Gvozdev1 1 Institute of Molecular Genetics, Russian Academy of Sciences, Moscow, Russia;2Belozersky Institute of Physico-Chemical Biology, Center of Molecular Medicine, Moscow State Univ
Trang 1CK2btes gene encodes a testis-specific isoform of the regulatory
Alla I Kalmykova1, Yuri Y Shevelyov1, Oksana O Polesskaya1,*, Anna A Dobritsa1,†,
Alexandra G Evstafieva2, Brigitte Boldyreff3, Olaf-Georg Issinger3and Vladimir A Gvozdev1
1
Institute of Molecular Genetics, Russian Academy of Sciences, Moscow, Russia;2Belozersky Institute of Physico-Chemical Biology, Center of Molecular Medicine, Moscow State University, Russia;3Department of Biochemistry and Molecular Biology,
University of Southern Denmark, Odense, Denmark
An earlier described CK2btes gene of Drosophila
melano-gasteris shown to encode a male germline specific isoform of
regulatory b subunit of casein kinase 2 Western-analysis
using anti-CK2btes Ig revealed CK2btes protein in
Drosophila testes extract Expression of a CK2btes–
b-galactosidase fusion protein driven by the CK2btes
pro-moter was found in transgenic flies at postmitotic stages of
spermatogenesis Examination of biochemical
characteris-tics of a recombinant CK2btes protein expressed in
Escherichia colirevealed properties similar to those of CK2b:
(a) CK2btes protein stimulates CK2a catalytic activity
toward synthetic peptide; (b) it inhibits phosphorylation of
calmodulin and mediates stimulation of CK2a by polylysine;
(c) it is able to form (CK2btes)2 dimers, as well as (CK2a)2(CK2btes)2tetramers Using the yeast two-hybrid system and coimmunoprecipitation analysis of protein extract from Drosophila testes, we demonstrated an associ-ation between CK2btes and CK2a Northern-analysis has shown that another regulatory (b¢) subunit found recently in
D melanogaster genome is also testis-specific Thus, we describe the first example of two tissue-specific regulatory subunits of CK2 which might serve to provide CK2 sub-strate recognition during spermatogenesis
Keywords: spermatogenesis; casein kinase 2; CK2 b subunit; CK2btes; testes
Protein kinase CK2 is involved in such general cell processes
as cell cycle regulation, transcriptional control, signal
transduction, development and proliferation [1–4] More
than 160 different proteins serve as substrates for CK2
Phosphorylation by CK2 has been found to affect activity
of such Drosophila proteins pivotal for realization of early
developmental program, as Cut-homeodeomain protein,
Cactus and Antennapedia [5–7] A CK2 holoenzyme
consists of two a- (or a¢-) and two b subunits The a subunit
of CK2 possesses catalytic activity and the regulatory
b subunit was shown to enhance stability of the holoenzyme,
activate CK2a and provide substrate specificity and CK2
targeting in cells In spite of CK2b being ubiquitously
represented among eukaryotes, it is far less conserved in
comparison with the catalytic CK2a This fact might be
explained by a wide spectrum of substrates and partner
proteins interacting with CK2b as a regulatory subunit
Moreover, other functions, besides being a part of the CK2 holoenzyme, can be ascribed to the b subunit For example,
it has been demonstrated that the CK2 b subunit is involved
in the regulation of catalytic activity of two other protein kinases (A-raf and Mos kinases [8–10]) The conclusion that CK2b has a more general functions is supported by the fact that significant imbalance of its amount in respect to
a subunit is found in tumor cells and some mammalian tissues such as testicles [11, 12]
Recently it was shown that the CK2 activity as well as the CK2 protein level are mostly elevated in rat and mouse testicles [12, 13] An important role for the CK2 activity in spermatogenesis was clearly shown by a Ôknock-outÕ of the CK2a¢ gene in mice resulting in a male sterile phenotype [14] Spermatogenesis is a complex differentiation process comprising mitotic and meiotic divisions of germline stem cells followed by sperm morphogenesis This process is known to have a lot of common features in Drosophila and mammals [15] However, genetic control and molecular mechanisms of both Drosophila and mammalian sperma-togenesis are still poorly understood
The genomes of most eukaryotes including mammals carry a single gene encoding b subunit of CK2 Only Saccharomyces cerevisiae and Arabidopsis thaliana are known to have two and three isoforms of CK2b, respect-ively [16, 17] Recently, we have described in Drosophila the SSL gene [18], later renamed CK2btes [19], as a first candidate on the role of a tissue-specific isoform of the CK2 regulatory subunit This gene is expressed exclusively in testes and encodes a protein sharing 45% amino-acid identity with the ubiquitous Drosophila b subunit Another potential Drosophila tissue-specific CK2 regulatory subunit (b¢) was identified in the yeast two-hybrid screen where
Correspondence to Y Y Shevelyov, Department of Molecular
Genetics of Animals, Institute of Molecular Genetics, 123182,
Kurchatov Sq 2, Moscow, Russia Fax: + 7 095 1960221;
Tel.: + 7 095 1961909; E-mail: shevelev@img.ras.ru
Abbreviations: CK2, casein kinase 2; CK2b, CK2 b subunit; CK2a,
CK2 a subunit; IP, immunoprecipitation; RNAi, RNA interference;
dsRNA, double stranded RNA; X-gal, 5-bromo-4-chloro-3-indolyl
b-galactopyranoside.
*Present address: Molecular Neurobiology Branch, NIDA, NIH, 5500
Nathan Shock Drive, Baltimore, MD, 21224, USA.
Present address: Department of Molecular, Cellular and
Develop-mental Biology, Yale University, New Haven, CT 06520, USA.
