Eur. J. Biochem. 241, 272-279 (1996) 0 FEBS 1996

Cloning, expression and properties of the a’ subunit of casein kinase 2 from zebrafish (Danio rerio) Marcelo ANTONELLI’, JosC L. DANIOTTI’, Daniel ROJO’, Catherine C. ALLENDE’ and Jorge E. ALLENDE’

’ Departamento de Bioquimica, Facultad de Medicina Universidad de Chile, Santiago, Chile ’ Departamento de Biologia, Facultad de Ciencias, Universidad de Chile, Santiago, Chile

(Received 25 June 1996) - EJB 96 0939/4

The protein kinase casein kinase 2 (CK2) is ubiquitous in eukaryotic cells and is apparently involved in the control of cell division. The holoenzyme is a tetramer composed of two catalytic subunits ( a and/ or a’) and regulatory subunits @). The a and a’ subunits are encoded by different genes but are very similar in amino acid sequence, except that (I’ is normally considerably shorter. There have been extensive biochemical studies with recombinant n and p subunits of many species, but only one previous description of the activity of an isolated recombinant a’ subunit from human CK2 (Bodenbach, L., Fauss, J., Robitzki, A., Krehan, A., Lorenz, P., Lozeman, F. J. & Pyerin, W. (1994) Recombinant human casein kinase IT. A study with the complete set of subunits (a , a‘, and p), site-directed autophosphorylation mutants and a bicistronically expressed holoenzyme, Eur: J. Biochem. 220, 263 -273). In the present work, the isolation and bacterial expression of a cDNA coding for the a’ subunit of zebrafish (Danio rerio) is reported. The clone covers the complete coding region that generates a protein of 348 amino acids that is 86% identical to the a‘ subunits of human and chicken, and 82% identical to the sequenced portion of the CK2a subunit of zebrafish. The recombinant a’ subunit has apparent K,,, values for ATP (6 pM), GTP (20 pM), casein (2.0 mg/ml) and the model peptide RRRDDDSEDD (0.3 mM) which are very similar to those of the recombinant a subunit of Xenopus laevis. The a’ subunit k,;,, was 7.2 min-’ which is again similar to that of Xenopus laevis a subunit (7.5 min-’). The a’ subunit also behaved similarly to CK2n with regard to optimal concentrations for Mg” or Mntz and to the inhibition by heparin and the poly(GluxOTyrZO) pep- tide. However a’ kinase activity was less sensitive to poly(U) inhibition than a, it was more heat stable than n, and a‘ was slightly more sensitive to KC1 inhibition than a. The difference in salt sensitivity, however, was enhanced by the presence of the regulatory p subunit which shifted the optimal salt concen- tration of the phosphorylating activity. The aipZ holoenzyme was inhibited by KCI concentrations above 100 mM, while the a& enzyme was stimulted by KC1 concentrations up to 150 mM and required 180 mM for inhibition.

Another important difference between a and a’ is seen in the degree of the stimulation of casein phosphorylation activity in the presence of the regulatory subunit. When assayed at 100 mM KCI stoichiometric amounts of CK2p produced maximal stimulation of both a’ (D. rerin) and n ( X . laevis), however the activity levels with a’ were stimulated 20-fold by while the addition of [I stimulated 1x ( X . luevis) only 7 -8-fold.

Keywords: casein kinase 2; protein kinase CK2; zebrafish (Danio rerio); a’ subunit of CK2; /? subunit of CK2.

Protein kinase CK2 (also known as casein kinase 2 or CKII) is a Ser/Thr protein kinase that is ubiquitous in eukaryotic cells and that is present both in nuclei and cytoplasm (for review see [l -31). CK2 is a heterotetramer with a composition of az/lz, aa’ pz, or a;pz, although higher order polymers have also been de- tected [4, 51. The a and a’ subunits contain the catalytic domain of the protein and are active by themselves, while the p subunits are regulatory, affecting positively or negatively the catalytic ac-

Correspondence to J . E. Allende, Casilla 70086, Santiago 7, Chile Fax: +56 2 731 6320. Abbreviations. CK2, protein lunase CK2 or casein kinase 2; CK2a,

the a subunit of protein kinase CK2; CK2a’, the a’ subunit of protein kinase CK2; CK2P, the /I subunit of protein kinase CK2.

Enzyme. Protein kinase CK2 (EC 2.7.1.37). Note. The novel sequence data published here have been submitted

to the EMBL sequence data bank and are available under accession number X99964.

tivity and also influencing the stability and substrate specificity of the a-type subunits.

CK2 is in the same branch of the protein kinase evolutionary tree as the cyclin-dependent kinases (cdk) and mitogen-activated protein kinases (MAPK), enzymes involved in cell cycle control 161. Consistent with this structural relatedness, there is much evidence that involves CK2 in the control of cell division. CK2 is known to phosphorylate more than 100 different proteins [3], many of which are involved in cell proliferation, such as RNA and DNA polymerases, DNA topoisomerases and ligases, tran- scription factors and oncogene products (SRF, CREB, JUN, MAX, MYC, MYB, large T of simian virus-40) and tumor-sup- presor proteins (p53 and Rb) 171. The level of the CK2 gene expression and activity has been observed to increase in trans- formed cells, tumors and embryonic cells compared with quies- cent cells and tissues [8-10]. Depletion of CK2 activity through the use of temperature-sensitive mutants 11 I], antisense oligo-

Antonelli et al. ( E m J . Biochem. 241) 273

nucleotide treatment [12], or microinjection of antibodies [I 31 causes cells to stop division in the GUS transition point.

