Insect Biochemistry and Molecular Biology 29 (1999) 215223

Protein phosphorylation during Rhodnius prolixus embryogenesis:

protein kinase casein kinase II activity
Eliane Fialho

a, b,*

, Hatisaburo Masuda b, Mario A.C. Silva-Neto

Departamento de Nutricao Basica e Experimental, Instituto de Nutricao Josue de Castro, Universidade Federal do Rio de Janeiro, UFRJ,
P.O. Box 68041, Rio de Janeiro CEP 21941-590 Rio de Janeiro, Brazil
Departamento de Bioqumica Medica, Instituto de Ciencias Biomedicas, Universidade Federal do Rio de Janeiro, UFRJ, P.O. Box 68041, Rio
de Janeiro CEP 21941-590 Rio de Janeiro, Brazil
Received 28 August 1998; received in revised form 27 November 1998; accepted 4 December 1998

Abstract
Protein kinase casein kinase II (CK II) activity was assayed during Rhodnius prolixus embryogenesis. Vitellin (VT) is the main
endogenous substrate during the whole development. It is maximally phosphorylated at the third day of embryogenesis by CK II
and then its phosphorylation decreases to a basal level by the time of first instar eclosion. When dephosphorylated casein was used
as an exogenous substrate a different profile of enzyme activity was obtained. CK II activity increases on day 1 after fertilization
and reaches a plateau on day 7 and its activity remains elevated until eclosion. Extracts obtained from oocytes or from 3-day old
eggs were fractionated through gel filtration chromatography. CK II activity was assayed in each fraction and the enzyme obtained
from the 3-day old eggs was shown to be three times more active than that obtained from oocytes, although the amount of enzyme
present in the fractions was the same. These enriched CK II fractions were assayed against different effectors, such as: cAMP, H8, H-89, calphostin C, sphingosine, polylysine and heparin. Heparin was the most effective one. When CK II activity was assayed
in non-fertilized eggs, no activation of the enzyme was observed when compared to fertilized eggs. These data indicate that CK
II is activated in a fertilization dependent process. The decrease in CK II activity against VT coincides with the beginning of VT
proteolysis processing suggesting a possible relationship between protein phosphorylation and yolk degradation. 1999 Elsevier
Science Ltd. All rights reserved.
Keywords: Casein kinase II; Embryogenesis; Rhodnius prolixus; Phosphorylation; Vitellin

1. Introduction
Protein kinases are known to promote the regulation
of countless intracellular processes which are fundamental for cell metabolism, growth and differentiation
(Hanks et al., 1988). Casein kinase II (CK II) is an
ubiquitous oligomeric enzyme that preferentially phosphorylates Ser/Thr residues in acidic stretches of its substrates. CK II is a cyclic nucleotide-independent
Ca2+/calmodulin-insensitive protein kinase which is

Abbreviations: CK II, casein kinase II; ECL, enhanced chemiluminescence; PAGE, polyacrylamide gel electrophoresis; PMSF, phenylmethylsulfonyl fluoride; SDS-PAGE, polyacrilamide gel electrophoresis in the presence of sodium dodecyl sulphate; TCA,
trichloroacetic acid; VT, Vitellin
* Corresponding author. Fax: +55-21-280-8343.
E-mail address: fialho@server.bioqmed.ufrj.br. (E. Fialho)

inhibited in vitro by low concentrations of heparin and

utilizes both ATP and GTP as phosphate donors
(Hathaway and Traugh, 1980; Agostinis et al., 1987).
This enzyme consists of two subunits, a (catalytic
subunit) and (regulatory subunit), which form a
tetrameric complex 2 2 (Pinna, 1990). This kinase
phosphorylates a broad spectrum of potential physiological substrates which include enzymes of intermediary
metabolism, cytoskeletal proteins, nuclear transcription
factors and proteins involved in cell cycle regulation
(Pinna, 1994). However, factors that may control CK II
activity in vivo are still unknown.
CK II regulation during development has not received
much attention by investigators despite the involvement
of this enzyme on cell proliferation and differentiation
(Ospina et al., 1992; Daz-Nido et al., 1994; Gruppuso
and Boylan, 1995). Glover et al. (1983) purified a CK
II activity from developing embryos of Drosophila mel-

