Eur. J. Biochem.

180,441 -448 (1989)

0FEBS 1989

Casein kinase I1 from Rana temporaria oocytes

Intracellular localization and activity during progesterone-induced maturation
Konstantin V. KANDROR I, Aleksej 0. BENUMOV2 and Aleksandr S. STEPANOV'
A. N . Bakh Institute of Biochemistry, USSR Academy of Sciences
' Department of Embryology, Biological Faculty, M. V. Lomonosov Moscow State University

(Reccived June 2l/November 8, 1988) - EJB 88 0734

A homogeneous preparation of casein kinase 11 has been isolated from the ribosome-free extracts of Rana
temporaria oocytes by means of chromatography on heparin-sephdrose, phosphocellulose and mono Q. The
enzyme consists of three subunits with molecular mass of 43 kDa, 41 kDa and 29 kDa. The protein kinase was
labelled with radioactive iodine and injected back into oocytes. As shown by histoautoradiography the enzyme
forms a diffuse ring aroung the nucleus in the oocyte cytoplasm. A part of casein kinase I1 is found in in-
formosomes. During oocytes maturation casein kinase I1 activity increases 7 h after progesterone administration
and at the final stages of maturation (20 - 23 h). Cycloheximide blocks the second augmentation of kinase activity
and does not influence the first one.

Casein kinases I1 are unique among the great number of
presently known protein kinases. On the one hand, these
enzymes are found in the cytoplasm and nuclei of all
eukaryotic cells studied and account for a great deal of the
total phosphoprotein transferase activity of cell extracts. On
the other hand, little is known about these enzymes besides
the most general characteristics obtained in vitro. Casein
kinases 11 (except for the enzymes from yeast and
myxomycetes) all share a common polypeptide structure and
consist of four subunits (a2p2 or aa'pz), with ala' having a
molecular mass of 35-44 kDa and p of 24-29 kDa. The
enzymatic properties of all the casein kinases I1 in vitro are
very similar [l]. However, the role of these enzymes in vivo as
well as their physiological substrates remains obscure.
Intracellular localization which often helps our under-
standing of the actual functions of an enzyme is investigated
in this paper. Protein kinases with similar properties are found
not only in cytoplasm, but in nuclei as well [2, 31. The present
authors have reported on the RNA-binding activity of casein
kinases 11, and Rittschoff & Traugh as well as Thoen et al.
have found the enzyme in mRNP or informosomes [4 - 71.
These and other data (such as the inhibitory effect of RNA
on the activity of this protein kinase and the influence of
phosphorylation on the RNA-binding activity of protein sub-
strates) have prompted the suggestion of participation of
casein kinases I1 in RNA-dependent processes [8, 91. This hy-
pothesis can be verified by following the fate of the enzyme
in the living cell. Giant cells, such as the oocytes of amphibia,
allow one to perform microsurgical operations and thus come
close to in vivo conditions.
A simple and convenient procedure developed for the iso-
lation of casein kinase I1 from the oocytes of the frog, Rana
temporaria, is described in the present paper. The preparation
obtained was labelled in vitro, without loss of protein kinase
Correspondence to K. V. Kandror, A. N. Bakh Institute of Bio-
chemistry, Leninsky prospekt 33, Moscow, USSR 117071
Enzyme. Casein kinase I1 (EC 2.7.1.-).
or RNA-binding activity, and injected back into oocytes. The
fate of the radiolabelled enzyme was followed by means of
histoautoradiography and biochemical techniques. It has been
established that this enzyme is concentrated in the cytoplasm
without penetrating into the nucleus, with about 20-30% of
soluble casein kinase I1 being found in informosomes.
The activity of casein kinase I1 towards endogenous sub-
strates sharply increases at early stages of progesterone-stimu-
lated maturation of the oocytes. Cycloheximide, while almost
completely blocking protein synthesis, does not influence pro-
tein kinase activation or protein phosphorylation. The enzyme
activity increases again at the final stages of maturation as a
result of de novo synthesis.
MATERIALS AND METHODS
Materials and buflers
[ Y - ~ ~ P ] A Tand
P Na'251 were obtained from Izotop
(USSR), ATP from Merck (FRG), casein from Calbiochem
(USA), heparin-Sepharose from Pharmacia (Sweden) and
phosphocellulose P-I 1 from Whatman (England).
The standard buffer contained 10 mM triethanolamine,
10 mM KC1, 0.1 M NaCl (unless otherwise indicated), 5 mM
MgC12, 1 mM EDTA, 6 mM 2-mercaptoethanol, 0.2 mM
phenylmethylsulfonyl fluoride, 10% glycerol, pH 7.8. The
buffer for radioactive labelling contained no NaCl, glycerol,
phenylmethylsulfonylfluoride and 2-mercaptoethanol. The
buffer for centrifugation in sucrose gradient contained 10 mM
triethanolamine, 0.1 M KCl, 5 mM MgCI2, 6 mM 2-mercap-
toethanol, 1 mM EDTA, 4% C H 2 0 , pH 7.8.
Oocyte preparation
Rana temporaria were injected with an aqueous suspension
of hypophysis (1 -4 hypophyses/female). 10-20 h later the
frogs were killed, ovulated oocytes were collected, washed
442