A related finding has been the discovery that the cattle para- site Theileria parva causes an increased expression of CK2a in bovine lymphocytes and transforms these cells [14]. This finding has been followed by experiments using transgenic mice in which the CK2a transgene is governed by an immunoglobulin promoter [IS]. The observation of high incidence of lymphomas in these animals and the finding that, for this effect, the CK2a gene can complement activated MYC has established the CK2a gene as a proto-oncogene.

The cDNAs coding for the a and B subunits of CK2 of many species have been cloned and sequenced [2, 31. These sequences demonstrated that the structures of both a and p subunits have been highly conserved throughout evolution, suggesting that the enzyme plays a key role in all eukarotic cells. Fewer reports have appeared for the isolation of the cDNAs coding for the a’ subunits, and include those for yeast 1161, chicken [17] and hu- man [I&!]. The sequences derived from these studies demonstrate that a‘, although encoded by different gene, is highly analogous to the a subunit, with the most important difference being that a’ is 41 amino acids shorter at the carboxyl terminus. While many recombinant a and /3 subunits of CK2 have been expressed in bacteria and bacculovirus [2, 31, there is only one report [I91 of the successful expression of the a’ subunit. In that report, qualitative data was presented about some of the properties of human recombinant a’.

The zebrafish (Danio verio) has become an important system to carry out genetics in vertebrate systems and to study embry- onic development 1201. Previously we reported the cloning and sequencing of cDNAs coding for the a and p subunits of CK2 from zebrafish and the finding that in situ hybridization de- mostrated high expression of the mRNAs for both subunits in the developing nervous system of early embryos of this species

In this report we present the cloning, sequence and bacterial expression of a cDNA coding for the a’ subunit of the protein kinase CK2 from zebrafish. The expression of the recombinant f i subunit of the same species is also reported. Some of the quan- titative biochemical characteristics of the a’ subunit are very similar to those described for a, however, the stimulation of a‘ catalytic activity by the p subunit consistently yielded higher activity than that obtained with recombinant a from Xenopus laevis. Also higher sensitivity of a’ to salt is detected, especially when the regulatory /I’ subunit is present to form the holoen- zyme. However, a’ appears to be more stable to heat denatura- tion than a.

121 1.

MATERIALS AND METHODS

Cloning of the a’cDNA from zebrafish. The cDNA coding for the a’ subunit was isolated from a i ZAP (Stratagene) cDNA library constructed by oligo (dT) priming of total RNA obtained from zebrafish embryos of 24-28 h postfertilization (kind gift of Dr R. Riggleman). The screening was carried out with a probe corresponding to 950 nucleotides of the coding sequence of sub- unit (I of X. laevis [22], labelled with ”P by random priming. One of several clones analyzed contained a 2450-bp insert corre- sponding to the complete coding sequence of the a‘ subunit of zebrafish protein kinase CK2.

Sequencing, expression and purification of zebrafish CK2 d. The DNA sequence of the clone containing the a’ cod- ing sequence was obtained by the dideoxynucleotide chain-ter- mination method 1231 using double-stranded plasmid DNA as a template for the T,-DNA polymerase. All synthetic primers used

for sequencing were synthesized by Oligopeptido, the core facil- ity of the University of Chile.

The complete coding sequence of the a‘ subunit was ampli- fied by PCR using primers 5’ T A T A G A A T T C T T A T G C C C G G T C C G G T G A G C A G 3’, and 5’T A T A T A G A A T T C C C T G C T T T T A G C C C A T C A T G 3‘.

These primers contained EcoRl sites that were used to sub- clone the amplified fragment in that site of the pT7-7 vector in the correct reading frame. The recombinant vectors were used to transform Escherichia coli BL-21 which was induced with isopropyl-thio-/3-D-galactoside (0.5 mM). The recombinant n’ subunit was purified as described for the recombinant Xenopus CK2a [24] using DEAE-cellulose and phosphocellulose column chromotography.

Expression of the /? subunit of zebrafish CK2. The com- plete coding region for the zebrafish CK2P isolated previously [211 was amplified by PCR using primers 5’ T A T A G G A T C C A T G A G T A G C T C A G A G G A A G T G T 3’, 5‘ T A T A C T C G A G T T T A G C G G A T G G C T T T G A C G 3’ which provided BamHl and Xhnl sites at the two ends of the amplified product.

After digestion with BamHl and Xhol, the PCR product was ligated to the pGEX4T3 vector. The recombinant vector was used to transform E. coli BB4, assaying for transformation of colonies containing the vector with the correct insert. A positive colony was grown and induced by isopropyl-thio-p-D-galacto- side, and the presence of a 52-kDa fusion protein (GT-CK2B was verified by means of SDS/PAGE. The fusion protein was purified by affinity chromatography on glutathione agarose and treated with thrombin to free the p subunit as previously de- scribed. [24, 251.

