
o casein kinases and histone HI kinase are different
enzymes. The nuclear extract from the non-
o
E hepatectomized liver or from the X-irradiated and
5
50 hepatectomized livercontainedcasein kinase 1 and
> /^ \ 2, but no histone HI kinase. The activity
> / \ phophorylating endogenous substrate was sub-
U tracted from that in the presence ofhistone HI for
<
each fraction. Phosphorylating activities of frac-
£ o II tions 8 to 9 or to 11 were lower in the presence of
Nz 100' 1C A1, 1 1 histone HI than in its absence, indicating that
histone HI inhibited the phosphorylation of the
endogenous substrate. The fractions, where the
activity was higher in the preence of histone HI
50 -
\ / Fig. 1. Gel filtration chromatography of the nuclear
//ya\•\ \ / \\ extracts from rat liver. Superose 12 attached to
FPLC system was used. (A) Partially hepatecto-
mized(2.54mgprotein). (B) partiallyirradiatedand
hepatectomized (2.58mg protein), and (C) non-
ii hepatectomized (2.58mgprotein). Histone HI ( ).
i i 1i0 12i 14 16 18i 20' casein (A.) and none (•) were used as substrate.
FRACTION NO- Activity in 25/A of the eluate was shown in the
figure Protein contents.
Tablk2. Protein Kinases in the Nuclear Extract of Rat Liver
X No. of Activities (nmol/10minimg protein)
Rat
rays Exp. HK CKI CK1I
Non-HPX 3 0.58±0.09 10.87 ' 0.78 3.88+0.54
HPX 2 2.31 ±0 4(v 17.52+4.78 4.74+1.42
HPX + 3 0.62+0.36 15.30+ l.38+ 4.16+1.33
The nuclearextract from the non-hepatectomized(non-HPX)orthehepatectomized (HPX) ratwasused
for FPLC. Protein kinase activities and protein contents ofeach fraction were measured and summed
up. HK; histone HI kinase. CKI and CKII; casein kinase 1 and 2 respectively. The asterisks indicate
a significant difference from the non-hepatectomized (P<0.05).
HI Kinase in Regenerating Liver Nuclei 1005
than in its absence, were regarded to contain Table3. Inhibition by histone HI of the phos-
histone HI kinase and their differences are sum- phorylating activity ofthe nuclear extract with or
med up (Table 2). Nuclear histone HI kinase without casein
activity of the hepatectomized liver was higher histone HI (mg/ml reactionmixture)
than either the non-hepatectomized or the X ray- Enzyme Casein
0.5 1.0
irradiated and hepatectomized liver. Partial in-
activation of the histone HI kinase however was nmol/10min/mg protein
noticed on the fractions after chromatography, Fr. 8 + 5.50 6.56 3.32
-
since the specific activity after chromatography 2.54 1.62 1.18
was similar to that before chromatography (com- Fr. 10 + 10.98 9.41 5.04
-
pareTable2withTable 1). The reasonofinactiva- 1.63 1.91 1.33
tion is unknown, but instability of growth associ- Fr. 15 -+ 37.17 17.32 16.73
ated histone kinase after chromatography was re- 0.80 1.33 1.98
ported [6, 15]. The values with casein are not corrected for 3~P
Apparent molecular weight of three protein uptakewithoutcasein. Fraction(Fr.) 10andFr. 15
kinases were calculated from chromatography of contain casein kinase 2 and casein kinase 1 respec-
tively.
the standard proteins; ferritin, catalase, aldolase,
bovine serum albumin, egg albumin, chymotrypsi-
nogen A and cytochrome c. Some ofthe standard
DISCUSSION
proteins, especially ferritin, showed peakds with
smaller molecular weight after chromatography. Phosphorylation of histone HI is closely related
DNA
This would be due to depolymerization of the to the initiation of synthesis ofregenerating
polymerized form of the proteins in the elution liver [2, 3]. However, it is still not known which
buffercontaining2-mercaptoethanol. Thesepeaks enzyme is responsible for the phosphorylation.
with smaller molecular weight were also used for Although cAMP-dependent protein kinase was
calculation. The obtained regression line was found in the nuclei [11], the enzyme was not
log(molecular weight)=5.32—0.137X(retention related with phosphorylation of histone HI at the
time in min); x——0.9646. The calculated sizes of time of DNA synthesis [4] and was removed from
the protein kinases were 381 kDa (casein kinase the nuclear extract in the present experiments
2), 92kDa (histone HI kinase), and 12kDa (ca- through treatment with cAMP and ammonium
sein kinase 1), respectively (data not shown). sulfate precipitation [5, 6]. Protein kinase C and
The results shown in Figure 1 indicated that Ca2+/calmodulin-dependent protein kinase II are
histone HI inhibited phosphorylation of endoge- also reported in rat liver nuclei [9, 10], but the
nous substrate by the kinase eluted simultaneous- present assay condition was not favorable for the
ly. Phosphorylation of endogenous substrate and Ca-dependent kinases, since the assay medium
casein was then measured in the presence of contained no Ca2+ but EDTA. The inhibitors of
different concentrations of histone HI, since ca- the protein kinase C did not inhibit the nuclear
sein kinases were the protein kinase found in the kinase [4]. These protein kinases, therefore, will
nuclei in the present condition except for histone not be considered further in this article.
