Chinese Journal of Biochemistry and Molecular Biology CK2 252

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1 ISSN CN ΠQ Chinese Journal of Biochemistry and Molecular Biology (9) : CK ) 3 3, 1),2) 3 3, 1),2) 3, 1) ( 1), ; 2), ) CK2 Π. CK2, pact2 cdna CK2. CK2, 8, (UBA52) cdna (100 %). GST pull2down CK2 UBA52., 252 (UBA52) CK2,,. CK2 ; ; ; 252 (UBA52) ; GST pull2down Q ; R ; Q94611 Identification of Ubiquitin252 Amino Acid Fusion Protein as Interacting Partner of Protein Kinase CK2 Subunit by Two2hybrid Screening CHEN Bi 1) 3 3, LUO Yan2Ting 1),2) 3 3, LIU Xin2Guang 1),2) 3, LIANG Nian2Ci 1) ( 1) Institute of Biochemistry and Molecular Biology, Guangdong Medical College, Zhanjiang , China ; 2) Aging Institute of Guangdong Medical College, Dongguan , Guangdong, China) Abstract Protein kinase CK2 is a ubiquitous eukaryotic messenger2independent protein serineπthreonine kinase. To investigate the role of CK2 in spermatogenesis, a bait plasmid has been constructed by subcloning the human CK2 cdna fragment into the yeast two2hybrid vector p GBKT7. Both of the bait p GBKT72HCK and a human testis cdna library vectors were then cotransformed into AH109 competent cells to screen the interacting protein of human CK2 subunit by yeast two2hybrid system. The result showed that eight positive clones were selected for interaction with human CK2 subunit. One of the potential binding partners showed high homology to human ubiquitin252 amino acid fusion protein gene (100 %). GST pull2down assay confirmed that CK2 interacted with human ubiquitin252 amino acid fusion protein in vitro. The interaction between CK2 and UBA52 was demonstrated both in a yeast two2hybrid system and in an in vitro assay. The importance of such interaction in spermatogenesis is unclear. Further investigation is required to address this : ; : (973 ) (No. 2007CB507403) ; (No ) ; (No ), ; (No. 06Z015) Tel : ,E2mail : com 3 3 Received :January 2,2008 ; Accepted :April 29,2008 Supported by Major State Basic Research Development Program of China (973 Program,No. 2007CB507403) ; National Natural Science Foundation of China (No ) ; Guangdong Natural Science Foundation(No ) ; Key Foundation of Natural Science Research for Guangdong Universities (No. 06Z015) and Science Technology Program of Bid Invitation of Zhanjiang (No. ZZ0605) 3 Corresponding author Tel : , E2mail : com 3 3 The two authors contributed equally to this work

2 9 : CK issue. Key words human protein kinase CK2 ; yeast two2hybrid system ; testis ; human ubiquitin252 amino acid fusion protein (UBA52) ; GST pull2down CK2, 2 (casein kinase 2 or ) Π, 2 ( ) 2 ( ) ( ). CK2, [1 3 ]. CK2 [4 ]. Xu [5 ] Csnk2a2, CK2. :,. CK2,., Csnk2a2 CK2, CK2. : CK2,. : Csnk2a2 - Π-,, 30 %,. Litchfield [3,6 ],CK2 (caspase) Bax P53, CK2.,CK2, CK2.. CK2, cdna CK2, GST pull2 down p GEX24T21, CK2 cdna p GBKT72HCK ( Kan r Trp) [8,9 ], E. BL21 (DE3). coil DH5 JM109, pact2 ( Amp r Leu) AH109 GAL4 DNA2BD cdna Clontech. Taq DNA, Xho, BamH,TA pmd182t ( ), Alu Promega, Hae. TRIzol TM,RT Invitrogen. X2 2gal Sigma. ProFound Pull2Down GST Protein :Protein Interaction Kit Pierce. CK2 [10 ]. CK2. ECL. NC, Bio Basic p GBKT72HCK (BD) p GBKT72HCK [8,9 ], AH109,. Fas2FasL,. Sertoli Fas2FasL,caspase28., MATCHMAKER cdna (AD) CK2 Bid (Bcl22 ),Bid Clontech AD. caspase, [3,5,7 ]. Bcl2 PEG dsdna p GBKT72HCK2, ds cdna pact2 DNA LiAc AH Ade - ΠHis - ΠLeu - ΠTrp - SD mm, 2. Clontech. Clontech., DNA ( AD BD ), JM109. Amp LB AD BD (AD