The expression and purification of the Xenopus laevis a and /? subunits of CK2. The expression and purification of CK2a and /3 subunits from X. laevis were carried out as de- scribed previously [24]. The preparation of CK2a used in the present study had a specific activity of 200 nmol ”P incorpo- rated into casein. min-’ . mg-‘.

The assay for protein kinase activity of the a and a’ sub- units and of the reconstituted holoenzyme. The standard assay mix contained in a total volume of 30 p1 8 mM MgC1,; 50 niM Hepes pH 7 5 ; 0.5 mM dithiothreitol; 50 pM [y-’zP]ATP(500- 2500 cpdpmol) ; 5 g/ml dephosphorylated p-casein ; 50 mM KC1 (or as indicated) and usually 1.2 pmol a’(or a) subunit. The incubation was carried out at 30°C for 10 min. The reaction was terminated and the samples were processed by means of a filter paper technique as described previously [24]. When subunit /{ of CK2 was added to the assay mixture, the salt concentration was increased to 100 mM KCl.

Initial velocity measurements were performed in duplicate under the standard conditions except for the varied substrate concentrations which were ATP, 0.02-500 pM, GTP 4.7- 550 pM, casein 0.05-15 mg and peptide, 0.01 -1 mM.

All determinations were carried out in duplicate and the val- ues presented have been corrected for those obtained in the ab- sence of enzyme.

Materials. [y-”P] ATP (4500 Ci/mmol) was purchased from ICN [y-”P]-GTP (600 Ci/mmol) was prepared in our laboratory by the method of Walseth and Johnson [26]. Dephosphorylated p casein, (referred to as casein throughout), Calmodulin, heparin, salmon sperm DNA, polyuridylic acid, poly(GluX”Tyr2”) of average M , 46 000, and glutathione-agarose were from Sigma. Restriction enzymes were from Promega. The peptide RRRDD- DSEDD was synthesized by Oligopeptido, the core facility of the University of Chile. All other reagents were purchased from Merck, Germany.