HI kinase (Table 3). Histone HI inhibited phos- Thepresentresultsshowthatthe nuclearextract
porylationofcaseinbycaseinkinase 1 (fraction 15) of the regenerating liver contained two kinds of
or 2 (fraction 10). Phosphorylating activity of casein kinases and one histone HI kinase. The
fraction 8 which contained endogenous substrate extract seemed to contain another protein kinase
was activated with casein and the activity was which was associated with endogenous substrate.
inhibited by histone HI irrespective ofaddition of This will be a casein kinase as discussed later.
casein. Two kindsofcasein 1 (NI) and2(Nil), although
the molecular weight of casein kinases in the
presentresults are not consistent with the reported
1006 K. Asai\
values (e.g., 50 and 133 kDa [8] or35 and200kDa The present results coincide with the previous
[15] respectively). In the present paper however resultsthat the enzyme responsible for phosphory-
they are called casein kinase 1 and 2. Activities of lation of histone HI will be a growth associated
casein kinases increased a little in the regenerating histone kinase ofLangan [4]. Apparent molecular
liver and Xrays enough to inhibit phosphorylation mass ofthe kinase was 92 kDa. Molecular weight
of histone HI scarcely affected the increase. A of growth associated histone kinase is not re-
HMG
nuclear non-histone protein, 14 is phos- ported, but that of catalytic subunit is 67,000 [6].
phorylated with casein kinase 2 [8, 16] and X rays Cyclin-p34cdc2 protein kinase is specific for histone
HMG
do not affect phosphorylation of 14 [3]. HI and is thought to be the same as growth
These are in consistent with the results that X rays associated histone kinase [20, 21]. Association of
scarcely affected the activity of casein kinase. p34cdc2 with cyclin, 62 kDa, will constitute a com-
Both casein kinase 1 and 2 are inhibited by plex of 96kDa, a similar value to the present
histone HI. An endogenous inhibitor protein of results, although apparent molecular mass of cyc-
casein kinasewasreportedpreviously, whichwasa lin-p34cdc2 complex is evaluated as 220 kDa from
25 kDa protein found in high mobility group chromatography with Superose 12 [21]. Nuclear
(HMG) protein fraction ofrat liver, but it was not localization ofthe kinase has been shown [22. 23].
characterized further [17]. However, histone HI Although thecyclin-p34cdc2playa role in the G2to
HMG M
was not removed from their fraction, which transition of the cell cycle, the same enzyme or
usuallycontained histone HI, before separation of its homologue is claimed to participate for the Gl
the inhibitor [17]. The present results suggest that toS transition [24-26]. Thusthe histone HI kinase
this endogenous inhibitor protein of casein kinase found in the nuclei will be growth associated
is histone HI. Inhibition by histone HI ofnuclear histone kinase or a kinase related to it, which will
casein kinase may have physiological significance play a role in phosphorylation ofhistone HI at the
DNA
forregulatingphosphorylation ofnuclearproteins, onset of synthesis.
although more experiments should be carried out The mechanism how X rays inhibit appearance
before concludinghistone HI asa natural inhibitor ofthe histone HI kinase inthe nuclei isnotknown.
of nuclear casein kinase. It is not known whether X rays affect directly the
A protein kinase is associated with endogenous appearance of histone HI kinase in the nuclei.
substrate andthat isthecauseofhigh phosphoryla- Instability of growth-associated histone HI kinase
tion rate without added substrate (Table 1). The has been reported [6, 15, 27]. Activity of p34
enzyme wasable tophosphorylatecasein (oritwas protein kinase can be controlled by binding of
activated with casein) and it was inhibited by cyclin which is usually synthesized in each cell
histone HI. It was reported that casein kinase 2in cycle, and the activity can be changed depending
the nuclear extract was associated firmly with high on the phosphorylation state ofthese two proteins
molecular weight protein such as nucleolin or [28, 29]. On the other hand, synthesis of p34cdc2
progesterone receptor [18, 19]. Hence, the en- during the Gl phase of stimulated lymphocytes
zyme phosphorylating endogenous substrate may was observed [25]. Hence X rays may suppress
be casein kinase 2. appearance of the histone HI kinase in the nuclei
Histone HI kinasewasfound in the regenerating by changing the cellular metabolism resulting in
nuclei, while it was not observed in the non- inactivation ofthe kinase, or by inhibiting denovo
hepatectomized nuclei. X irradiation inhibited the synthesis of protein(s) constituting the kinase.
appearence ofthe histone HI kinase activity in the
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