A
100 ii 2 1 200 1.0

-
k
1
0
m
I:
a N
-
6
0 50 1 0.5%
Z
0
0

0 0 0
5 10 15 20 25
Fraction number

0.1
I
-
0
U
z

02

Fig. 1. Isohtion of cusein kinuse 11.(A) Chromatography on heparin-Sepharose; (B) chromatography on phosphocellulose P-I1; (C) chromatog-
raphy on mono Q. Volumes of the columns were 50 ml (A), 15 ml (B) and 1 ml (C). Gradients: 300 mlO.1-0.8 M NaCl (A); 80 ml 0.2-
1.0 M NaCl (B); 30 ml 0.1 -0.6 M NaCl (C).Elution rate: 6 ml/h (A), 10 ml/h (B), 60 ml/h (C). 20 p1 of each fraction were incubated with
60 pg dephosphorylated casein and 0.1 mM [y-3zP]ATP(100 cpm/pm) for 1 h at 22°C. Total volume of incubation mixture was 100 pl;
(0-0) protein kinase activity; (----) A Z e 0 ;(A-A) concentration of NaCl

thoroughly with Ringer solution and standard buffer, then
used for isolation of casein kinase 11.
Fully grown oocytes were isolated by treating the ovaries
of Rana temporaria with collagenase (Merck, FRG) at a con-
centration of 2 mg/ml in 0.1 M sodium phosphate, pH 7.5
with vigorous shaking.
Enzymatic assays
Protein kinase assay was carried out in standard buffer
(100-
(final volume 100 pl) in the presence of [ Y - ~ ~ P I A T P
1000 cpm/pmol) usually for 1 h at 22OC. 60 pg dephos-
phorylated casein were added to incubation mixture, if indi-
cated. The reaction was stopped by the addition of 4-5 ml
ice-cold 5% trichloroacetic acid. The acid-precipitated ma-
terial was applied to glass filters (GF/A, Whatman), washed
with 5% trichloroacetic acid and dried.
To evaluate ATPase activity, protein samples were incu-
bated with 0.1 mM [y-32P]ATP(200 cpm/pmol) in a total vol-
ume of 100 pl for 1 h at room temperature. Then, 1 ml of a
10% suspension of Norit A charcoal in 0.1 M sodium phos-
phate, pH 6.0, was added. The mixture was vigorously mixed
for 10 min, charcoal was settled by centrifugation and
Cherenkov radioactivity in the supernatant was counted.
Each experimental point represents the mean value of three
independent measurements. Phosphatase activity was assayed
as described earlier [7].
Purification of casein kinase 11
60 - 100 ml of oocytes from Rana temporaria, ovulated in
vivo, were washed with 3 1 Ringer solution and standard
buffer, transferred to centrifugation tubes and disrupted by
centrifugation (30000 x g, 20 min). The supernatant fraction
was collected avoiding the lipid layer, filtered (if necessary)
on a glass filter or passed through a Sephadex G-25 column.
The solution obtained was applied to a 50-ml heparin-
Sepharose column. The column was thoroughly washed with
standard buffer and eluted with a linear gradient of NaCl
(0.1 -0.8 M) at a flow rate as high as 6 ml/h. Fig. 1 A presents