214 Antoiielli et al. ( E m J . Binchem. 241)

A GACGAGGTGCTATCCTAGGGGTGATATCCGCCTACCTGTCAGmATCCCTCGATCACTTACT

CAATCT AATTGGTOGATCTTAGAffiAAC~ACACTCCATTTGAAGATTAATffi~TC

TAGATTCGCGTGTATffiAACTIGTGGTOGCCAATAAPACCAACCCATCGATACCGTTAGCmA

GTTAGCGGCAGTTATCCAGAGGGAACGATTTAGAAGAGAGCCCGCTGAACCGCTGTAAWACT

ATTATA~GCCAAV+GCGATGATCAW+CGC~ACTCCCAGAGACAATCGATCAACTCGA

CATTTCGGCKGCTTCACGTIGTDrrPATG?TCCGTATi"ACCGCGTAACGA~TTCAGCGAGCTI'G

ACAGGTCTG~CTCAA~CTCTCTCTTTGCATCAATGTCTATTTAAATGCAGA

A T A G G A G A A C T G C G A C A G G C A A T C G C A T A T G C A T A T A T T A C G A ~ ~ M T T G m A ~

G T C f f i A C A T A ~ A T T A ~ C ~ C f f C G A C T G A C A T T A T G ~ T C C

~ A ~ C A R T I V I R A T A C A C T G G C G O T C C A ~ G A A P A T A A G C T G ~ C T G A A C C G ~ A

CGCCCTTTGCITI?TGCTTXGCTITTAGTOGCGCGACGATAAGCGCGAGGTCATGCGCGCGC

TGGCEAGCAGCTGTCAATCACAATIGTTCGCGAGAGATCGCACACTGACGACGGCTIGCAAG

CGCGTIGTTlTCCGGTACCACAGTGCnTCTGTAGTGlTTCACAAV+GAGG~AACTI'GTCGC

TGTffiCTTGGAORATCTffiTAGTAGTAGGTOGAGAGAAGGCGGATCAACTGGWCCCGCC

A T G C C C G G T C C G G T G A G C A G C A A A G C A C G G G T G T A T G C A

M P G P V S S K A R V Y A D A N T V K S K

GAATACTGGGATTATGAAGCACATGTGCCTAG~AGCAACCA~~~A'EACTACCAGC~TA

E Y W D Y E A H V P S W S N Q D D Y Q L V

CGGAAGCTCGGAAGAGGCAAATACAGTGAAGTITITGAGGCCATCAAC4TCAACAGCAAffiAC

R K L G R G K Y S E V F E A I N I N S N D

AGAGTAGTI'GTWTACTAAAGCCTGTTAAGAAGAAGAAGATTAAACGTGAAATWTT

R V V V K I L K P V K K K K I K R E I K I

TTGGAGAACTIGAGAGGAGGAACCAACATCATTffiTCPTGTAGACACAGTGAAAGACC~lV

L E N L R G G T N I I R L V D T V K D P V

TCTAGAACGCCAGCCCTfDTGTTTGAGTACATAAATAACACAGATTTCAAffiATCTCTATCAA

S R T P A L V F E Y I N N T D F K D L Y Q

AGACTAACAGACmGPICATCffiATRTACTTGTATGAACTTCTGAAGGCGCTGGATTAffCT

R L T D F D I R F Y L Y E L L K A L D Y S

CATAGCA%GGCATCATGCATCGCGACGTCAAGCWCATAATGTCATGATAGACCACCAAATG

H S M G I M H R D V K P H N V M I D H Q M

A G A A A G T T G C G O C C T T G C T G A G ' I T C T A T C A C C C T G C A C A G G A G T A C A A T

R K L R L I D W G L A E F Y H P A Q E Y N

GTAAGGGTI'GCTTCAAGATCATATAAGGGACCAGAACIGCTAGTAGACTATCAGATGTATGAC

V R V A S R S Y K G P E L L V D Y Q M Y D

TACAGTCTCXACATGTGGAGCTTAGGCTGTATGTTAGCCAGTATGAT~CAGAAAGAGCCA

Y S L D M W S L G C M L A S M I F Q K E P

TTTTTCCA~C~GACRTGACCAGCTOGTCAGAATCGC~GGTTCTTOGAACCGAA

F F H G Q D N Y D Q L V R I A K V L G T E

GAGCTGTTTAGCTACCTGAACAAGTATCACATTGAGCTGGACACAffiCTTCAAAGATCTTCTC

63

126

189

252

315

378

441

504

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630

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E L F S Y L N K Y H I E L D T R F K D L L

f f i C C A G C A G A C A C G C ? + A A C G C % G G A G b A C T T I ' G T T C T T

G Q Q T R K R W E N F V L P E N Q H L V S

CCAGAAGCTCTGGATCCTGGATAAACTGCTGCGTTACGATCATCAGCAGAGACTGACTGCT

P E A L D L L D K L L R Y D K Q Q R L T A

CAAGAGGCCATOGAACACCClTACTTCTATCCTGTGTTGAAGGGACAGCCTCTGTCAAACTCA

Q E A M E H P Y F Y P V L K G Q P L S N S

GAAGGCGCACTGGCAATAAGCAGffCAACCACAACATGA%GGCTAAPAGCAffi~C~

E G A L A I S S S T T T

TTACCTCAATGTGTCGTGCAGAGACATGCTGCCTTTCAGGTGTGAAGCTTCAGTTTA~AG

AAAGAAGTTATTAAGCAACACAAGACTETACAGAATGAACACAAWCTGTAGTCCCTCTC

CTACATTCACTTIGTATTAGTGCTGCTTGCCATTGTTATCTAGGTACCAAACTOGTGTI?ITCT

CCTCCCTCCACCCCATCTCTCTCTCTCTCmCTCTCTCCCTTI'GAGTTGGTCATCTCCATTKG

~ A A C R C T A ~ T A T T A A ~ C ~ A A A G A A T G C C T G C T C ~ C

CTCAATGATTCTCAGGCTGTCEACGGSGCTCCTCATCCTOGTIGCATTATGTACTG

GAAGATI'GTGTI'GTTCAATGACTlGCCCTGCTGATATGAGCTCAGATTATAAACTATAGTATT

GTCAGGTATGGAGGCTTTACTGCAATTTATTGAAPATAAGATATATTAAATCAAACCGTTFIT

P

882

945

1008

1071

a' D.rer. MPGPVS -SKARWAD~KSKEYYEAHVPSWSNQDDYQLVRKLGRGK 49

a'H.sap. . . . . AAG.R ..... EV.SLR.R.... ........ G. . . . . . . . . . . . . . . 50

a'G.ga1. .... AAG.R . . . . . EV.SLR.R . . . . . . . . . . . . G............... 50

a' D.rer. YSEVFEAININSNDRVVVKILKPVKKKKKIKREIKILENLRGGTNIIRLVE 99

a'H.sap. . . . . . . . . . . T..E . . . . . . . . . . . . . . . . . . V.............K.I. 100

a'G.gal. . . . . . . . . . . T..E..................V.............N.I. 100

a' D.rcr. TVKDPVSRTPALVFEYINFKDLYQRLTDFDIRFYLYELLKALDYSHS 149

a'H.sap. . . . . . . . K...............Q...I.........M.........C.. 150

e'G.ga1. . . . . . . . K...............Q...I.........M.........C.. 150

a' D.rer. M G I M H R D V K P H N V M I D H Q M R K L R L I D W G L A E F Y H P A Q E Y 199

a'H.sap. .................................................. 200

u'G.ga1. . . . . . . . . . . . . . . . . . . QK . . . . . . . . . . . . . . . . . . . . . . . . . . YF.. 200

a'D.rcr. PELLVDYQKYDYSWMWSLGCMLASMIFQKEPFFHGQDNYDQLVRIA~ 249

a'H.sap. . . . . . . . . . . . . . . . . . . . . . . . . . . . . RR.. _ . . . . . . . . . . . . . . . . 250

ol'G.gal. ............................ R.. . . . . . . . . . . . . . . . . . . . 250

Fig. 1. Sequence of cDNA clone coding for the c1' subunit of zebrafish CK2. (A) Deoxynucleotide and derived amino acid sequence of the cDNA clone coding for a' subunit of protein lunase CK2 of zebrafish (D. rerio). (B) Comparison of the amino acid sequence of zebrafish (D. rcrio) with the known sequence of the a' subunits of human ( H o m ~ supien.r) and chicken (Ca1Iu.r gallus).