(Received 25 September 2001, revised 7 December 2001, accepted 14
January 2002)
Trang 2CK2a was used as a bait [20] In this work we present
compelling evidence that the CK2btes protein serves as a
tissue-specific isoform of the CK2 regulatory subunit in
Drosophilamale germline
E X P E R I M E N T A L P R O C E D U R E S
Plasmid constructions
PCRs were performed according to the recommendations
of the manufacturer using GeneAmp XL PCR Kit (Perkin
Elmer, Branchburg, NJ, USA) containing high fidelity
mixture of DNA-polymerases
CK2btes and CK2a expression constructs: An 850 bp
BamHI–SalI fragment of the CK2btes cDNA #112 (this
cDNA sequence, cloned in the pBlueScript SK- vector,
contains no poly(A) tail and corresponds to nucleotides 72–
840 of the SSL (CK2btes) cDNA #911 sequence deposited
in GeneBank under accession number L42285, see also [18])
was subcloned in the pQE 30 expression vector (Stratagene,
La Jolla, CA, USA) The recombinant protein with the
N-terminal His6-tag comprises the whole CK2btes ORF
except for the 11 amino acids at the N-terminus
A 1011-bp fragment of D melanogaster CK2a gene
comprising the whole ORF region was PCR-amplified from
Drosophila genomic DNA using the following pair of
primers: 5¢-CAGGATCCATGACACTTCCTAGTGCG
GCTCGC-3¢ and 5¢-CCAAGCTTTTATTGCTGATTAT
TGGGATTCATTTGACCA-3¢ (the gene encoding the
DrosophilaCK2 a subunit does not contain introns in the
coding region [21]) The BamHI–HindIII digested PCR
fragment was subcloned in the pQE 30 vector
CK2b¢ probe for Northern-analysis The161 bp
3¢-frag-ment of the CK2b¢ gene was PCR-amplified from Drosophila
genomic DNA using primers 5¢-ATAAGCTTGCTTT
AGTGCCCACTTATTCGAAAAG-3¢ HindIII–BamHI
digested PCR product was cloned into the pBlueScript
SK-vector and then recloned by KpnI–BamHI into the pTZ19R
vector In vitro transcription was performed for 1 h at 37°C
in the buffer containing 40 mMTris/HCl, pH 7.5, 60 mM
MgCl2, 5 mMNaCl, 10 mMdithiothreitol, 0.5 mMof each of
the ATP, GTP, CTP, 100 ng of the linearized plasmid DNA,
20–100 lCi [a-32P]UTP, 2–5 units of T7 RNA polymerase
(Gibco BRL, Life Technologies, CA, USA), 25 U of RNAse
inhibitor (Gibco BRL, Life Technologies, CA, USA)
Constructs for P-element transformation
To make the CK2btes–b-galactosidase fusion construct, a
934-bp fragment of the CK2btes gene including the 161 bp
of promoter region linked to the whole ORF was
PCR-amplified from the DNA of the cosmid clone #9 [18] using
the following pair of primers: 5¢-GACTGCAGTGAAGG
GCATCGAGTCCTCGGG-3¢ and 5¢-GAGGATCCGG
GACATTCCTTAGCCAGGAGGG-3¢ To make the
b-galactosidase expressing construct, a 173-bp PCR
frag-ment of the CK2btes gene including the 161 bp of promoter
region joined with the first 12 bp of the ORF region was
amplified from the DNA of the cosmid clone #9 using the
same direct primer as for the CK2btes–b-galactosidase
fusion construct and the following reverse primer:
5¢-CTGGATCCGGACACGACATGCTCACTCGAA TAA-3¢ Both PstI–BamHI digested PCR fragments were cloned in frame, with the b-galactosidase ORF devoid of the ATG, into the pCaSpeR-bgal vector [22]
To generate the CK2btes ÔantisenseÕ construct, the XhoI fragment of the CK2btes cDNA #421 corresponding to the 12–700 bp region of the sequence of cDNA #911 (one XhoI site in the cDNA #421 is located in the MCS of BlueScript SK- vector, and another XhoI site is located in the adaptor sequence at the opposite side of insert) was cloned into the modified testis vector kindly provided by H.D Hoyle (University of Indiana, Bloomington, IN, USA) [23] This vector carries the regulatory region of the b2-tubulin gene driving testis-specific expression of any gene substituting the b2-tubulin ORF The regulatory region had been cloned upstream of the mini-white gene in the pCaSpeR4 vector The modification of the vector includes the insertion in its EcoRI cloning site of the MCS polylinker, which may now
be used for cloning with EcoRI, XhoI and KpnI The ÔantisenseÕ orientation of the CK2btes cDNA relative to the b2-tubulin promoter was verified by restriction digestion Constructs for the yeast two-hybrid system assay
To make CK2a AD- and BD- constructs, the whole CK2a ORF region was amplified from Drosophila genomic DNA using the following pair of primers: 5¢-CAGAATTCA TGACACTTCCTAGTGCGGCTCGC-3¢ and 5¢-CTG GATCCTTATTGCTGATTATTGGGATTCATTTGA CCA-3¢ EcoRI–BamHI digested PCR product was cloned
as a fusion with the GAL4 activation domain in the pGAD424 vector, or as a fusion with the GAL4 DNA-binding domain in the pGBT9 vector (Clontech, La Jolla,
CA, USA)
To prepare CK2btes AD- and BD- constructs, the whole CK2btes ORF region was amplified from the cDNA #911 using the following pair of primers: 5¢-CTGGATCCCT ATGTCGTGTCCCAGGAGCATCGAG-3¢ and 5¢-GTC TGCAGTTAAAAATTCGGGACATTCCTTAGCCA GG-3¢ BamHI–PstI digested PCR product was cloned as a fusion with GAL4bd in the pAS2-1 vector (Clontech, La Jolla, CA, USA) The CK2btes ORF was excised from the pAS2-1 plasmid by joint BamHI and PstI digestion, the PstI end was blunted by T4 DNA polymerase, and fragment was cloned in the BamHI–XhoI digested (the XhoI end was also blunted) pACT2 vector (Clontech, La Jolla, CA, USA) as a fusion with GAL4ad
To prepare CK2b AD- and BD- constructs, the whole CK2b ORF region was amplified from the pEV55Dmb plasmid DNA (kindly provided by C.V.C Glover (Uni-versity of Georgia, Athens, GA, USA), it contains a full size cDNA of D melanogaster b subunit [24]) using the follow-ing pair of primers: 5¢-CAGGATCCCTATGAGCAGC TCCGAGGAAGTCTCCT-3¢ and 5¢-CTGTCGACTTA GTTTTTCGCTCGTAGTGGCATTTTAAAATTGGCT GC-3¢ BamHI–SalI digested PCR fragment was cloned into BamHI–SalI digested pAS2-1 vector, or into BamHI– XhoI digested pACT2 vector