Table 1. Purification of casein kinase II
Activity of casein kinase 11: 1 unit is defined as pmol 32P incor-
porated/60 Fg dephosphorylated casein during incubation in the stan-
dard protein kinase assay (described in Materials and Methods)
-
Step Total Total Specific
protein activity activity
( x 30-6) ( x 10-6)
mg unit/h unit h-
mg - '
Ribosome-frce extract 2000 - -
Heparin-Sepharose 7.04 1.37 0.19
Heparin-Sep harose
(after dilution) 7.04 1.13 0.16
Phosphocellulose 0.95 1.04 1.09
Phosphocellulose
(after dilution) 0.95 0.40 0.42
Mono Q 0.15 0.20 1.33
a typical elution profile for chromatography on heparin-
Sepharose. The peak of protein kinase activity is seen to be
eluted by 0.5 M NaCl. The fractions containing protein kinase
activity were pooled, diluted to a NaCl concentration of 0.2 M
and applied to a 15-ml phosphocellulose P-11 column. Fig. 1B
shows the corresponding elution profile. As was the case at
the first step, the enzyme was eluted at a high ionic strength
(0.5 - 0.6 M), which gave a fair separation from other proteins
and a high degree of purity. The final step involved FPLC on
mono Q columns (Pharmacia). This procedure requires the
preparation to be desalted. Although casein kinase I1 readily
undergoes inactivation on dialysis, gel filtration and dilution,
we employed gradual dilution of the preparation with a buffer
of low ionic strength, thus resigning ourselves to inevitable
losses (Table 1). Chromatography on mono Q was performed
at a rate of 1 ml/min at the room temperature using the buffer
for radioactive labelling. The total gradient (0.1 -0.6 M
NaCl) volume was 30 ml. Fig. 1 C outlines a typical elution
profile with the maximum protein kinase activity coinciding
with the major peak of absorbance at 280 nm.
Radioactive labelling of casein kinase II
The preparation obtained was labelled with radioactive
iodine by the convcntional technique with the use of
chlorainine T [lo]. The labelled preparation was immediately
applied to a I-ml heparin-Sepharose or poly(U)-Sepharose
column and washed with standard buffer overnight. When
radioactivity in the eluate fell to background level, the labelled
enzyme was eluted with 0.6 M KCl. Ten fractions were col-
lected, and the total and trichloroacetic-acid-precipitated
radioactivity was counted in each of them. The values
obtained were the same, thus pointing to essentially complete
removal of the unbound radioactivity (Fig. 2). The radioac-
tivity of samples labelled with iodine was measured on a
Berthold model MAG510 counter.
Oocyte maturation and protein kinase activity
The individual fully grown oocytes were washed thor-
oughly with Ringer solution and transferred to Petri dishes
on the basis of 2 ml oocytesi20 ml Ringer solution containing
0.01 M Hepes, pH 7.3, 150 pg progesterone (Merck, FRG).
Incubation lasted 7 h, 12 h, 16 h, 20 h or 23 h at 17-18°C.
Maturation controlled by the germinal vesicle breakdown
443
'
-
o 0 1 2 3 L 5 6 7 8 9 1 0
Fraction n u m b e r
Fig. 2.Rechromatograph))of 12sI-lubelledcasein kinase I1 on poly ( U ) -
Sepharose. 2 4 aliquots were taken from each fraction (total volume,
100 121) and their radioactivity was measured before (0--0) and
after (0-0) precipitation with 5% trichloroacetic acid
was complete between 16 h and 20 h. Control oocytes were
incubated under the same conditions without progesterone
for 7 h.
In some experiments, 40 - 50 pg/ml cycloheximide
(Merck, FRG) was added to the incubation medium. After
incubation, the oocytes were washed with Ringer solution
(3 x 30 ml), standard buffer (4 x 10 ml) and homogenized with
a pestle in a centrifuge tube in an ice bath. The homogenates
were centrifuged for 15 min at 20000 rpm. The supernatant
fractions were collected and stored in liquid nitrogen before
