Chinese Journal of Biochemistry and Molecular Biology CK2 252

ISSN 100727626 CN 1123870ΠQ Chinese Journal of Biochemistry and Molecular Biology 2008 9 24 (9) :826 832 CK2 252 1) 3 3, 1),2) 3 3, 1),2) 3, 1) ( 1), 524023 ; 2), 523808) CK2 Π. CK2, pact2 cdna CK2. CK2, 8, 1 252 (UBA52) cdna (100 %). GST pull2down CK2 UBA52., 252 (UBA52) CK2,,. CK2 ; ; ; 252 (UBA52) ; GST pull2down Q555 + 17 ; R33912 + 1 ; 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 524023, China ; 2) Aging Institute of Guangdong Medical College, Dongguan 523808, 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 : 2008201202 ; :2008204229 (973 ) (No. 2007CB507403) ; (No. 30672205) ; (No. 05011579), ; (No. 06Z015) 2006 3 Tel : 075922388582,E2mail : xgliu64 @126. 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. 30672205) ; Guangdong Natural Science Foundation(No. 05011579) ; 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 : 075922388582, E2mail : xgliu64 @126. com 3 3 The two authors contributed equally to this work

9 : CK2 252 827 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 ( ) ( 2 2 2 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. 1 1. 1 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. 1. 2 11211 p GBKT72HCK (BD) p GBKT72HCK [8,9 ], AH109,. Fas2FasL,. Sertoli Fas2FasL,caspase28., 11212 MATCHMAKER cdna (AD) CK2 Bid (Bcl22 ),Bid Clontech AD. caspase, [3,5,7 ]. Bcl2 PEG dsdna. 11213 p GBKT72HCK2, ds cdna pact2 DNA LiAc AH109. 5 Ade - ΠHis - ΠLeu - ΠTrp - SD. 30 1 mm, 2. Clontech. Clontech., DNA ( AD BD ), JM109. Amp LB AD BD (AD

828 24 Amp r,bd Kan r ). Amp LB, DNA, PEG( ). AD AH109. 011 g AD BD, 011 mg DNA LiAc ( ) AH109,.. 1. 2. 4 DNA, DNA, pact2. NCBI BLAST, BLASTN, cdna,. 1. 2. 5 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, 94 40 s, 62 30 s,72 1 min,32,72 10 min. 1. 2. 6 BamH ΠXho PCR, pmd182t TA. JM109. DNA, BamH ΠXho DNA. 252 DNA p GEX24T21, BamH ΠXho, BL21 (DE3)., p GEX24T212UBA52. 1. 2. 7 p GEX2 4T212UBA52 BL21 (DE3), Amp LB 37., 100 l 5 ml Amp LB, 37, A 595 018, IPTG( 018 mmolπl),37 4 h. 2 100 5 min. 0175 mm 12 %, 200 V,40 min,sds2page, R2250,. 1. 2. 8 GST pull2down : SDS2PAGE, (NC), NC BSA, CK2 ( ) ( ),ECL,,. 2 2. 1 3. 4 10 9 cfuπ g, Clontech 10 8 cfuπ g, 1166 gπ l, A 260 Π A 280 1188. 213 10 5 cfuπ g, 1115 10 6, 396, SDΠAde - ΠHis - ΠLeu - ΠTrp - 168 Ade +,His +,Leu +,Trp +. 2 168, 138,. 2. 2 PCR 138 1 24 4 SDΠAde - ΠHis - ΠLeu - ΠTrp -, 2, 3 4 6 10 12 14 18 21 23, 1 %, 1 DNA. 3 11 22,12 15,10 14,17 18, 21 23 PCR, PCR Alu Hae, 1 %, Fig. 1 (A, B).,3 11 22, 12 15, 10 14, 17 18, 21 23. 8. 4 6 10 14 16 PCR 220 bp, 21 23 PCR 300 bp, 3 11 22 12 15 PCR 500 bp, 17 18 PCR 750 bp.

9 : CK2 252 829 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 2. 3 8 8, AH109, SDΠLeu -, 2,. 2. 4 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, 16 460 bp 252 ( homo sapiens ubiquitin252 amino acid fusion

830 24 protein adrenal mrna) 37 481 bp 100 %. 2. 6 GST pull2down 21611 pmd218t2uba52 p GEX24T212UBA52 RNA RT2PCR 252 cdna, 1 DNA, 500 bp 250 bp (Fig. 2, 1), 387 bp. pmd218t2uba52 BamH ΠXho, 2 693 bp 387 bp (Fig13A). p GEX24T212UBA52 BamH Π Xho, 4 950 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 21612 252 p GEX24T21 p GEX24T212 UBA52 BL21 (DE3), IPTG 27 kd ( Fig. 4, 2) 41 kd ( Fig. 4, 4), p GEX24T21 252. IPTG (Fig. 4, 1 3), p GEX24T2 1. 21613 GST pull2down Fig. 5A ( 2), SDS2PAGE GST2 UBA52(41 kd) CK2 (38 kd), CK2 GST UBA52, 2 GST, CK2 252. Western, (CK2 38 kd), Fig. 5B., GST2UBA52 CK2, Western,UBA52 CK2,. 3 Field Song [11 ] (yeast two2hybrid system),,

9 : CK2 252 831 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 -. 2 138, 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,. 2007 hprp3p [12 ] Rio1 [13 ]. 1975 Goldstein,,, (Ubiquitin). UBA52 52 60S 76 128 [14 ]. UBA52. 2006,Mart nez2heredia [15 ] 252,. [14 ],

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