Protein purification, generation of antibodies Expression and purification of recombinant proteins from
E coli using Ni2+/nitrilotriacetic acid resin (Qiagen Inc.,
Trang 3CA, USA) were performed according to the Stratagene
protocols Drosophila CK2a and CK2btes recombinant
proteins purified under nondenaturing conditions were used
in the in vitro assays (measurement of CK2a activity, gel
filtration experiments) Human CK2a and Drosophila
CK2btes proteins purified from E coli inclusion bodies
under denaturing conditions were used for the generation of
antibodies in rabbits The specificities of isolated antisera
were tested by Western analysis
RNA isolation and Northern-analysis
Total RNA was isolated by guanidinium thiocyanate
extraction [25] from embryos, pupae, larvae, females, male
carcasses and testes of gt wastrain, fractionated by
electro-phoresis in denaturing formaldehyde-agarose gel and
transferred to a nylon HyBond-N filter (Amersham, Little
Chalfont, UK) Filter prehybridization, hybridization and
washing were performed according to standard protocols
[26] As a control for the RNA loading, hybridization of the
same filter with the rp49 probe [27] was performed
CK2 activity test
The equimolar mixture of CK2a and CK2btes proteins
purified under nondenaturing conditions or the CK2a
protein alone were assayed for the CK2 phosphorylation
activity using a synthetic peptide RRRDDDSDDD as a
substrate The reaction was carried out in the buffer (45 mM
Tris/HCl, pH 8.0, 5 mMMgCl2, 1 mMdithiothreitol, 50 lM
ATP, 2 lCiÆmL)1 [c-32P]ATP (3000 CiÆmmol)1), 200 lM
peptide) containing different NaCl concentrations (from
0 mMto 200 mM) at 37°C for 5 min The reaction aliquots
were loaded onto P81 phosphocellulose paper, washed with
85 mMphosphoric acid, and incorporated radioactivity was
measured by the liquid scintillation counter
For the phosphorylation of calmodulin (kindly provided
by N B Gusev, Moscow State University) the aliquots of
fractions after gel filtration assay containing 50 ng of
CK2a either alone or in combination with equimolar amount
of CK2btes protein were used The reaction was carried out
in 50 mMTris/HCl, pH 8.0, 10 mMMgCl2, 150 mMNaCl,
20 lM ATP, 10 lCiÆmL)1 [c-32P]ATP (3000 CiÆmmol)1),
10 lMcalmodulin at 37°C for 15 min Polylysine (Sigma,
St Louis, MI, USA) at concentration 100 lgÆmL)1 was
added where necessary The reaction was stopped by
cooling in ice, and the samples were subjected to 15%
SDS/PAGE The gels were dried and autoradiographed
Assays for detection of protein-protein contacts
in yeast two-hybrid system
Protein–protein interactions were assayed using three
different approaches For the b-galactosidase filter assay,
single colonies cotransformed with AD- and BD- constructs
were picked and transferred to a Whatman no 5 paper,
which was further incubated on a fresh plate for 2–3 days
The filters were frozen in liquid nitrogen, layered over a
second filter prewetted with Z-buffer (16.1 gÆL)1 Na2
H-PO4Æ7H2O, 5.5 gÆL)1 NaH2PO4ÆH2O, 0.75 gÆL)1 KCl,
0.246 g L)1 MgSO4· 7H2O), which contained 0.27 mL
2-mercaptoethanol and 1.67 mL
5-bromo-4-chloro-3-indo-lyl b-galactopyranoside (X-gal; 20 mgÆmL)1 in
dimethyl-formamide) per 100 mL Incubation was performed at
30°C for up to 12 h
For the liquid assay 5 mL cultures with synthetic medium were inoculated with single colonies cotransformed with AD- and BD- constructs and were grown until
D600 1 Each culture (1 mL) was transferred to a microcentrifuge tube and centrifuged for 5 s The yeast pellet was dissolved in 100 lL of Z buffer and frozen in liquid nitrogen After thawing, 700 lL of Z buffer with mercaptoethanol and 160 lL O-nitrophenyl-b-D -galacto-side (4 mgÆmL)1in Z buffer) were added and the reaction was incubated for 1 h at 30°C The reaction was stopped
by addition of 400 lL 1M Na2CO3 After centrifugation for 10 min at 13 400 g, the A420was measured b-Galac-tosidase activity was calculated in Miller units according to the formula: units¼ 1000 · A420/(culture volume in ml· incubation time in min· D600)
In addition, the ability of the yeast strain HF7c (carrying HIS3reporter gene) being cotransformed with AD- and BD- constructs to grow on the medium without histidine was used to verify protein–protein interactions
Immunoprecipitation
A total of 100 hand-dissected pairs of testes were homo-genized in the buffer containing 50 mMTris/HCl, pH 8.0,
150 mM NaCl, 0.05% Nonidet P40, cocktail of protease inhibitors After 3 h of incubation at 4°C followed by
15 min centrifugation at 4000 g, crude extract was fivefold diluted with IP buffer (50 mMTris/HCl, pH 8.0, 150 mM NaCl, 0.05% NP40, 5 mM EDTA, 0.2% BSA, 0.02% NaN3, cocktail of protease inhibitors) Immunoprecipita-tion was carried out over night at 4°C with 1 lL of anti-DmCK2a Ig, kindly provided by C.V.C Glover The complex was precipitated by incubation with protein A–Sepharose (4 Fast Flow, Pharmacia Biotech, Uppsala, Sweden) for 1 h at 4°C Immunoprecipitate was washed fourfold with 0.5 mL IP buffer and then fractionated on the 8% SDS/PAGE followed by Western analyses with poly-clonal anti-(b-galactosidase) Ig (ICN Pharmaceuticals inc., Costa Mesa, CA, USA)
Western blot analysis For the detection of CK2btes and CK2a proteins in testes extracts by Western analysis the following antibodies were used: anti-CK2btes nonpurified serum at a dilution of