0965-1748/99/$ - see front matter 1999 Elsevier Science Ltd. All rights reserved.
PII: S 0 9 6 5 - 1 7 4 8 ( 9 8 ) 0 0 1 2 9 - 5

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E. Fialho et al. / Insect Biochemistry and Molecular Biology 29 (1999) 215223

anogaster but no studies on the regulation of this enzyme

were conducted. Schneider et al. (1986) were the first to
show an increase in CK II activity during mouse
embryogenesis and in cultured tumoral cells. In cytosolic
extracts of Caenorhabditis elegans CK II activity is 3 to
10 fold lower than that found in embryos, and variations
in the activity are also associated with the mRNA levels
of subunit (Hu and Rubin, 1990). In Dictyostelium
discoideum CK II was purified and its regulation during
cell proliferation and differentiation was worked out
(Ospina et al., 1992). Daz-Nido et al. (1994); Gruppuso
and Boylan (1995) working in mouse neocortex and hepatic extracts also investigated CK II activity during
embryogenesis.
Insect eggs contain organelles called yolk spheres,
filled with vitellin (VT) a phosphorylated lipoglycoprotein which is the major source of amino acids for embryo
development. In the oocytes of the blood sucking bug
Rhodnius prolixus, VT accounts for 80% of total oocyte
proteins and is utilized during embryogenesis (Masuda
and Oliveira, 1985; Oliveira et al., 1986; Oliveira et al.,
1989). VT phosphorylation in insect oocytes and ovaries
was previously demonstrated by several studies (Glover
et al., 1983; Takahashi, 1983; Birnbaum et al., 1987;
Silva-Neto and Oliveira, 1993). In those reports protein
kinases from different classes were characterized such as
cyclic nucleotide dependent protein kinases (Takahashi,
1983; Edelman et al., 1987) and CK II (Glover et al.,
1983; Birnbaum et al., 1987; Issinger, 1993; Silva-Neto
and Oliveira, 1993). In Rhodnius CK II is the main protein kinase able to phosphorylate VT in chorionated
oocytes (Silva-Neto and Oliveira, 1993).
In order to analyse the activity of CK II during
embryogenesis and its possible relationship with VT
phosphorylation, in this paper CK II activity was measured in the eggs during the days following oviposition.
Here we show that VT is maximally phosphorylated by
CK II activity at the third day after oviposition, and also
that the activation of this enzyme is strictly dependent
on fertilization. A further decrease in the phosphorylation level of VT is attributed not to a decrease in the
CK II activity but to the interaction between these molecules. A possible relationship between protein phosphorylation and yolk degradation is discussed.

2. Materials and methods

2.1.

Chemicals

Soybean trypsin inhibitor, lima bean trypsin inhibitor,

leupeptin, benzamidine, antipain, PMSF, EDTA, EGTA,
sodium azide, bovine serum albumine, heparin, ATP,
cAMP, molecular weight standards, sphingosine, polylysine, glicine, acrylamide, bis-acrylamide, TEMED,
Dowex 1X10 beds were from Sigma Chemical Com-

pany (St. Louis, Mo, USA). Calphostin C, H-8 and H89 were purchased from CALBIOCHEM. Polyclonal
antibody against subunit from Upstate Biotechnology
Incorporated. Labeled nucleotide (-32P-ATP) was prepared according to Maia et al. (1983). Casein mixture
(Merck Darmstadt, Germany) was chemically dephosphorylated according to Reimann et al. (1971). All other
chemicals were from analytical grade.
2.2.