3 Amp r,bd Kan r ). Amp LB, DNA, PEG( ). AD AH g AD BD, 011 mg DNA LiAc ( ) AH109, DNA, DNA, pact2. NCBI BLAST, BLASTN, cdna, RNA 252 cdna Trizol A549 RNA, Olig dt, SuperScript First2Strand System for RT2PCR, 252 cdna. PCR ( P1) : 5 2AGG GAT CCA TGC ATC TTT GTGAAG ACCC2 3, 5 BamH ; (P2) : 5 2CCGCTCGAGTTATTTGACC TTC TTC TTGGG2 3, 5 Xho, 387 bp. PCR : 95 3 min, s, s,72 1 min,32,72 10 min BamH ΠXho PCR, pmd182t TA. JM109. DNA, BamH ΠXho DNA. 252 DNA p GEX24T21, BamH ΠXho, BL21 (DE3)., p GEX24T212UBA p GEX2 4T212UBA52 BL21 (DE3), Amp LB 37., 100 l 5 ml Amp LB, 37, A , IPTG( 018 mmolπl),37 4 h min mm 12 %, 200 V,40 min,sds2page, R2250, GST pull2down : SDS2PAGE, (NC), NC BSA, CK2 ( ) ( ),ECL,, cfuπ g, Clontech 10 8 cfuπ g, 1166 gπ l, A 260 Π A cfuπ g, , 396, SDΠAde - ΠHis - ΠLeu - ΠTrp Ade +,His +,Leu +,Trp , 138, PCR SDΠAde - ΠHis - ΠLeu - ΠTrp -, 2, , 1 %, 1 DNA ,12 15,10 14,17 18, PCR, PCR Alu Hae, 1 %, Fig. 1 (A, B)., , 12 15, 10 14, 17 18, PCR 220 bp, PCR 300 bp, PCR 500 bp, PCR 750 bp.

4 9 : CK Fig. 1 The electrophoresis analysis of the positive clones PCR products A bait plasmid has been constructed by subcloning the human CK2 cdna fragment into the yeast two2hybrid vector pgbkt7. The bait pgbkt72hck and a human testis cdna library vectors were then cotransformed into AH109 competent cells to screen the interacting protein, which can bind to protein of human CK2 subunit, by yeast two2hybrid system. Some of screening clones were analyzed by restriction enzymes. (A) Five microlitres of clones PCR products were digested with Alu. Lanes 1 14 showed the different clones (clone code : 4,6,3,11,22,12,15,10,14,16,17,18,21,23). The clones were visualized by electrophoresis using 1 % agarose gels staining with SYBR, respectively. The fragment of cutting clone 4,6,10, 14,16 was about 220 bp. The fragment of clone 21,23 was about 300 bp. The fragment of clone 3,11,22,12,15 was about 500 bp. The fragment of clone 17,18 was about 750 bp. M was 250 bp DNA ladder marker ; (B) Five microlitres of clone PCR products were digested with Hae. Lanes 1 14 showed the different clones(same as A). These clones were visualized by electrophoresis using 1 % agarose gels staining with SYBR, respectively. The fragment of digested clone 4,6,10,14,16 was about 220 bp. The fragment of clone 21,23 was about 300 bp. The fragment of clone 3,11,22,12,15 was about 500 bp. The fragment of clone 17,18 was about 750 bp. M was 250 bp DNA ladder marker , AH109, SDΠLeu -, 2, Table 1,, AD (pact22 library cdna) BD Ade - ΠHis - ΠLeu - ΠTrp -, Lac Z +, p GBKT72HCK2 pact22library cdna. Table 1 Result of retestion by cotransformation Group Plasmid 1 Plasmid 2 (DNA2BD) (AD) SD selection medium Lac phenotype Positive control pcl1 2AdeΠ2HisΠ2LeuΠ2Trp Blue (positive) Negative control pgbkt72hck 2AdeΠ2HisΠ2LeuΠ2Trp White (negative) Experiment pgbkt72hck pact22library cdna 2AdeΠ2HisΠ2LeuΠ2Trp Blue (positive) 2. 5 DNA, CK2 8, CK2 491 bp cdna, NCBI blast, bp 252 ( homo sapiens ubiquitin252 amino acid fusion