1 : 5000; and anti-(Drosophila CK2a) serum, kindly provi-ded by C.V.C Glover, at a dilution of 1 : 5000 For detection of CK2a in gel filtration assay rabbit anti-(human CK2a) polyclonal IgG was used Alkaline-phosphatase-conjugated anti-(rabbit IgG) Ig (Sigma, St Louis, MI, USA) was used as a secondary reagent
Samples were resolved by electrophoresis in SDS/PAGE and blotted onto Hybond-C membrane (Amersham, Little Chalfont, UK) Blots were developed using the CDP-star detection system (Tropix, Bedford, MA, USA) according to the recommendations of the manufacturer
Gel-filtration experiments Proteins were passed through the Pharmacia SMART system chromatographic Superose 6 column in the buffer
Trang 4(25 mMTris/HCl, pH 8.5, 1MNaCl) in the flow rate regime
(40 lLÆmin)1)
P-element transformation
Transgenic lines were generated using standard P-element
mediated germline transformation technique [28] with
Df(1)w67c23(2), y strain and the pTURBO transposase
source Three transformant lines were established for the
b-galactosidase bearing construct, two lines for the
CK2btes-b-galactosidase fusion construct, and one line for the
ÔantisenseÕ CK2btes construct
Histochemical staining of tissues
For the b-galactosidase staining, testes from adult
Drosophilamales, as well as carcasses, were hand-dissected,
fixed in 2% glutaraldehyde in KCl/NaCl/Pibuffer (8 mM
Na2HPO4, 137 mM NaCl, 0.5 mM MgCl2, 1.6 mM
KH2PO4, 2.7 mMKCl, pH 8.0) for 30 min, washed twice
in KCl/NaCl/Pi buffer and stained with 0.25% X-gal at
37°C for 1.5 h in the buffer containing 150 mM NaCl,
10 mM NaH2PO4, pH 7.5, 1 mM MgCl2, 3.1 mM
K3[FeII(CN)6], 3.1 mMK4[FeIII(CN)6]
R E S U L T S
CK2btes protein is generated inDrosophila testes
at postmitotic stages of spermatogenesis
Previously we have revealed the CK2btes transcripts in
Drosophilatestes only [18] To detect the CK2btes protein
in testes, we raised rabbit polyclonal antibodies against a
recombinant CK2btes protein purified from E coli These
antibodies recognize a protein with mobility of
approxi-mately 30 kDa in testes extract, but do not reveal any
specific signal in the corresponding region in the extracts
from males with removed testes and from ovaries
(Fig 1A) The electrophoretic mobility of the recognized
protein in testes extract is slightly different from that
expected for the protein with the calculated molecular mass
of 25 kDa The recombinant CK2btes protein purified
from E coli during electrophoresis also runs slower than
expected The retardation might be due to the peculiarities
of amino-acids content of the CK2btes protein A similar
gel retardation was seen in case of ubiquitous Drosophila
CK2b when a 24.8-kDa protein runs as a 28-kDa one [24]
The generated antibodies are expected not to cross react
noticeably with the b and b¢ subunits of CK2 because these
subunits are rather divergent from the CK2btes protein
(45% and 46% of identity, respectively [18,20]) Thus, we
conclude that CK2btes protein is expressed in Drosophila
testes and we are able to detect it using the anti-CK2btes
Ig
To study spatial expression pattern of the CK2btes
protein in the male germline, we generated transgenic flies
expressing either the b-galactosidase protein alone or the
CK2btes–b-galactosidase fusion protein, both being under
the control of the CK2btes promoter (Fig 1B)
Expres-sion of the reporter genes was monitored by histochemical
X-gal staining of whole adult testes Both constructs give
the same X-gal staining pattern at premeiotic and
postmeiotic stages of spermatogenesis (Fig 1C) No
b-galactosidase activity was revealed in the apical part
of a testis where mitotic divisions take place Other Drosophilamale tissues were not stained also (not shown) This expression pattern suggests that CK2btes protein is expressed only at postmitotic stages of male germline, and
it resembles that of other Drosophila male germline specifically expressed genes, such as b2-tubulin, dhod, Sdic and others [29–31]
Recombinant CK2btes protein stimulates catalytic activity of recombinant CK2 a subunit towards
a synthetic peptide substrate The CK2 holoenzyme is known to be a heterotetramer of
a2b2structure The b subunit is catalyticaly inactive by itself but it specifically stimulates the phosphorylation activity of CK2a 5- to 10-fold [1] To test the CK2btes protein for its ability to stimulate catalytic activity of CK2a, Drosophila CK2btes and CK2a recombinant proteins were expressed
in E coli Proteins purified under nondenaturation condi-tions were used for the CK2 activity assay In the reac-tion buffer without NaCl, the CK2btes protein 2.5-fold
Fig 1 Expression of the CK2btes protein in testes (A) Detection of CK2btes protein in Drosophila tissues Western analysis using poly-clonal anti-CK2btes Ig: R, recombinant CK2btes protein purified from E coli; T, protein extract from 7 pairs of adult testes; C, protein extract from three male carcasses with removed testes; O, protein extract from seven pairs of ovaries Molecular mass markers are shown
to the left (B) Diagram of microinjected constructs CK2btes region is black, b-galactosidase region is gray (C) X-gal staining (dark region)
of testes from a transgenic fly line carrying the CK2btes–b-galactosi-dase fusion construct under the control of the CK2btes promoter region The arrow marks the tip of the testis, no b-galactosidase staining of somatic tissues was observed (not shown).