Insects, oocytes and eggs

Insects were taken from a colony of R. prolixus maintained at 28C and 70% relative humidity. Animals were
fed on rabbit blood at 3 week intervals. Normal and nonfertilized mated females fed on rabbit blood six days
beforehand were allowed to lay eggs for a period of 6
hours. These eggs were grouped and kept for the desired
length of time under the same conditions used for rearing
the colony. All days, in the same time, fertilized and
non-fertilized eggs were stored at 18C until the end of
embryogenesis. Fertilized and non-fertilized chorionated
oocytes were dissected from vitellogenic females in the
same day, washed with 0.15 M NaCl at 4C to remove
ovarian debris and stored at 18C. The oocytes and
eggs were homogenized in 20 mM Tris-HCl pH: 8.4,
150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 0.02%
NaN3, and a mixure of protease inhibitors with final concentrations of 0.05 mg/ml antipain, 0.05 mg/ml leupeptin, 0.05 mg/ml soybean trypsin inhibitor, 0.05 mg/ml
lima bean trypsin inhibitor and 1 mM benzamidine.
Homogenates were centrifuged at 11,000g for 10 min
at 4C. Floating lipids and pellets were discarded and
supernatants were used for protein kinase assays and
protein measurements.
2.3.

Protein kinase assays

Unless otherwise stated reaction mixtures contained

final concentrations of 50 mM Tris-HCl pH 8.0, 150 mM
NaCl, 10 mM MgCl2, 1 mM EDTA, 1 mM EGTA, 500
M -32P-ATP (500 cpms/picomol) and 0.1 mg/ml of
supernatant protein from oocytes or laid eggs in a final
volume of 0.1 ml. In some experiments dephosphorylated casein, 5 mg/ml was used as an exogenous substrate
and 0.025 mg/ml of oocyte or laid eggs protein. Heparin
was added in a final concentration of 1 g/ml. Reactions
were started by the addition of 32P-ATP and incubated
for 30 min. at 37C. Two aliquots of 25 l were analysed
by TCA-precipitable protein (Sahal and YamagushiFujita, 1987). Other conditions as described in SilvaNeto and Oliveira (1993). Protein was determined using
bovine serum albumin as standard as modified by Lowry
et al. (1951).

E. Fialho et al. / Insect Biochemistry and Molecular Biology 29 (1999) 215223

2.4.

Gel filtration chromatography on S 200 HR

Samples of supernatants from oocytes and third day

were obtained as described above. Gel filtration chromatography was then performed at 4C with a flux of 15
ml/hour in a 841.5 cm column of Sephacryl S 200 HR,
previously equilibrated with 20 mM Tris-HCl pH 8.0,
1.0 M NaCl, 10 mM NaF, 1 mM EDTA, 1 mM EGTA
and, 0.02% NaN3. Aliquots of 20 l from each fraction
were assayed for protein kinase activity using the standard reaction medium described above without NaCl and
with dephosphorylated casein as exogenous substrate. In
some experiments where stated c-AMP, H-8, H-89, calphostin C, sphingosine, polylysine, heparin were added
to the assay.
2.5.

SDS-polyacrylamide gel electrophoresis (PAGE)

Polyacrylamide gels (15150.1 cm) were run in the

presence of SDS (Laemmli, 1970) at a constant current
of 20 mA. Gels were stained, dried, and exposed to Xray film at 70C. The following molecular mass standards were used: myosin (205 kDa), -galactosidase
(116 kDa), phosphorylase b (98 kDa), albumin (66 kDa),
ovalbumin (45 kDa), glyceraldehyde-3-phosphate
dehydrogenase (36 kDa), carbonic anhydrase (29 kDa),
and cytochrome c (12.3 kDa).
2.6.

217

Western blottings

Aliquots from gel filtration with the maximal activity

of protein kinase were separated by SDS-PAGE (7.5%)
and transferred at 100 mA for 60 minutes to a nitrocellulose membrane. Membranes were probed with a polyclonal antibody raised against a polypeptide of the catalytic site of the subunit of human casein kinase II and
developed using the ECL system. Other conditions as
described in (Towbin et al., 1979).

3. Results
3.1. CK II activity during Rhodnius prolixus
embryogenesis
When CK II activity was assayed against endogenous
substrates of oocytes and developing eggs, we noticed
an increase of three fold by the third day of embryogenesis (Fig. 1). This activity decreases up to the time of
eclosion of the first instar larvae at day 15 after oviposition (Fig. 1). Protein kinase activity was strongly
sensitive to heparin indicating that the major enzyme
activity involved with development is CK II. In vitro
phosphorylated substrates were identified by SDS-PAGE
(Fig. 2A) and by autoradiography (Fig. 2B). As it can
be seen in Fig. 2B, vitellin or the 103 and 56 kDa VT