Antonelli et al. ( E m J. Binchern. 241) 275

a’ D.rer. G T E E L P S Y L N K Y H I E L D T R F K D L L G Q Q T R K R W B N F Y L L D 2 99

a’H.sap. . . . . . YG . . K . . . . D..PH.N.I..HS........IHS..R......... 300

o’G.gd. . . D . . YG . . K.......PH.N.I...HS.......IHS..R......V.. 300

ff ’ D.rer. L L D K L L R Y D H Q Q R L T A Q ~ H P Y F Y P Y L K G Q P L S N S E G A 348

o’H.sap. . . . . . . . . . . . . . . . . K . . . . . . . . . . . V.E.SQPCADN.V.S.G L.AAR 350

a’G.gal. . . . . . . . . . . . . . . . . K . . . . . . . . . . . V . E . S Q P S . . N . V . S . G L . . . 350

Fig. 1. (Continued). Fig. 3. Autophosphorylation of CK2 with a or a’ catalytic subunit. 3 pmol recombinant CK2a’ (lanes 1-6) or CK2u (lanes 7- 12) was incu- bated with 50 pM [y-’2P]ATP (specific activity 4000 cpdpmol) for 1 h at 30°C in the presence (+) or absence (-) of 12.5 pM polylysine. Additional conditions for autophosphorylation were as follows: 50 mM Hepes, pH 7.5, 8 mM MgCl,, 0.5 mM dithiothreitol and 50 mM KCI (lanes 1-3 and 7-9), or as above but with 100 mM KCI and 3 pmol CK2P (lanes 4-6 and 10-12). Products were analyzed by 13% SDS/ PAGE and autoradiography. Arrows indicate the migration of the CK2 subunits. Numbers indicate the positions of molecular mass markers.

Fig. 2. Expression and purification of the recombinant CK2a’ sub- unit of zebrafish. The cDNA coding for a’ was subcloned in the expres- sion vector pT7-7 and expressed in E. coli as detailed in Materials and Methods. E. coli extracts and purified fractions were analyzed on 10% SDS/PAGE, and stained with Coomassie Blue. Lanes: 1, whole extract of E. coli BL21 (D53) transformed with a’ recombinant vector pT7-7 and induced with isopropyl-thio-P-D-galactoside; 2, supernatant fraction of the E. coli extract after centrifugation at 20000 g for 30 min; 3, active kinase fractions pooled after DEAE-cellulose chromatography; 4, pooled active fractions after phosphocellulose chromatography. Numbers corre- spond to the positions of standard molecular-mass markers. CK2a’ is approximately 40 kDa.

RESULTS

Screening of a cDNA library of zebrafish with a PCR-ampli- fied fragment corresponding to 950 nucleotides of the coding region of CK2a of X . laevis [22] yielded several clones, one of which was found to contain the complete coding sequence corresponding to the a’ subunit of zebrafish CK2. The nucleotide sequence and the derived amino acid sequence of the coded pro- tein are presented in Fig. 1A. The protein encoded corresponds to a peptide of 348 amino acids that has all the conserved se- quence features of a typical protein kinase [6]. The calculated isoelectric point of this protein is 8.33. However, the recombi- nant protein which contains four extra amino acids originating from the vector has an isoelectric point of 8,7. The isoelectric points of human (I and a’ subunits are 7.8 and 9.0, respectively [19]. Fig. 1 B compares the derived amino acid sequence of the (I’ subunit of zebrafish with the sequences of the human and chicken a‘ subunits. The zebrafish protein is two amino acids shorter than the other animal a’ subunits and is 86% identical to these subunits along the first 331 amino acids, the last few amino acid5 containing proportionately more differences. Over the same stretch of amino acids, the a’ is 83% identical to the a subunit of human CK2, which is 43 amino acids longer. This same conserved region is 82% identical to the X . laevis a sub- unit. Comparison of the amino acid sequence of the a’ zebrafish

subunit with the 298 amino acids available for the (x subunit of the same species yields 82% identity, with the a subunit contain- ing 10 additional amino acids at its carboxyl terminus.

The cDNA coding for the a‘ subunit of zebrafish was intro- duced into the pT7.7 expression vector and the recombinant pro- tein was expressed in E. coli. After purification on DEAE-cellu- lose and phosphocellulose as described [21], the specific activity of the CK2a’ in different preparations was 180-200 nmol ”P incorporated into casein . min-’ . mg-’, a range similar to that reported for CK2a. Fig. 2 shows the protein patterns for different stages of purification.

The cDNA coding for the zebrafish CK2P previously re- ported [21] was also expressed in E. coli using the pGEX-2T vector and the recombinant protein was purified exactly as de- scribed previously with the subunit of X . laevis [24] (data not shown).

The autophosphorylation of a (D. uerio) and a‘ ( X . luevis) was assayed in the presence and absence of the P subunit (X . luevis) and polylysine (Fig. 3).