Trang 5stimulates the CK2a activity (Fig 2) Recombinant human
b subunit at the same conditions activates Drosophila
CK2a twofold (not shown) While the CK2a activity is
practically independent of NaCl concentration in the
absence of CK2btes, it is increased 5.5 times under
physiological conditions (150 mM NaCl) in the presence
of the equimolar amount of CK2btes (Fig 2) It was
shown that the regulatory b subunit of D melanogaster
CK2 purified from baculovirus expression system
en-hanced the activity of catalytic a subunit towards synthetic
peptide fivefold [24] Therefore, CK2btes protein stimulates
CK2a activity in vitro at the optimal NaCl concentration
approximately to the same extent as the ubiquitous
b subunit
Recombinant CK2btes protein inhibits the ability
of CK2 a subunit to phosphorylate calmodulin
and this inhibition can be overcome by the polylysine
It was shown that in contrast to the stimulatory effect on
phosphorylation of majority of substrates, b subunit from
Drosophila, as well as from mammals, suppresses the
calmodulin phosphorylation by the CK2 a subunit [32,
33] Polybasic compounds such as polylysine and protamine
abolish this inhibition We asked whether the CK2btes
protein behaves similarly in respect to calmodulin
phos-phorylation by a subunit As shown in Fig 3 (lanes 3 and
5), calmodulin is phosphorylated by recombinant
Drosophi-la CK2a, whereas the addition of equimolar amount of
CK2btes results in less efficient incorporation of
radio-activity in this substrate The addition of polylysine
practically has no effect on the phosphorylation of
calmodulin by free a subunit (Fig 3, lane 4), but drastically
stimulates activity of the equimolar mixture of a with btes
(Fig 3, lane 6) Thus, CK2btes protein, such as canonical
b subunit, mediates stimulation of CK2 by polylysine
Recombinant CK2btes protein forms tetrameric complexes with CK2 a subunitin vitro
To elucidate the structure of CK2a–CK2btes complexes in vitro, recombinant CK2a and CK2btes proteins, purified under native conditions, were analyzed separately, or in the equimolar mixture, in gel-filtration experiments Proteins eluted from the column were detected by Western blot analysis using (CK2btes) Ig and polyclonal anti-(human CK2a) Ig that also recognizes the Drosophila CK2a Figure 4 shows the results of Western analysis of fractions 15–20 with a protein marker range from 158 kDa (fraction 16, IgG) to 17 kDa (fraction 19, myoglobin) It is seen that CK2btes protein is mainly eluted in the fraction 18 marked with ovalbumin possessing a molecular mass of
44 kDa The appearance of CK2btes in this fraction indicates that most of the protein molecules are associated
in the (CK2btes)2homodimers with a calculated molecular mass of 50 kDa When CK2a and CK2btes molecules were mixed together before passing through the column each type of subunits was mainly detected in the fraction 17 where protein complexes of a larger size (less than 158 kDa, but more than 44 kDa) were eluted This elution profile most likely reflects the proposed (CK2a)2(CK2btes)2 tetr-amer structure with the predicted molecular mass of
130 kDa The ability to dimerize and to form heterotetra-metic complexes with the a subunit are the canonical features of the regulatory subunit of CK2
CK2btes protein interacts with CK2 a subunit
in yeast two-hybrid system
To examine whether the CK2 a subunit is able to interact with the CK2btes protein in vivo, two-hybrid system experiements were carried out This system was designed
to test protein–protein interactions in yeast cells The PCR-amplified ORF regions of both a subunit and CK2btes cDNAs were cloned into the two-hybrid system vectors pACT2 (or pGAD424) and pAS2-1 (or pGBT9) as the
Fig 2 CK2a phosporylation activity dependence on the NaCl
concen-tration in the presence (open circles) or absence (filled circles) of
equi-molar quantity of CK2btes recombinant protein The equiequi-molar mixture
of CK2a and CK2btes recombinant proteins purified from E coli
under nondenaturing conditions or the CK2a protein alone were
as-sayed for the CK2 phosphorylation activity using a synthetic peptide
RRRDDDSDDD as a substrate The reaction was carried out in the
buffer containing different NaCl concentrations (from 0 m M to
200 m M ).