Fig. 1. CK II activity during Rhodnius prolixus embryogenesis. CK

II activity was assayed in oocytes and eggs supernatants using 32PATP as described under Materials and methods in the absence (open
circles --) or presence (closed triangles --) of heparin 1 g/ml.
After 30 min of reaction two aliquots were removed from each reaction
medium spotted on Whatmann 3 MM filter papers which were then
precipitated in cold TCA, washed and dried. Radioactivity associated
with the phosphorylation of endogenous substrate was estimated by
liquid scintillation counting. Each point represent the meanS.E. of
three independent experiments.

polypeptides produced during its programmed proteolysis (Oliveira et al., 1989) are phosphorylated by CK II.
The identity of VT proteolytic products was determined
through western blotting (data not shown) as described
by Oliveira et al. (1989).
3.2. CK II activity assayed with dephosphorylated
casein
Since vitellin is under proteolytic attack during most
part of egg development, the decrease of its in vitro
phosphorylation after day 3 (Fig. 1) could be related to
the disappearance of CK II sites in the molecule. In order
to determine the levels of enzymatic activity in this period we decided to use casein as an exogenous substrate.
Under this condition, Fig. 3 shows that CK II activity
slightly decreases after fertilization (day 0). After day 0
CK II activity increases up to day 7 reaching a plateau,
and the enzyme stays activated up to the end of
embryogenesis. This material was also submitted to
SDS-PAGE followed by autoradiography as can be seen
on Fig. 3B. This result shows that the decrease on CK
II activity against endogenous substrates (Fig. 1) is
related to the lack of phosphorylation sites on VT molecule as a result of its proteolytic processing during
development. Dephosphorylated casein is a substrate for
several protein kinases. Although CK II is the main protein kinase acting on this substrate under our experimental conditions as judged by its marked heparin sensitivity shown in Fig. 3A, and also by autoradiography
Fig. 3C.

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E. Fialho et al. / Insect Biochemistry and Molecular Biology 29 (1999) 215223

Fig. 2. SDS-PAGE analysis of CK II activity against endogenous substrates. Samples from each reaction medium assayed in the absence of
heparin on Fig. 1 were subjected to SDS-PAGE on a 7.5% polyacrylamide separating gel. A: gel stained with Coomassie Blue; B: autoradiogram
of A. Lane sd: high molecular mass standards. The molecular mass of each standard polypeptide is indicated at the left of the figure; lane Oc.:
oocyte supernatant; lane 014: supernatants obtained from eggs at different days of embryogenesis. The numbers correspond to the day after
oviposition. Open arrows at the right side indicate the position of polypeptides derived from vitellin subunits. Arrowheads on panel B indicate the
position of main phosphorylated polypeptides derived from vitellin subunits.

3.3. CK II activity of oocytes and 3-day old eggs

after gel filtration chromatography
CK II is a protein kinase with unknown physiological
regulators. Several polycationic low molecular weight
molecules are able to increase its activity in vitro. The
activation of CK II during Rhodnius development could
be related simply to the presence of positive low molecular effectors such as polyamines (Birnbaum et al., 1987).
In order to check this hypothesis, oocyte and 3-day old
eggs extracts were submitted to gel filtration chromatography as shown on Fig. 4. The amount of protein used
from each source is nearly the same as it is shown by
absorbance at 280 nm (Fig. 4, panels A and B). When
fractions were assayed for protein kinase activity with
dephosphorylated casein as substrate we observed that
CK II remained activated after the third day of
embryogenesis (Fig. 4, panels A and B). This increase
in activity can not be attributed to the synthesis of low

molecular weight effectors following fertilization since

they were separated by the gel filtration chromatography.
The association of those molecules with the holoenzyme
is not probable since a high concentration of NaCl was
used in the chromatography in order to disrupt noncovalent interactions.
3.4. Western-blot analysis of fractions of oocyte and
3-day old eggs enriched with CK II
The increase in the CK II activity on day three after
oviposition could be due to an increase in the synthesis
of the enzyme. In order to test this possibility the peak
of CK II activity obtained from the oocyte or the eggs
on its third day, were submitted to SDS-PAGE (Fig. 4C),
transferred to a nitrocellulose membrane and reacted
with a polyclonal antibody raised against the subunit
of human CK II (Fig. 4D). This figure shows that the
amount of CK II protein found in the oocyte or in the