The autoradiography shows that a’ either alone, or as a part of the holoenzyme, is appreciably less phosphorylated than a. Both subunits cause a very marked phosphorylation of P. In this experiment it is seen that as observed previously [27], polylysine causes an inhibition of phosphorylation.

The biochemical properties of the zebrafish CK2a’ were characterized. Table 1 presents the kinetic constants of CK2a’ obtained with different substrates. When the values obtained for the a’ subunit of zebrafish are compared with the values deter- mined for the recombinant a subunit of X . laevis very small differences are observed. The K,,, of a’ is approximately 7.2 min-I, which is very similar to the value observed for the recombinant X . laevis (x.

The inhibition of CK2d by different concentrations of hepa- rin, DNA and poly-(Glu””Tyr20) was also very similar to that observed with recombinant a of X . laevis (data not shown). However, the inhibition observed with polyuridylic acid was sig- nificantly different (Fig. 4). This polynucleotide has been pre- viously shown to be a strong inhibitor of purified CK2 holoen- zyme [28]. The a‘ subunit of zebrafish is less sensitive to the effect of this polynucleotide, the Zso values for a’ being 2.6-fold higher than for a.

The heat stability of a has been shown to be greatly increased by the presence of p (21. When a comparison between the kinetics of inactivation of a and a’ at 45 “C is made, a signifi-

276 Antonelli et al. (EUK J. Biochern. 241)

Table 1. Comparison of kinetic parameters of the recombinant cata- lytic subunits, a from ( X . Zaevis) and a‘ from D. rerio. Kinetic con- stants were determined by regression analysis of double-reciprocal plots obtained from initial velocity data using a broad range of substrate con- centrations, using the program MICROCAL. The catalytic constants k,,,,, values are averages of three determinations performed under the standard conditions described in material and methods with casein and ATP as substrates. These k,,l, values were 7.2 min-’ and 7.5 min-’ for D. rerio CK2a and X . laevis CK2u, respectively.

~~

Substrate _______

Apparent K, , , of

CK2a’ (D. rerio) -

CK2a (X . laevis) _______

.._ - PM

~

ATP 6 7 GTP 20 19 Casein 2.0 mglml 2 5 mglml RRRDDDSEDD 310 230

1

i 0 20 40 60 80 100

Poly U (pg/ml) Fig. 4. Inhibition of CK2a and CK2a’, by polyuridylic acid. Zebrafish recombinant CK2a’ (A) or CK2a (0) or from X . laevis was incubated under standard conditions as given in Materials and Methods in the pres- ence of the indicated concentrations of polyuridylic acid. 100% activity in the absence of poly(U) for CK2u’ was 6 pmol ’*P. min ’ incorporated and for CK2a was 5.0 pmol . min-I.

cant difference is observed (Fig. 5). The X. laevis a subunit is considerably more labile than the D. rerio a’ subunit. The time required for 50% inactivation of a is approximately 3 min, while the equivalent time for u’ is 7.5 min. In both cases, the presence of the p subunit has a dramatic stabilizing effect.

One of the important characteristics of the activity of the a subunit of CK2 is that it is inhibited by physiological concentra- tions of salt (NaCI or KCI). This inhibition is altered by the presence of the p subunit, which shifts the optimal concentration of salt to 100 mM or over [29]. Comparison of the effect of different concentrations of KCI on the activity of u and u’ in the presence or absence of the p subunit yields some interesting differences (Fig. 6). In the absence of p, the a’ subunit appears to be slightly more sensitive to KCI concentration. Especially significant are the differences of inhibition observed between

1

time (min) Fig. 5. Comparative heat stability of CK2a (X. Zaevis) and CK2a’ (0. rerio). The enzyme subunits (8 Fg/ml) were incubated at 45°C for the times indicated then immediately assayed for 10 min at 30°C for CK2 kinase activity as given in Materials and Methods. CK2/j subunit when present at 45°C was 7 pglml. The incubations at 45°C contained a’ (A), a (0). u’ and /? (A) or (x and ,8 (e). The 100% activity at zero time, in pmol ’2P incorporationlmin, were 3.2 and 4.3 for (L’ and (x and 64 anti 38 for a’/l and ab, respectively.

70 mM and 120 mM KCl. These differences are considerably enhanced when the assay is carried out in the presence of the / j subunit. With recombinant a of Xenopus, p causes a shift in the optimal salt concentration from about 25 mM to over 100 mM KCI, reaching values 40% higher than those seen with the low- est salt concentration. Inhibition by salt is only observed over 180 mM KCI. This behavior is typical of other (I subunits studied. In the case of the recombinant (I‘ however, p has a dif- ferent effect. Very little stimulation is observed by increasing the concentration from 50 mM to 200 mM KCI and clear inhibi- tion is observed at 135 mM KCI. The differences in the effect of salt make it difficult to compare the stimulation of p on the activity of the two types of catalytic subunits. These experi- ments, however, were carried out at 100 mM KCI because this concentration is close to optimal for the activity of the holoen.- zyme with both types of subunits.