Fig 3 Effect of polylysine on the phosphorylation of calmodulin by catalytic subunit or by holoenzyme reconstituted from CK2a and CK2btes proteins Calmodulin was phosphorylated in the presence (lanes 2, 4, 6) or absence (lanes 1, 3, 5) of polylysine by either catalytic subunit alone (lanes 3, 4), or by equimolar mixture of CK2a and CK2btes proteins (lanes 5, 6) Lanes 1 and 2, no CK2a was added Samples were electrophoresed in 15% SDS/PAGE and autoradio-graphed The arrows indicate the position of calmodulin, which runs as
a doublet.
Trang 6fusions with GAL4-activator (AD), or GAL4-binding (BD)
domains Besides, the ubiquitous Drosophila CK2 b subunit
was cloned in both AD- and BD-vectors To assay
interactions, different combinations of AD- and
BD-constructs were cotransformed into SFY526 and HF7c
yeast strains carrying lacZ or HIS3 reporter genes,
respect-ively, under the control of the GAL4-binding sites When
protein interactions take place, the reporters proteins are
expressed and this expression can be monitored by X-gal
staining or the cell growth on medium without histidine
The filter and liquid b-galactosidase assays, as well as the
growth on His–selection medium were carried out in order
to detect and quantify the strength of an interaction The
results of these experiments are presented in Table 1 The
pronounced b-galactosidase activity in cells cotransformed with CK2a(BD) and CK2btes(AD) constructs, as well as the cell growth on the medium without histidine indicate that CK2btes protein does interact with the CK2 a subunit
in yeast cells Moreover, the strength of such interaction is nearly the same as in the case of a/b CK2 interaction (124
vs 156 Miller units, Table 1) We also observed the nearly equal ability of different b subunits to form dimers com-posed of two b subunits, of two btes subunits and, of the mixture of b/btes subunits These interactions are weaker than interaction of a subunit with btes or b subunit, but nevertheless, they are quite significant The two-hybrid system data give clear evidence that the Drosophila catalytic CK2 a subunit is able to interact with the CK2btes protein
in yeast cells It is also seen from the obtained results that CK2btes protein might compose homodimeric as well as heterodimeric (with ubiquitous b) structures which are well known to be the prerequisite for the CK2 holoenzyme formation
CK2btes protein is coimmunoprecipitated with CK2 a subunit inDrosophila testes extracts
To demonstrate the association of CK2btes and CK2a in vivo in Drosophila testes, coimmunoprecipitation ments were performed The main difficulty in these experi-ments was the insufficient avidity of polyclonal antibodies directed against CK2btes protein as well as those directed against the D melanogaster CK2 a subunit (the latter were kindly provided by C.V.C Glover) This problem was circumvented by the use of the transgenic flies expressing the fusion CK2btes–b-galactosidase protein in testes Anti-(CK2a) Ig were used for IP of protein complexes from testes extracts of two transgenic lines, one of which expressed the fusion CK2btes–b-galactosidase protein and the other, used
as a negative control, expressed b-galactosidase alone (the structure of transgenic constructs is depicted on Fig 1B) The IP complexes were bound to protein-A–Sepharose, washed and separated by SDS/PAGE The immunostaining
of Western blot was performed by commercially available, high affinity anti-(b-galactosidase) Ig
Anti-(b-galactosidase) Ig staining revealed single bands of different mobility in testes extracts of transgenic flies (Fig 5, lanes 1, 3), corresponding to the b-galactosidase or the CK2btes–b-galactosidase fusion protein, respectively Lanes
Fig 4 Analysis of oligomerization status of the CK2a and CK2btes
proteins by gel filtration The CK2a and CK2btes proteins alone or in
the equimolar mixture were passed through the Pharmacia SMART
system chromatographic Superose 6 column and fractions were
ana-lysed by Western blotting using anti-CK2a or anti-CK2btes Ig
Posi-tions of the corresponding protein markers run in parallel are
designated by arrows.
Table 1 CK2 subunits interactions in two-hybrid system The interactions were determined by growth on His–medium (activation of HIS reporter gene) and quantitative and qualitative assays for b-galactosidase (activation of LacZ reporter gene) BD, pGBT9; BD*, pAS2-1; AD, pGAD424; AD**, pACT2 Activity values are given as mean values ± standard deviation from two to four different experiments.