E. Fialho et al. / Insect Biochemistry and Molecular Biology 29 (1999) 215223

219

Fig. 3. CK II activity assayed with dephosphorylated casein. CK II activity was assayed in oocytes and eggs supernatants. A: Phosphorylation
of casein (5 mg/ml) with 32P-ATP as described under Materials and methods in the absence (open circles --) or in the presence (closed triangles -) of 1 g/ml heparin. After 30 minutes of reaction two aliquots were removed from each reaction medium spotted on Whatmann 3 MM filter
papers which were then precipitated in TCA, washed and dried. Radioactivity associated with the phosphorylation was estimated by liquid scintillation counting. Each point represent the meanS.E. of three independent experiments. B: Samples from each day assayed against dephosphorylated
casein in the presence or in the absence of heparin were subjected to SDS-PAGE on a 15% polyacrylamide separating gel. Autoradiogram of the
phosphorylation reaction in the absence of heparin and C: Autoradiogram of the phosphorylation reaction in the presence of heparin. Lane Ooc.,
Oocyte supernatant; lane 013 eggs supernatants from different days of embryogenesis. The numbers correspond to the day of embryo development.
Arrows point to main casein polypeptides.

eggs in its third day are very similar. This result strengthens the possibility that the increase in CK II activity
may be due to a possible activation by covalent modification following fertilization.
3.5. CK II activity in the presence of protein kinase
effectors
CK II regulation by covalent modification through
phosphorylation-dephosphorylation mechanisms was
reported by several authors (Meggio et al., 1983; Agostinis et al., 1987; Ackerman et al., 1990; Mulner-Lorillon
et al., 1990; Litchfield et al., 1991). The presence of comigrating protein kinases could contribute to the total
CK II activity in the enriched CK II fractions obtained
on S 200 HR. We decided then to test enzyme fractions
against several well known modulators of protein phosphorylation. CK II activity from both oocyte and third
day old eggs is not extensively affected neither by cyclic
nucleotide dependent protein kinase modulators such as
c-AMP, H-8, H-89 nor by protein kinase C effectors
such as sphingosine and calphostin C (Table 1). Polylysine, an commonly used activator of casein kinases did
not induce a significative change on the activity of the
enzyme obtained from oocytes but induced a 40%
increase in the activity of the enzyme obtained from
three day old eggs. Heparin classically known as an
inhibitor of CK II induced a dramatic change in casein
phosphorylation. These results demonstrate that CK II
activation at day three is not due to any co-migrating
protein kinase activity.
3.6.

CK II activity in unfertilized eggs

The above set of data indicate that oocyte CK II is

under regulation after fertilization. Since insects lay their

eggs the process of oviposition could be responsible for

CK II activation. Non-mated females of Rhodnius produce and lay unfertilized eggs (Davey, 1967). These
eggs were then assayed for CK II activity during several
days after oviposition. Fig. 5 shows that CK II activity
remains unchanged even when these unfertilized eggs
are left to stand until 13 days after they were laid.
Besides that a marked heparin inhibition was observed
in all days tested. This material was also analysed by
SDS-PAGE (Fig. 6A) followed by autoradiography (Fig.
6B). Although VT is still the most phosphorylated substrate this molecule does not suffer the proteolytic processing observed in developing eggs.

4.

Discussion

In the present study we show that CK II is activated

up to the third day of embryogenesis but its activity in
vitro, against endogenous substrates, decreases and
reaches a basal level by the fourteenth day after oviposition (Fig. 1). Since vitellin suffers proteolytic attack
following fertilization (Fig. 2) it seems reasonable to
believe that the fall on CK II activity, after 3 days of
embryogenesis, is related to the disappearance of its consensus phosphorylation sequences on VT molecule. This
hypothesis was strongly supported when CK II activity
was assayed in oocytes and eggs under development,
using casein (Fig. 3) as substrate. The CK II activity
increased from day 1 to day 7 when it reached a plateau
and remained activated up to the time of eclosion of the
first instar larvae. Oviposition of unfertilized eggs did
not result in CK II activation as shown in Fig. 5, nor to
VT degradation (Fig. 6). Taken together these data suggest that CK II activity is, in fact, activated following
fertilization and the decrease in the phosphorylation