being used for preparation of ribosome-free extracts (90 min
at 40000 rpm).
Ribosome-free extracts of oocytes at different stages of
maturation were applied to I-ml heparin-Sepharose columns
(Pharmacia, Sweden). The columns were washed with 20-
50 in1 of standard buffer and the adsorbed material eluted
with standard buffer containing 0.7 M NaCl. The protein
kinase activity in the obtained protein fractions was measured
as previously described.
Injection of labelled casein kinase II into oocytes,fractionation
of the injected oocytes and histoautoradiography
Radioactive casein kinase I1 was injected into oocytes by
means of a pneumatic micromanipulator [l 11in a dose of 70 nl
(about 1000 cpm, 3 -4 ng protein)/cell. Siliconized capillaries
were used for convenience and to minimize adsorption.
Centrifugation in sucrose and CsCl gradients was carried out
in a model L5-50 Beckman centrifuge (USA) [ll]. In the
latter case the samples were preliminarily dialysed against the
centrifugation buffer (200 - 300 vol., 3 or 4 times).
To perform histoautoradiography, I6 h after the injection
operated (experiment) and intact (control) oocytes were fixed
in freshly prepared Bouin's solution for 12 h at 20°C. Picric
acid was then washed off with 4% C H 2 0 (pH 7.2) and the
cells were placed in 10% aqueous glycerol. Seven days later
the oocytes were dehydrated with butanol and embedded
in paraffin. Using a Reichert-Jung 3020/biocut microtome,
sagittal sections of 5 pm were cut, mounted on slides, dried
at 37°C for three days and treated with 15% perhydrol (pH
7.2 - 7.4) completely remove of the pigment. After being
444
Fig. 3. Electrophoresis of casein kinase II. (A) 0.7 pg casein kinase I1
stained with Coomassie blue R-250. (B) 0.36 pg in v i m phos-
phorylated casein kinase I1 (1.2 pg casein kinase I1 was incubated
with 0.1 mM [y-32P]ATPwith specific activity 1000 cpm/pm for 1 h
at 22°C in a total volume of 50 pl). (C) '251-iabelledcasein kinase I1
(SO000 cpm).(D) '251-labelledcaseinkinase I1 (40000 cpm) wasiiicu-
bated with 0.1 mM [Y-~'P]ATP(I000 cpm/pm) and 30 pg dephos-
phorylated casein for 1 h at 22°C in a total volume of 50 pl. Only
15 pl of incubation mixture was electrophoresed. B, C and D are
autoradiographsof the dried gels. Exposure time was 24 h. The arrows
indicate the positions of standards: phosphorylase b (94 kDa), bovine
serum albumin (67 kDa), ovalbumin (43 kDa), carbonic anhydrase
(30 kDa), trypsin inhibitor (20.1 kDa)
washed and dried the samples were dipped in type-M
radiosensitive emulsion (Gosniikhimfotoproekt) and exposed
for 30days at 4°C. Autoradiographs were developed in
amidol developer for 3.5 min at I S T , fixed in 30% sodium
thiosulfate for 3.5 min at 18"C, rinsed in running tap water
and stained with haematoxylin according to Carazzi. Perma-
nent specimens, when prepared, were examined in a Opton
Standard microscope. Localization of the protein kinase was
determined from the presence of reduced silver grains over
the nuclei and cytoplasm of the oocytes.
Other methods
Protein content was determined by the method of Schaff-
ner and Weissmann [12]. Electrophoresis was performed by
the method of Laemmli [13]. Proteins of molecular mass 14-
94 kDa (Pharmacia calibration kit) were used as standards.
Autoradiography involved the exposure of X-ray film
(Kodak, USA) to dried gels. The exposure time is to be speci-
fied below.
RESULTS
Isolation and radioactive labelling of casein kinase I f
Casein kinase I1 has been isolated from ribosome-free ex-
tracts of ovulated Rana temporaria oocytes as it is described
in Materials and Methods. Electrophoresis according to
Laemmli demonstrated the purified enzyme to consist of three
polypeptide chains (a, a' and p) with molecular mass of