5 protein adrenal mrna) bp 100 % GST pull2down pmd218t2uba52 p GEX24T212UBA52 RNA RT2PCR 252 cdna, 1 DNA, 500 bp 250 bp (Fig. 2, 1), 387 bp. pmd218t2uba52 BamH ΠXho, bp 387 bp (Fig13A). p GEX24T212UBA52 BamH Π Xho, bp 387 bp (Fig. 3B),,. Fig. 2 Agarose gel analysis of human ubiquitin252 amino acid fusion protein( UBA52) RT2PCR Ten microlitres of UBA52 RT2PCR products were visualized by electrophoresis using 2 % agarose gels staining with SYBR. M: DL2000 marker ; 1 : UBA52 PCR product Fig. 3 Identification recombinant plasmids of pmd218t2uba52 and pgex24t212uba52 (A) Ten microlitres of recombinant plasmids of pmd218t2uba52 digested by BamH and Xho were subjected to 1 % agarose gel. Lane 1 was the insert of UBA52 about 387 bp. Lane M was 200 bp ladder marker ; (B) Ten microlitres of recombinant plasmids of pgex24t212uba52 were digested by BamH (lane 1), BamH ΠXho (lane 2), respectively. The products of UBA52 (lane 3) and the empty vector of pgex24t21 (lane 4) were visualized by electrophoresis using 1 % agarose gels staining with SYBR. The insert size was about 387 bp. M 1 was DL 2000 marker ; M 2 was DNAΠHind marker p GEX24T21 p GEX24T212 UBA52 BL21 (DE3), IPTG 27 kd ( Fig. 4, 2) 41 kd ( Fig. 4, 4), p GEX24T IPTG (Fig. 4, 1 3), p GEX24T GST pull2down Fig. 5A ( 2), SDS2PAGE GST2 UBA52(41 kd) CK2 (38 kd), CK2 GST UBA52, 2 GST, CK Western, (CK2 38 kd), Fig. 5B., GST2UBA52 CK2, Western,UBA52 CK2,. 3 Field Song [11 ] (yeast two2hybrid system),,

6 9 : CK Fig. 4 SDS2PAGE analysis expression of recombinant plasmid pgex24t21 and pgex24t212uba 52 in BL21 ( DE3) bacteria Recombinant plasmid pgex24t21 and pgex24t212uba52 were transformed into E. coli BL21 (DE3) respectively. induced with 0. 8 mmolπl E. coli BL21 (DE3) cells were lysed after being IPTG for 0, 4 hours and then analyzed by 12 % SDS2PAGE. There were very strong expression of pgex24t21 and pgex24t212uba52 induced with 018 mmolπl IPTG for 4 hours (Lane 2, 4) and very weak expression for 0 hour (Lane 1, 3). M was protein marker,., DNA2AD cdna, pact22cdna, p GBKT72HCK ( Lac Z, HIS3, ADE ),. Lac Z, X2 2gal IPTG, SDΠAde - ΠHis - ΠLeu - ΠTrp , 1 24., GST.,., CK2 CK2, Fig. 5 In vitro interaction of GST2UBA52 and CK2 (A) Bait protein ( GST2UBA52) was immobilized glutathione, prey protein ( CK2 ) was bound to immobilized bait protein, incubated for 1 hour at room temperature and then unbound protein was washed away. Protein complex was eluted after incubation. GST2tagged bait protein(plus bait, minus prey) was used as positive control (lane 1), a non2treated gel control (plus prey, minus bait) was used as negative control ( lane 3) and incubated proteins(lane 2) were separated by using 12 % SDS2 PAGE and then were analyzed by Western blot using by CK2 polyclonal antibody(b,lane 2). Purified CK2 [10 ] was used as positive control (B, Lane 1), M: protein marker CK2. CK2, hprp3p [12 ] Rio1 [13 ] Goldstein,,, (Ubiquitin). UBA S [14 ]. UBA ,Mart nez2heredia [15 ] 252,. [14 ],