Fig. 7 shows the effect of addition of increasing amounts of the p subunit to the recombinant a’ of zebrafish and of (1 of X . laevis at 100 mM KCI. Although the original specific activities of the isolated a and a’ subunits at 40 mM KCI were similar, the activity in the absence of /l at 100 mM was lower for a’ due to its higher sensitivity to salt. Increasing the amounts of recombi- nant /i caused a gradual stimulation of the activity of both cata- lytic subunits. Maximum stimulation is attained at a stoichiomet- ric ratio of 1 /I subunithubunit a or a’. A significantly higher activity ( ~ 3 0 % higher) is reached with the a‘ subunit of zebra- fish. The stimulation by p of u from X. laevis is of the order of sevenfold, while the stimulation of the u‘ subunit of zebrafish i:i about 20-fold. This result is representative of those obtained in four experiments performed which gave very similar stirnulation values; the addition of p to a’ always resulted in significantly higher activity than that obtained with a and the stimulation of u‘ by /? of X. laevis was always 15-fold or higher.

Antonelli et al. (Eur: J. Aioclzeni. 241) 277

100

80

60

40 CI

o\" 20 Y

)r

>

0 14C

cu 12c Y

t .- .- t o a

0 1oc

8C

6C

4c

2c

( 50 100 I50 200 250 300

KCI (mM)

Fig. 6. The effect of KCI concentration on the activity of CK2u and CK2a'. Assays were performed under standard conditions except for the use of different KCl concentration. (A) Assays of catalytic subunit alone where 100% activity was set as that obtained at the lowest salt concen- tration used (18 mM) and corresponded for a to 6.5 pmollmin-' X. luevis CK2n (0) and 7.5 pmollmin-' for D. rerio CK2a' (A) of "P incorpo- rated into casein substrate. (B) Assay of the same catalytic subunits with 5 pmol CK2B ( X . laevis), where the lowest salt concentration used was 30 mM and 100% activity for CK2a2/3, (0) was 26 pmol/min and for CK2a',J2 (A) was 88 pmollmin-' of "P incorporated into casein.

Recombinant /? subunit of zebrafish, which differs from the X. laevis CK2b in only three amino acids at its carboxy end [21], is also able to stimulate the homologous a' subunit, stimu- lation of 13-fold and 20-fold being observed with different prep- arations (data not shown).

With some substrates, the presence of the /? subunit has an inhibitory effect on the CK2a catalytic subunit activity in con- trast to the stimulatory effect which is seen when casein is used as substrate. One such substrate is calmodulin [3]. In Fig. 8 it is shown that both n and a' subunits phosphorylate calmodulin. Some trace impurities in the calmodulin preparation are also phosphorylated. The phosphorylation of calmodulin is almost completely eliminated by the presence of the CK2/j subunit, with both types of CK2a subunits.

70

60

50

40

30

20

10

0

A A

0 1 2 3 4

Beta subunit (pmol)

I ' I ' I ' I ' I I

0 1 2 3 4

Be ta subunit ( p m o l ) Fig. 7. Activation of CK2u and CK2a' by different concentrations of recombinant CK2P subunit of X . laevis. The phosphorylation of casein by 1.2 pmol of X . luevis CK2a (0) and 1.2 pmol of D. rerio CK2a' (A) was assayed as described in Materials and Methods at a 100 mM KC1 concentration in the presence of different amounts of recombinant X . laevis CK2P subunit. The insert shows stimulation (-fold) for the same experiment.

Fig. 8. Phosphorylation of calmodulin by CK2d (0. rerio) and CK2a (X. luevis) subunits. The standard kinase assay as described in Materials and Methods was carried out using 1.2 pmol of catalytic subunit, either CK2a' (D. rerio) or CK2a (X . laevis) as indicated and 100 pmol bovine brain calmodulin (lanes 1-4) or without exogenous substrate (lanes 5 - 8). The CK2P subunit (1.6 pmol) was added as indicated. Phosphopro- teins were resolved on 13% SDS/polyacrylamide gels and visualized by autoradiography. The migration of calmodulin (17 kDa) is indicated by an arrow. Molecular-mass markers migrated as indicated on the right.

fish [21], identify this cDNA as coding for an a' subunit. Despite the similarity of the a' sequence to that of the a subunits, some structural differences may be significant. Its theoretical isoelec- tric point is of 8.33 while the values for the recombinant human and X . luevis a subunits are 7.8 and 7.6, respectively. The human a' subunit was also found to have an alkaline isoelectric point [19]. Another difference that may be relevant is that a region of a that contains threonines that are in context for CK2 phosphory-

DISCUSSION The a' protein encoded by the isolated cDNA clone from

zebrafish is 86% identical to the a' subunit of human and chicken and two amino acids shorter than these polypeptides. The length of the coding region (348 amino acids) and compari- son of the deduced sequence with the a and a' subunits of other species [2] and with the part known for the a subunit of zebra-

278 Antonelli et al. (Eur: J. Biochem. 241)

lation and which therefore could be sites for autophosphor- ylation (T127LTDYD132), is changed in the zebrafish a’ to RLTDFD. The change of TI27 to R not only eliminates this residue as a potential site of phosphorylation but also may nega- tively affect the phosphorylation of the T129 [30]. In agreement with this hypothesis, in the experiments in which we compared the autophosphorylation of a ( X . laevis) with that of a’ (D. re- rio), the intensity of the labellings of a was consistently stronger than for n’. The actual sites of phosphorylation of the a and the a’ subunits are presently unknown.