43 kDa, 41 kDa and 29 kDa (Fig. 3A). When the enzyme was
incubated with [ Y - ~ ~ P I Athe
T Plabel was incorporated into the
light subunit (p) (Fig. 3B). It has been shown earlier in this
laboratory that the protein kinase belonging to the RNA- or
heparin-binding proteins from the oocytes of Rana temporaria
possesses all the properties of casein kinase I1 [7, 101 in vitro.
40pg isolated enzyme was labelled with Na1251 and
chloramine T. The specific radioactivity of protein kinase
preparations labelled by this technique was lo5- lo6 cpm/pg
protein. An electrophoretic pattern of the labelled enzyme is
presented in Fig. 3C. In spite of the higher sensitivity provided
by the autoradiography of iodine-labelled preparations com-
pared to Coomassie staining, no components other than the
three polypeptide chains of the protein kinase were detected.
Repeat chromatography on heparin- or poly(U)-
Sepharose (see Materials and Methods) allows the removal of
not only excess label but also of damaged enzyme molecules
which have lost the RNA-binding activity in the course of
isolation and labelling. Control experiments demonstrated
that the labelled enzyme retained the ability to phosphorylate
casein (Fig. 3D). Thus, the preparations of labelled casein
kinase I1 exerted both a RNA-binding and an enzymatic ac-
tivity.
Injection of 1251-labelledcasein kinase II into oocytes
and evaluation of its intracellular localization
Immediately after repeat chromatography, the labelled
protein kinase was diluted to a KCl concentration of 0.1 5 -
0.2 M and injected into 100 - 200 oocytes of Rand temporaria
(70 nl/cell), ovulated in vivo, by means of a pneumatic micro-
manipulator using silicinized capillaries.
The oocytes were incubated in Ringer solution containing
0.01 M Hepes (pH 7.3), penicillin and streptomycin for 16 -
18 h. The damaged cells (accounting for not more than 3%
of the total) were then discarded and the rest washed with
standard buffer, homogenized, then centrifuged at 30000 g
for 30 min. Not less than two thirds of the injected label was
found in the pellet. Supernatant was collected; an aliquot was
analyzed with the help of electrophoresis to determine if there
was any metabolic degradation of exogenous radioactive en-
zyme during the incubation. Since the electrophoretic mobility
of protein kinase subunits did not alter, one may hope that
the enzyme kept its native activity after injection. The rest of
the supernatant fraction was applied to sucrose gradients
(15 - 30%) based on standard buffer containing 4% C H 2 0
for fixation of ribonucleoproteins. Centrifugation was per-
formed for 3 h at 39000 rpm in a SW-50 rotor at 4'C. A
typical pattern obtained is given in Fig. 4A. There is a distinct
peak in the 50 - 70 S range where frog oocyte informosomes
normally concentrate [ll]. It was concluded on the basis of
three experiments that 20 - 30% of the label contained in the
mitochondrion-free extract was incorporated into the par-
'
ticles. The addition of 2sI-labelled casein kinase I1 to oocytes
during homogenization does not lead to its incorporation into
informosomes, which indicates that specific incorporation
rather than adsorption takes place (Fig. 4B).
To ensure that the particles with which exogenous casein
kinase I1 is associated represent true informosomes, centrifu-
gation in CsCl density gradient was employed. lnformosomes
are known to have a specific protein:RNA ratio of 3 : l (by
mass) corresponding to a CsCl density of about 1.4 g/cm3
[14]. The fractions of the sucrose gradient corresponding to
the 50 - 70s range were pooled and, after sucrose reinoval by
dialysis, were centrifuged in a CsCl density gradient for 24 h
at 40000 rpm and 4°C in an SW-50 rotor. The results are
 445