Type of interaction Filter b-galactosidase assay Growth on His–medium
b-Galactosidase activity (Miller units)
CK2a(BD): CK2btes(AD**) Blue Yes 124 ± 12
CK2a(BD): CK2a(AD) White Weak growth 0.05 ± 0.02
CK2b(BD*): CK2btes(AD**) Blue Yes 3.1 ± 0.4
CK2b(BD*): CK2b(AD**) Blue Yes 2.1 ± 0.1
CK2btes(BD*): CK2btes(AD**) Blue Yes 1.8 ± 0.3
CK2btes(BD*): (AD**) White No 0.05 ± 0.04
Trang 72 and 4 show the results of precipitation Antibodies against
CK2a precipitate the protein complex containing the
CK2btes–b-galactosidase fusion protein, but not the
b-galactosidase alone Clearly, the precipitated complex is
formed due to the association between CK2a and CK2btes
These complexes are not the result of nonspecific
aggrega-tion of over-expressed CK2btes protein with a subunit, but
rather they reflect the physiological situation, because
Northern-analysis has shown that the
CK2btes–b-galac-tosidase transgene was transcribed several times less
efficiently than the endogenous CK2btes gene (not shown)
Thus, CK2btes protein is a part of the CK2 holoenzyme in
Drosophilatestes
Drosophila b¢ subunit of CK2 is also testis-specific
Another Drosophila CK2 regulatory subunit (b¢) was
recently identified in the yeast two-hybrid screen of a
Drosophilaembryo cDNA library where CK2a was used as
a bait [20] However, its profile of expression was not
determined Using Northern analysis we have examined its
tissue-specific and developmental pattern of transcription
and, to our surprise, found the abundant transcript of
b¢ subunit only in testes (Fig 6) In our experiments no
mRNA in embryos, pupae, larvae, male carcasses and
females was detected, althougt we cannot exclude the
presence of some minor transcripts in these tissues or
stages of Drosophila development Consequently, CK2b¢ is
likely to be another testis-specific regulatory subunit in
Drosophila
D I S C U S S I O N
Our previous studies [18, 19] have shown that the SSL gene,
later renamed CK2btes, is a candidate for being a
testis-specific regulatory subunit of CK2: CK2btes transcripts,
encoding a putative protein with 45% identity to CK2
b subunit, were revealed in Drosophila testes only The
degree of sequence identity between Drosophila CK2btes
protein and b subunit was noticeably lower than among
b subunits from different organisms (chicken, mouse and
human sequences are 100% identical, Drosophila and
human sequences are 88% identical [16]), but still at the
same level as between S cerevisiae b and b¢ subunits (45%,
[34]) Therefore, it was likely but not strikingly obvious that
CK2btes protein functions as a regulatory subunit of CK2 during Drosophila spermatogenesis The data of this work provide direct evidence that the CK2btes gene encodes a male germline-specific protein possessing typical properties
of the b subunit of CK2 The CK2btes protein is able to bind the CK2 a subunit and to stimulate its phosphorylation activity towards a synthetic peptide in the in vitro experi-ments Like the canonical b subunit [32, 33], the CK2btes protein negatively regulates the CK2 catalytic activity toward calmodulin and this suppression is overcome by polylysine The CK2btes binding with a subunit occurs in yeast cells as was shown by registration of strong CK2a– CK2btes interaction in the two-hybrid system experiments The CK2btes protein forms homodimer molecules in vitro and in vivo, in yeast cells It is known [35] that the CK2b dimer serves as a precursor of the formation of the CK2 holoenzyme tetrameric structure This (CK2a)2(CK2btes)2 complex has been detected during our gel filtration experi-ments Finally, coimmunoprecipitation analysis corrobor-ates the association between CK2btes and CK2a in Drosophila testes extracts Therefore, CK2btes protein is indeed a testis-specific isoform of the CK2 regulatory subunit
The determined crystal structure of human CK2 holo-enzyme [36] allowed authors to identify amino-acid residues
in the b subunit participating in the b–b and b–a intersub-unit contacts The analysis of conservation of these residues
in the CK2btes sequence has shown that only 19 out of 39 residues contacting between two b subunits, and 12 out of
22 residues contacting between b–a are kept intact in the btes sequence This observation underlines the idea that probably not all contacting residues in the CK2 holoenzyme are important for the strong subunit interactions Detailed analysis is necessary to elucidate amino-acid residues in the regulatory subunit, which are crucial and sufficient to form CK2 holoenzyme
Mutational analysis [37] of functionally important domains in the b subunit has shown that the acidic region
Fig 5 Immunoprecipitation of the CK2btes–b-galactosidase fusion
protein from testes extract by anti-CK2a Ig Protein extracts from testes
of transgenic males expressing b-galactosidase alone (lanes 1) or
CK2btes–b-galactosidase fusion protein (lane 3) were precipitated by
anti-CK2a Ig The IP complexes were bound to protein-A–Sepharose,
washed, separated by SDS/PAGE followed by Western analysis and
immunostaining with polyclonal anti-(b-galactosidase) Ig (lanes 2 and
4, respectively) T, testes extract; IP, immunoprecipitated complexes
bound to protein-A–Sepharose after washing Positions of CK2btes–
b-galactosidase or b-galactosidase alone are indicated to the right.
Fig 6 Testis-specific transciption of CK2b¢ gene Approximately equal amounts of total RNA isolated from carcasses (male body remnants after removal of testes), testes, embryos, larvae, pupae and females were electrophoresed in 1% formaldehyde gel, blotted to Hybond-N membrane, and hybridized with either CK2b¢ probe (upper panel) or rp49 probe (lower panel) Hybridization signal with the CK2b¢ probe was detected only in the testes RNA.