7 832 24,,.,,. GST pull2 down 252 CK2,,. ( References) [ 1 ] Seldin D C, Landesman2Bollag E, Farago M, et al. CK2 as a positive regulator of Wnt signalling and tumourigenesis[j ]. Mol Cell Biochem, 2005, 274(122) :63267 [ 2 ] Ahmad K A, Wang G, Slaton J, et al. Targeting CK2 for cancer therapy [J ]. Anticancer Drugs, 2005, 16(10) : [ 3 ] Duncan J S, Litchfield D W. Too much of a good thing : the role of protein kinase CK2 in tumorigenesis and prospects for therapeutic inhibition of CK2 [J ]. Biochim Biophys Acta, 2008, 1784 (1) : [ 4 ] French A C, Luscher B, Litchfield D W, et al. Development of a stabilized form of the regulatory CK2 subunit that inhibits cell proliferation[j ]. J Biol Chem, 2007, 282(40) : [ 5 ] Xu X, Toselli P A, Russell L D, et al. Globozoospermia in mice lacking the casein kinase II alpha catalytic subunit[j ]. Nat Genet, 1999,23 (1) : [ 6 ] Litchfield D W. Protein kinase CK2 : structure, regulation and role in cellular decisions of life and death [ J ]. Biochem J, 2003, 369 (Pt1) :1215 [ 7 ] Escalier D, Silvius D, Xu X, et al. Spermatogenesis of mice lacking CK2alpha : failure of germ cell survival and characteristic modifications of the spermatid nucleus [J ]. Mol Reprod Dev, 2003, 66(2) : [ 8 ],,,. p GBKT72hCK2 [J ]. (Liang J Y, Huang G H, Liu X G, et al. Construction of shuttling expression plasmid p GBKT72hCK2A in yeast two2hybrid system[j ]. J Guangdong Med College),2004, 22 (4) : [ 9 ],,,. A CK2 [J ]. (Huang G H, Liang J Y, Chen B, et al. Aldolase A interacts with protein kinase CK2 subunit[j ]. Chin J Biochem Mol Biol), 2004, 20(5) : [10 ],,,. CK2 [J ]. (Chen X W, Liu X G, Liang J Y, et al. Cloning, expression of human protein kinase CK2 subunit in E. coli and purification and characterization of recombinant protein [J ]. Chin J Biochem Mol Biol),2004, 20(4) : [11 ] Fields S, Song O. A novel genetic system to detect protein2protein interactions[j ]. Nature,1989, 340(6230) : [12 ] Lehnert S, Ggtz C, Kartarius S, et al. Protein kinase CK2 interacts with the splicing factor hprp3p[j ]. Oncogene, 2008,27 (17) : [13 ] Angermayr M, Hochleitner E, Lottspeich F, et al. Protein kinase CK2 activates the atypical Rio1p kinase and promotes its cell2cycle phase2dependent degradation in yeast[j ]. FEBS J, 2007, 274(17) : [14 ] Ciechanover A. The ubiquitin proteasome pathway : on protein death and cell life [J ]. EMBO J, 1998, 17(24) : [15 ] Mart nez2heredia J, Estanyol J M, Ballesc J L, et al.. Proteomic identification of human sperm proteins [ J ]. Proteomics, 2006, 6 (15) :