C
~

06 10
A 0
150 -
0.1
0
E
600 0.2 30
\
m
2;
c

- I
~

In

k .6 6
m
mG100 c
C
N 0

/fir
x
0
I 3
m

5ot 0
.L

200 0.01 10

0 0 0
. ** 1.2

10 20 10 20 30 -0 10 20 30
Fraction number Fraction number Fraction number
Fig. 4. Subcellular localisation of casein kinase II by density-gradient centrifugation. (A) Sedimentation distribution in the sucrosc gradient of
'Z51-labelledcasein kinase 11 after injection into oocytes and incubation for 18 h, or (B) addition to oocytes during homogenization. The
4.8-ml gradients were loaded with 0.2 ml oocyte extract containing '251-labelled casein kinase 11. Centrifugation was performed for 3 h at
39000 rprn and 4°C in SW-50 rotor of L5-50 Beckman ultracentrifuge. (C) Density distribution in CsCl gradient of the particles associated
with 1251-labelledcasein kinase 11. Centrifugation was performed for 24 h a t 40000 rpm and 4°C in the same rotor. (0-0) Radioactivity;
(----) A Z a 0 ;(/'--A) density ofCsCl solution

presented in Fig. 4C. Most of the radioactive material is seen
to have a buoyant density of 1.38 g/cm3, which corresponds
well to informosomes.
Enzymes showing the properties of casein kinase I1 are
found both in the nuclei and cytoplasm of eukaryotic cells
[2, 31. It was of interest to find out to which of the subpopu-
lations the isolated enzyme belongs. With this end in view, the
preparation of labelled protein kinase was injected into 30
fully grown oocytes of Rana temporaria. 24 h later, the cells
were fixed and sections prepared which were then covered
with emulsion and exposed for 30 days. The autoradiographs
obtained are presented in Fig. 5A, B. It was observed that the
label concentrated in the cytoplasm and did not penetrate the
nucleus after a 24-h incubation. Moreover, '251-labelledcasein
kinase I1 was not distributed evently in the cytoplasm but
rather formed a diffuse concentric ring. The cytoplasm can be
divided into three zones with respect to protein kinase content
(see Fig. 5B). The peripheral zone (including the cortical
layer) contains no radioactivity. Next to it is a zone rich in
the protein kinase. Finally, the central area around the nucleus
contains a small quantity of the label. Autoradiography does
not allow one to reveal the structures with which casein
kinase I1 is associated and the reason for such an unusual
intracellular distribution of the enzyme. A more definite
answer may, probably, be given by electron microscopy.
Activity ojcasein kinase II
during progesterone-induced maturation of the oocytes
It is well-known that administration of progesterone to
fully grown oocytes leads to considerable changes in their
physiology and morphology. The molecular mechanisms of
these events are not yet sufficiently understood, but evidence
has been found to suggest that phosphorylation/dephos-
phorylation processes play the key role in the control of matu-
ration [15]. It is notable that augmentation of protein synthesis
during maturation is controlled at a post-transcriptional level
and results from the specific unmasking of mRNA stored in
the oocyte cytoplasm [16]. The translational status of mRNA
may be regulated by interaction with certain proteins. These
proteins form the pool of free RNA-binding proteins, which
is now under intensive investigation (for review see [14, 16-
181). RNA-binding proteins are usually isolated with the help
of chromatography on different immobilized polyanions
[RNA, poly(U), heparin]. It has been shown that RNA-bind-
ing proteins of amphibian oocytes contain protein kinase
activity [7, 81, related to casein kinase 11 and phosphorylation
substrates.
We have isolated this protein fraction from oocytes at
different maturation stages (see Materials and Methods) and
analysed their polypeptide compositions (Fig. 6A) and en-
dogenous protein kinase activity (Fig. 6 B). Incorporation of