The bacterial expression of the recombinant zebrafish a’ and subunits have been achieved using methods vcry similar to

those used with the Xrnopus subunits 1241. The availability of these purified subunits has provided the opportunity to assay their activity and to study some of their characteristics. This is especially important for the a’ subunit since there is very little quantitative information about its enzymatic properties.

The results presented above confirm and significantly extend those presented previously with the human a‘ subunit [19]. The kinetic parameters of zebrafish a’ are very similar to those of CK2u subunits that have been characterized previously. The ap- parent K,,, values for the four substrates tested are not signifi- cantly different from those of the recombinant Xetzopus a sub- unit or the a subunits of other species. The k, , , for the recombi- nant zebrafish a’ and Xerzopus luevis a are also very similar to each other and are i n the same range as those reported for CK2a by other researchers [31]. The optimal concentrations of Mg” (8 mM) or Mn” ( 5 mM) required for (K or a‘ activity are also similar. Both catalytic subunits also behaved in a similar fashion with respect to the effect of the p subunit on the phosphorylation of calmodulin, since the presence of the regulatory subunit elim- inates calmodulin phosphorylation by both CK2a and a‘.

Again, no significant differences are found in the inhibition of a’ and a by heparin, unfractionated DNA or poly(Clu8”Tyr”’), yet another polyanionic inhibitor, poly(U), is significantly less efficient with a‘ compared with n. Poly(U) is a much stronger inhibitor of the holoenzyme CK2 than poly(C) or poly(A) and, therefore, its inhibitory effect is not only due to its polyanionic character but must also involve some specificity in its interaction with the catalytic subunit 1281.

The heat stability of a‘ appears to be appreciably greater than that of a. For both catalytic subunits, however, the addition of p causes a very large increase in their stability.

The most interesting differences noted between a and a’ are related to the effect of salt and to the activation by subunit p.

The catalytic subunit is inhibited by salt concentrations in the physiological range, the n’ subunit being somewhat more sensitive to salt concentration than a. The difference in salt sen- sitivity is considerably enhanced by the presence of the p sub- unit. The CK2 uzp2 holoenzyme has a clear optimal salt concen- tration around 100 mM and requires concentrations of 180 mM to be inhibited. The cx:/J’. holoenzyme, however, is not signifi- cantly stimulated by increasing the KC1 concentration from 40 mM to 100 mM and is inhibited by higher concentrations.

subunit in altering the sensitivity of CK2a to physiological salt concentrations has been analyzed by another laboratory [32], however, the recent studies from Glov- er’s laboratory 1331 may be particularly relevant in that strains of Saccharomyces cerevisiae which have deletions of the two genes coding for CK2p subunit are specifically sensitive to high salt concentrations.

The differences in the sensitivity to salt between holoen- zymes containing either n or a’ may be due to the formation of higher-order aggregates which have been shown to occur in vitro depending on the salt concentration and which apparently influ- ence the activity of the enzyme. [51.

The importance of the

Another important difference between the two subunits is the effect of the addition of on the activity of the two different catalytic subunits. Consistently higher levels of activity were ob- served when [J’ was added to the a‘ subunit compared with a. The differences ranged from 20-50%. The stoichiometry of /3 to a or a’ was very similar, attaining maximum activation at ratios to a (or a‘)//l of 1. Since these holoenzyme reconstitution experiments were performed at 100 mM KCl (a salt concentra- tion that significantly inhibited a’ more than a), the higher stimu- lation of a’ by p was compounded with the reversal of the salt inhibition caused by the regulatory subunit. This combination of effects resulted i n p-mediated stimulations of (K’ activity of over 20-fold, more than twice the effect obtained with the recombi- nant a subunit of Xenopus and other species [2, 31.

In the work of Bodenbach et al. 1191 it was also found that equivalent amounts of a’ combined with f i yielded 65% more enzymatic activity than a similar combination with a. These au- thors, however, explained this observation by a higher specific activity of the a’ subunit.

The existence of isoforms of proteins with very similar prop- erties always raises the teleological question of why this multi- plicity is necessary at all. Although this question cannot be eas- ily answered, it is pertinent to indicate that the a or (K’ genes seem to be differently expressed in various tissues. The a’ ex- pression is greatly enhanced in the nervous tissue during the period of synaptogenesis [34]. Also, large diffcrcnces in the levels of the transcripts coding for the (K or (1‘ of CK2 have been found in different tissues of the chick embryo [17]. It seems, therefore, that the factors that regulate the expresion of a and i f may be different and may be tissue specific and the existence of two genes may facilitate the task of adapting the levels of the enzyme to the specific requirements of each cell type. Presently, there are no indications as to the nature of the promoters of these genes or to the factors that may interact with these promoter elements.

This work was supportcd by grants from (he Council for Tobacco Research, the International Centre for Genetic Engineering and Biotech- nology (ICGEB) and Fondo Nacional de Ciencia y Tecnologia-Chile. J. L. D. was a postdoctoral fellow of the ICGEB.

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