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E. Fialho et al. / Insect Biochemistry and Molecular Biology 29 (1999) 215223

Table 1
CK II activity in the presence of protein kinase effectorsa
Effector

Oocyte

Third day

Control
cAMP
Polylysine
H-8
Calphostin C
Sphingosine
Heparin

1.000.02
1.010.06
1.260.02
1.050.03
0.800.06
0.990.07
0.130.06

2.630.01
2.850.03
4.490.04
2.880.08
2.920.04
3.250.06
0.060.01

Fractions of oocyte or 3-days old eggs enriched with CK II were

both assayed against several protein kinase effectors using dephosphorylated casein as substrate as described in Materials and methods.
The values obtained with Control (without any effector) were considered 1 as reference; c-AMP 10 M; polylysine 0.1 mg/ml; H-8 1
M; H-89 0.1 M;calphostin C 0.1 M; sphingosine 60 M and heparin 1 g/ml. Other experimental conditions were as described in
Materials and methods. Each point is the meanS.E. of three independent experiments

Fig. 4. CK II activity after gel filtration chromatography and Western-blot analysis on enriched fractions of oocyte and 3-day old eggs.
Extracts of oocyte A; and third day old eggs B; were separately chromatographed on a S200 HR gel filtration column (1.584 cm). A: shows
the elution profile of oocyte column (open circles --) and CK II
activity of each fraction assayed using dephosphorylated casein as substrate as described on Material and Methods (closed circles --). B:
shows the elution profile of third day old eggs column (open circles -) and CK II activity of each fraction assayed using dephosphorylated
casein as substrate as described on Materials and methods (closed
circles --). Inset C: fractions with maximal CK II activity were submitted to SDS-PAGE (7.5%) and stained with Coomassie blue; Rhodnius Lipophorin was included as a negative control (lane 1). Fraction
obtained from oocyte preparation (lane 2) and fractions from 3-day
old eggs (lane 3). Inset D: the gel on C was transferred to nitrocellulose
membrane and reacted with a polyclonal antibody against a catalytic
subunit of human CK II. Molecular mass standards of Vitellin are
indicated at left of Inset C.

level of endogenous substrate can not be attributed to

the down regulation of the enzyme since it remained
activated as judged by the experiment of (Fig. 3).
Besides that, the increase in its activity also occurs even
when the enzyme is fractionated (Filhol et al., 1991b;
Glover, 1986) and separated from low molecular weight
effectors eventually present, such as polyamines (Fig. 4).
We also demonstrated that CK II protein levels are similar in the oocytes and third day old eggs (Fig. 4). A
synthetic peptide with a single phosphorylated site (RR-R-E-E-E-T-E-E-E) was also used as a substrate for
CK II (Kuenzel and Krebs, 1985) and the activity from
day 3 was also greater than the oocyte (data not shown).
This data suggested that the increase in CK II activity

Fig. 5. CK II activity in unfertilized eggs. CK II activity was assayed

in oocytes and non-fertilized eggs supernatants using the standard reaction media described under Materials and methods in the presence
(closed triangles --) and in the absence (open circles --) of heparin
1 g/ml. After 30 minutes of reaction two aliquots were removed from
each reaction medium spotted on Whatmann 3 MM filter papers which
were then precipitated in TCA, washed and dried. Radioactivity associated with the phosphorylation of endogenous substrate was stimated
by liquid scintillation counting. Each point represent the meanS.E. of
three independent experiments.

is related to some unknown covalent modification of the

enzyme. We decided then to investigate the presence of
co-migrating protein kinases on these gel filtration
enriched fractions. As Table 1 shows neither cyclic AMP
dependent nor protein kinase C effectors exerted any
dramatic effect on the CK II activity.
The above set of data indicated that upon fertilization
CK II activity in Rhodnius is positively regulated by
some kind of covalent modification of the enzyme. This
modification probably takes place during the first hours
of development. In fact, CK II activation occurs by different mechanisms such as the presence of positive