32P into the protein fractions analysed varied considerably
during maturation (see also Table 2). Phosphorylation in-
creased 10-fold, 7 h after progesterone administration then
decreased. A subsequent increase in phosphorylation (2 -
4-fold) was observed during the destruction of the germinal
vesicle.
As has already been mentioned, casein kinase 11is the only
protein kinase in the fraction of RNA- or heparin-binding
proteins. However, if ATPases and phosphatases are present
the results obtained might be due to changes in the activities of
these enzymes rather than protein kinase activity. Therefore,
ATPase and phosphatase activities were assayed in all the
fractions and in no case were they found to be significantly
different from background values (data not shown). Thus,
446
Fig. 5. Sagittalsection offully grown Rana temporaria oocytes injected with '251-labelledcasein kinase IIand incuhatedfov 24 h. (A) Distribution
of labelled enzyme between oocyte nucleus and cytoplasm. (B) A fragment of another oocyte at greater magnification
ATPases and phosphatases are either absent in the fractions
of heparin-binding proteins or inactive under the conditions
employed.
One can suggest that the increased protein kinase activity
was due to the de novo biosynthesis of RNA-binding proteins
such as the enzyme itself or its endogenous substrates. Indeed,
newly synthesized peptides appear in this protein fraction
during oocyte maturation. However, no favoured biosynthesis
of proteins serving as substrates for the protein kinase con-
cerned was observed.
The addition of cycloheximide suppressed label incorpo-
ration into trichloroacetic-acid-precipitatedmaterial by 90 -
95% while practically not affecting oocyte-membrane per-
meability to radioactive percursors (results not shown). As
expected, cycloheximide also inhibits the biosynthesis of
heparin-binding proteins in oocytes. However, a burst in the
phosphorylation of heparin-binding proteins (activation of
casein kinase 11) was again observed 7 h after oocyte stimu-
lation with progesterone in spite of translation being almost
completely inhibited. The subsequent decrease in the enzyme
activity was not associated with protein biosynthesis since
cycloheximide addition, at the time when the activity was
maximal (7 h), failed to prevent this decrease. Protein kinase
activation, observed at late maturation stages, when the ger-
minal vesicle is destroyed, was blocked by cycloheximide and
may thus be due to the de n o w enzyme synthesis. The same
is shown by the results of the preparative casein kinase I1
isolation from fully grown and mature oocytes from Ranu
temporaria, showing the latter to contain much more enzyme.
Thus, the activation and subsequent inhibition of protein
kinase at early stages of oocyte maturation are due to vari-
ation of the activity of the pre-existing enzyme and occur at
the post-translational level. The alternative explanation of
activation as being due to the de novo synthesis of the enzyme
or its protein substrates is ruled out completely by the results
obtained. At final maturation stages, however, the de novo
synthesis of casein kinase I1 does take place.
DISCUSSION
Highly purified preparations of casein kinase I1 have been
isolated from different tissues. When this article was under
preparation we learned about the work of Mulner-Lorillon et
al. concerning the isolation and properties of casein kinase I1
from the oocytes of another amphibian, Xenopus lnevis [19].
The results obtained by this group are rather close to our
data; however, we have never seen inhibition of amphibian
casein kinase I1 by Ca" (Kandror and Stepanov, unpub-
lished results), and the fl subunit of this enzyme represents the
single polypeptide in our preparations. We think that our
procedure of isolation is more convenient (only three steps)
 447