Trang 8(residues 55–64), which is highly conservative among
b subunits from different organisms, is responsible for the
downregulation of catalytic activity of a subunit toward
calmodulin and for the activation by polybasic compounds
The examination of amino-acid alignment in the acidic
region of three Drosophila CK2 regulatory subunits (Fig 7)
reveals the lack of two charged residues (Glu57 and Asp60)
in the btes sequence, which might be responsible for less
pronounced CK2btes-mediated effects on the calmodulin
phosphorylation by an a subunit The b¢ subunit has more
significantly reduced negative charge density in the acidic
region than btes subunit (Fig 7), but it is still able both to
suppress calmodulin phosphorylation and mediate
activa-tion by polylysine and protamine [20] Further structural
studies are required to unravel mechanism of this
regula-tion
In the previous studies it was shown that Drosophila
possesses tandemly repeated Stellate genes encoding a
protein with striking sequence similarity to the CK2
b subunit [38] Moreover, in the in vitro assay it was
demonstrated that the Stellate protein, although used in
at least 10-fold molar excess, was able to bind to and
stimulate the phosphorylation activity of the CK2 a subunit
[39] The functional homology of the Stellate protein with
the CK2 b subunit rises the formal possibility that the
Stellate protein may take part in the CK2 regulation
Nevertheless, all experimental evidences have shown that
the Stellate protein is absent in normal males [38, 39]
Stellategenes are expressed only in testes of the X0 males, or
males lacking the cry locus on the Y-chromosome In this
case the accumulated Stellate protein forms proteinaceous
crystals in primary spermatocytes of the cry-deficient males,
thus disturbing the spermatogenesis Therefore, Stellate
protein could not be viewed as an additional testis-specific
isoform of the CK2 regulatory subunit in normal males
The CK2 regulatory subunit is ubiquitous among
eukaryotes, but an amino-acid sequence of different b
sub-units is far less conservative as compared to the CK2
a subunits This fact might be referred to the supposed
function of b subunit as the regulator of substrate specificity
and targeting of the CK2 holoenzyme in cells It is suggested
that greater variability and specificity is required for the
realization of these functions Recent discovery of two
distinct b subunit genes in S cerevisiae and three genes in
A thalianarises a possibility that different b subunits may
serve to provide different substrate specificity or targeting of
the CK2 holoenzyme in cells [16, 17] Our data extend this
suggestion showing that some b subunits are specialized for
a specific tissue It seems likely that Drosophila CK2btes and
CK2 b¢ subunit genes were evolutionary adapted for
spermatogenesis
As was shown earlier [12], quantity of the b subunit reaches its maximum in the testicles of mammals, as compared to other tissues On the other hand, in Drosophila the ubiquitous CK2 b subunit gene is poorly expressed in testes [18] Therefore, the supposed requirement of massive
b subunit production during spermatogenesis is resolved by different ways in Drosophila and in mammals: while mammals utilitize the upregulation of expression of a single
b subunit gene, the fruit fly has generated in evolution two specialized genes for this purpose
Despite the accumulation of large amount of information about it, CK2 remains an enigmatic enzyme Its un-doubtedly crucial role in signalling is based only on a variety of indirect observations, rather than on clear evidence of cause-and-effect relations Xu et al [14] have shown that the CK2 activity was essential for the spermato-genesis in mammals The gene Ôknock-outÕ of the CK2 a¢ subunit in mice resulted in male sterility without any other physiological defects In S cerevisiae, the deletions of both CK2 a and a¢ subunit genes appeared to be lethal [40], whereas, the disruption of CK2b, or CK2b¢, or both resulted in no phenotype or morphology alterations except the elevated sensitivity to salt concentration in the medium [34] Thus, the question concerning vital functions of the CK2 b subunits in higher eukaryotes is still open
We tried to address this issue on the model of spermato-genesis in Drosophila by making a Ôknock-downÕ of the CK2btes gene by means of the RNAi mechanism This approach has been applied recently in Drosophila for disruption of gene function as an alternative to the classical mutational analysis [41] To use such an approach, we generated transgenic flies transcribing in testes the ÔantisenseÕ CK2btes RNA under the control of the b2-tubulin promo-ter We hoped that this RNA would anneal in vivo to the CK2btes mRNA thus forming dsRNA, and that this would lead to the CK2btes mRNA degradation In fact, we observed a detectable decrease in the CK2btes mRNA and protein level (2–4 times lower) in testes of transgenic males when the Drosophila stock was maintained at 28°C, while
no effect on amount of RNA and protein was observed at
18°C (not shown) The example of temperature sensitivity
of the RNAi effect was already described in Drosophila [42] but the molecular mechanism underlying it is unclear Nevertheless, we were able to Ôknock-downÕ to some extent the CK2btes gene in Drosophila testes, althought it should
be mentioned that this effect was rather unreproducible These unreproducible variations in the degree of the CK2btes protein drop down did not allow us to make any conclusions concerning the influence of the CK2btes decrease on the Drosophila male fertility
Recent evidence for the existence of a ÔfreeÕ fraction of the CK2 b subunit in mouse testicles [12] implicates a new role for the b subunit in spermatogenesis, apart from the regulation of CK2 catalytic activity It is known that the CK2 b subunit might specifically, but with lower strength, interact with some partner proteins, other than CK2
a subunit These interactive partners are represented, for example, by A-raf and Mos kinases [8–10] If this is the case
in Drosophila, the achieved decrease of the CK2btes protein level in testes of transgenic males might be insufficient to affect the CK2 activity, as it could be compensated by the initial molar excess of total pool of b subunits over the CK2
a subunit Taking into account the CK2btes ability to form
Fig 7 Alignment of acidic region 55–64 in three Drosophila CK2
regulatory subunits The GenBank accession numbers for the
sequen-ces shown are the following: D melanogaster CK2b (M16535),
D melanogaster CK2b¢ (U51209), D melanogaster CK2btes
(L49382) Dashes indicate gaps introduced to improve the alignment.
Trang 9heterodimers with other b subunits shown in our yeast
two-hybrid system experiments, it is reasonable to suppose a
possibility of a replacement of one b subunit by another in
the case of a deficiency of any of the subunits, i.e a so called
ÔbypassÕ mechanism may operate in order to maintain
appropriate levels and targeting of CK2 activity in testes In
accordance with this hypothesis are our results showing that
two to fourfold downregulation of the CK2btes gene in
transgenic males does not lead to the noticeable decrease of
total CK2 activity in testes (not shown) Drosophila
CK2b-related genes expressed in testes undoubtedly require further
investigation as a system for understanding how evolution
of structural properties is responsible for subtle functional
differences between related genes
A C K N O W L E D G E M E N T S
We are grateful to Dr C.V.C Glover for providing us with the
pEV55Dmß plasmid and the anti-DmCK2a antiserum, and to Dr H.D.
Hoyle for providing the testis vector We would like to thank Prof N.B.
Gusev for providing calmodulin and for fruitful advice We thank B.
Guerra for the help with gel filtration experiments and M Silicheva for
technical assistance This work was supported by the Russian
Founda-tion for Basic Researches Grants # 00-15-97896 and # 03-04-48420, as
well as by a FEBS short-term fellowship to A I K and by an EMBO
short-term fellowship (ASTF 9160) to Y Y S.
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