E. Fialho et al. / Insect Biochemistry and Molecular Biology 29 (1999) 215223

221

Fig. 6. SDS-PAGE analysis of CK II activity against endogenous substrates in oocytes and unfertilized eggs. Samples from each reaction media
assayed with 32P-ATP in the absence of heparin on Fig. 5 were subjected to SDS-PAGE on a 7.5% polyacrylamide separating gel. A: show the
gel stained with Coomassie Blue; B: shows the 32P-labeled autoradiogram. Lane sd, high molecular mass standards; lane Oc., oocyte supernatant;
lane 013 non-fertilized eggs supernatants obtained from eggs at different days after oviposition. The numbers represent the day after oviposition.

effectors of the enzyme which bind to the subunit

(Tuazon and Traugh, 1991); autophosphorylation of its
subunits (Meggio et al., 1983; Edelman et al., 1987);
modulation of signal transduction pathways induced by
different ligands such as EGF, IGF-1, insulin and p34cdc2
(Ackerman et al., 1990; Litchfield et al., 1991; Sommercorn et al., 1987; Bosc et al., 1995, respectively), the
presence of DNA binding sperm proteins (histone and
protamine) that functions as a potent activators for CK
II in fertilized eggs (Ohtsuki et al., 1996) or simply by
an increase in its synthesis (Sommercorn et al., 1987).
EGF stimulation in cultured A-431 cells occurs through
direct phosphorylation of CK II by a yet not identified
protein kinase (Ackerman et al., 1990). Mulner-Lorillon
et al. (1990) and Litchfield et al. (1992) were the first
to demonstrate that p34cdc2, an enzyme deeply involved
in cell cycle control, could phosphorylate and modulate
CK II activity. Sanghera et al. (1992) showed that this
regulation can also be achieved by protein kinase C
dependent phosphorylation in sea urchin oocytes.
Although several reports are available in the literature

there is no exact definition of the effects protein phosphorylation may play in the regulation of this enzyme.
The role of protein phosphatases in this process is still
uncertain. Agostinis et al. (1987) also reported that some
categories of these enzymes are also able to dephosphorylate and activate CK II. The cdc25 protein contains
an intrinsic tyrosine phosphatase activity that directly
activates p34cdc2, this latter enzyme phosphorylates and
modulates CK II activity (Dunphy and Kumagai, 1991;
Gautier et al., 1991). The determination of the CK II
activation pathway requires the isolation and cloning of
these enzymes from several systems together with the
identification of its phosphorylation consensus
sequences. In Rhodnius eggs it will be necessary to
identify protein kinases and/or phosphatases activated
upon fertilization and verify whether the effect of these
enzymes modify the activity of CK II.
Finally an important point is the relationship of CK
II with its main endogenous substrate, vitellin. The
relationship of vitellin phosphorylation and vitellin
degradation deserves to be investigated. Several well

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E. Fialho et al. / Insect Biochemistry and Molecular Biology 29 (1999) 215223

known proteolytic systems were recently demonstrated

to be regulated by protein phosphorylation status such
as phosphoprotein Tau, arrestin, nucleolin and neurofilaments (Pant, 1988; Litersky and Jonhson, 1992; Azarian
et al., 1995). Takahashi (1983) was the first to demonstrate that during the development of Bombyx mori, cAMP-dependent protein kinase regulates the affinity of
proteases to vitellin. In Rhodnius eggs, experiments are
still required in order to demonstrate whether CK II
phosphorylation of vitellin occurs in a timetable dependent way associated with VT degradation by the growing
embryo, or not.

Acknowledgements
This work was supported by grants from Conselho
Nacional de Desenvolvimento Cientfico e Tecnologico,
the Financiadora de Estudos e Projetos, and the Programa de Apoio ao Desenvolvimento Cientfico e Tecnologico. We thank Lilian S.C. Gomes, Rosane O.M.M.
Costa, Helosa S.L. Coelho, Jose S. Lima Junior, and
Jose F. Souza Neto for their excellent technical assistance, and finally to Dr. Pedro Lagerblad Oliveira for
helpful comments.

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