Fig. 6. Electrophoresis qf in vitro phosphorylated heparin-binding proteins isolated from oocytes at diJj‘erent stages of maturation (euch track
contains 5 ggprotein). 30-40 pg of heparin-binding protein was incubated with 0.1 mM [Y-~’P]ATP (600 cpm/pm) for 1 h at 22°C in a total
volume of 100 PI. (A) The gel was stained with Coomassie blue R-250. (B) Autoradiograph of the dried del. Exposure time was seven days.
The arrows indicate the positions of standards (as in Fig. 3). For numbers of tracks see Table 2

Table 2. Casein kinase activity during maturation of Rana temporaria zyme from a mitochondrion-free extract. This data is not
oocytes surprising since Rittschof and Traugh, as well as Thoen et at.,
Progesterone was added at the start of the incubation. Control oocytes have already reported the discovery of casein kinase I1 among
were incubated without progesterone. Oocytes were incubated in
protein of mRNP from rabbit reticulocytes and cryptobiotic
cycloheximide for the periods shown, as described in Materials and
Methods. The numbers on the left are the lane numbers of the gel in gastrulae of Arteria salina [4, 51. However such a direct ap-
Fig. 6 proach (isolation and dissociation of mRNP to individual
components, and analysis of protein kinase activity among
No. lncubation Period in Specific phosphorylation obtained proteins) in this case is pregnant with artifacts since
time cycloheximide of heparin-binding proteins there are a lot of almost insurmountable difficulties which
hinder the isolation of pure informosomes [18]. In this paper
h pmol 32P/pgprotein we give independent arguments in favour of an informosome-
1 1 0 0.50
forming function of casein kinase I1 in vivo.
2 1 0 5.57 In no case did we see the incorporation of casein kinase I1
3 12 0 0.91 into ribosomes or polysomes. It may be that this enzyme (like
4 16 0 0.57 some other proteins) is specific for free cytoplasmic in-
5 20 0 1.20 formosomes and is absent from polysome-bound informo-
6 23 0 2.01 somes.
I 23 0-7 3.84 The biological meaning of casein kinase I1 being associ-
8 23 7-23 1.18 ated with informosomes is mysterious since RNA, like all
9 23 12-23 0.89
10 23 16-23 0.71
polyanions, is a potent inhibitor of this enzyme. The protein
kinase appears to be inactive when a part of informosomes.
It may be that the reversible complex formation with RNA is
needed for regulating enzyme activity by keeping the protein
and gives better results in terms of purity and specific activity kinase in the inactive state.
of the enzyme. Since no labelled casein kinase I1 is found in the oocyte
When ‘2s1-labelledcasein kinase I1 was injected back into nucleus we conclude that the enzyme, isolated as described
oocytes we found the major fraction of the label (2/3 or even above, is of cytoplasmic origin. Therefore, the results obtained
3/4 in some cases) in the pellet after low-speed centrifugation. argue against the existence of a common nuclear/cytoplasmic
This might be due to enzyme association with mitochondria pool of casein kinase I1 in the oocyte. Our observation con-
[20]. This point, however, calls for further investigation. firms the results of Baydoun et al. [21] who have shown that
A part of exogenous protein kinase is associated with cytoplasmic and nuclear type-I1 kinases are distinct enzymes
informosomes, which involves 20 - 30% of radiolabelled en- in spite of many common properties in vitro.
448
Activation of casein kinase I1 during progesterone-in-
duced maturation has been always reported for oocytes of
the starred sturgeon Acipencer stellatus Pall [22]. It may be
speculated that activation of this protein kinase accompanies
all the changes in cellular growth and proliferation, since it
is observed during transformation [23], differentiation [24],
embryogenesis [25] and mitogen administration [26].
As we noticed earlier [9], phosphorylation of RNA-bind-
ing proteins by casein kinase I1 decreases their affinity for
RNA. This may be the source of dissociation of proteins from
stored mRNA and unmasking of messages. So, the increase
in casein kinase I1 activity during oocyte maturation can be
considered as an intermediate step in an as-yet-unknown reac-
tion cascade, starting from progesterone reception by the
oocyte membrane and leading to the utilization of stored
mRNA.
The mechanism of enzyme activation is as yet unknown.
It may involve the dissociation or decomposition of a certain
inhibitor whose existence has been reported [27, 281. Also, we
observed the unaccountably spontaneous activation of the
protein kinase in the course of purification. Studies on the
mechanisms of this activation are in progress. It may well be
related to the regulation of casein kinase I1 activity in the
living cell. An increase in the amount of the protein kinase at
late stages of maturation may be necessary for its accumu-
lation and subsequent use in zygote division.
Surprisingly, we have found from, the recent article by
Mulner-Lorillon et al., that injections of casein kinase I1 block
maturation in Xenopus oocytes and suppress the burst of
phosphorylation in maturing oocytes [19]. From our point
of view this fact does not correspond well to our finding
concerning transitory activation of this enzyme at certain
stages of maturation. Undoubtedly, the fate, functions and
regulation of casein kinase 11 during oocyte maturation must
be studied further.
The authors express their deep gratitude to Professor A. S. Spirin
for constant attention and discussion of the results.
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