Absence of Caprin1 Results in Defects in Cellular Proliferation1

Cytoplasmic activation/proliferation-associated protein-1 (Caprin-1) is a cytoplasmic phosphoprotein that is the prototype of a novel family of highly conserved proteins. Its levels, except in the brain, are tightly correlated with cellular
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  of November 15, 2015.This information is current as Cellular ProliferationAbsence of Caprin-1 Results in Defects in Bin Wang, Muriel D. David and John W. Schrader 10.4049/jimmunol.175.7.42742005; 175:4274-4282; ;  J Immunol References, 18 of which you can access for free at: cites 29 articles This article Subscriptions is online at: The Journal of Immunology Information about subscribing to Permissions copyright permission requests at: Email Alerts free email-alerts when new articles cite this article. Sign up at: Print ISSN: 0022-1767 Online ISSN: 1550-6606. Immunologists All rights reserved.Copyright © 2005 by The American Association of 9650 Rockville Pike, Bethesda, MD 20814-3994.The American Association of Immunologists, Inc., is published twice each month by The Journal of Immunology   b  y g u e  s  t   on N o v e m b  e r 1  5  ,2  0 1  5 h  t   t   p :  /   /   w w w . j  i  mm un ol   . or  g /  D o wnl   o a  d  e  d f  r  om  b  y g u e  s  t   on N o v e m b  e r 1  5  ,2  0 1  5 h  t   t   p :  /   /   w w w . j  i  mm un ol   . or  g /  D o wnl   o a  d  e  d f  r  om   Absence of Caprin-1 Results in Defects inCellular Proliferation 1 Bin Wang, 2 Muriel D. David, 2 and John W. Schrader 3 Cytoplasmic activation/proliferation-associated protein-1 (Caprin-1) is a cytoplasmic phosphoprotein that is the prototype of anovel family of highly conserved proteins. Its levels, except in the brain, are tightly correlated with cellular proliferation. Wedisrupted  caprin-1  alleles in the chicken B lymphocyte line DT40 using homologous recombination. We readily obtained cloneswith one disrupted allele (31% of transfectants), but upon transfection of heterozygous cells we obtained a 10-fold lower frequencyof clones with disruption of the remaining allele. Clones of   caprin-1 -null DT40 cells exhibited marked reductions in their prolif-eration rate. To obviate the problem that we had selected for  caprin-1 -null clones with characteristics that partially compensatedfor the lack of Caprin-1, we generated clones of DT40 cells heterozygous for the  caprin-1  gene in which, during disruption of theremaining wild-type allele of the chicken  caprin-1  gene, the absence of endogenous Caprin-1 would be complemented by condi-tional expression of human Caprin-1. Suppression of expression of human Caprin-1 resulted in slowing of the proliferation rate,due to prolongation of the G 1  phase of the cell cycle, formally demonstrating that Caprin-1 was essential for normal cellularproliferation.  The Journal of Immunology,  2005, 175: 4274–4282. C ytoplasmic activation/proliferation-associated protein-1(Caprin-1) 4 and Caprin-2 form a novel family of proteinsthat are highly conserved in vertebrates (1). Except inadult brain, where Caprin-1 may serve a different function, levelsof Caprin-1 correlate with cellular proliferation, with high levelsoccurring in thymus and spleen, and low levels in kidney, liver,and muscle. Levels of Caprin-1 proteins increased when restingsplenic T or B lymphocytes were stimulated to divide. Likewise, inthymocytes, levels of Caprin-1 were high in the subpopulation of large, dividing cells and lower in the major population of small,nondividing thymocytes. Caprin-1 levels also increased when bonemarrow cells were stimulated to divide with hemopoietic growthfactors such as IL-3 or CSF-1. Conversely, levels of Caprin-1 de-creased when M1 monocytic leukemic cells were induced to dif-ferentiate to nondividing cells, and when IL-3-dependent cells andNIH-3T3 cells were deprived of IL-3 and serum, respectively (1).Although the expression patterns of Caprin-1 are consistent witha positive role for Caprin-1 in regulation of cellular proliferation,some evidence suggested a negative role. Thus, overexpression of a GFP-Caprin-1 fusion protein in NIH-3T3 cells resulted in a dose-dependent inhibition of cell division (1). Similar inhibition of pro-liferation was also seen when Caprin-1 was overexpressed in Baf/3cells (immature hemopoietic cells), WEHI-231 cells (immature Blymphocytes), or 293 HEK cells (embryonic kidney epithelial cells)(B. Grill, G. Wilson, and J. Schrader, unpublished data), indicatingthat this inhibitory effect was not cell-type specific. Interestingly, Ca-prin-2 (also called EEG1) mRNA increased in erythroid cells as theyneared terminal differentiation, and overexpression of Caprin-2 inChinese hamster ovary cells decreased cellular proliferation and in-duced apoptosis (2), leading to the proposal that it functions as aproapoptotic inhibitor of the cell cycle. Taken together, these ob-servations would be consistent with a role for Caprin-1 and Ca-prin-2 as suppressors of cell proliferation.To resolve the question of whether Caprin-1 is a positive ornegative regulator of cell proliferation, we undertook the genera-tion of   caprin-1 -null cells by gene targeting, using the chicken Blymphocyte line DT40, which exhibits an unusually high rate of homologous recombination (3–10). Materials and Methods Cell culture DT40 cells were maintained in log-phase proliferation at densities between10 4 and 10 6 cells/ml, in RPMI 1640 (Invitrogen Life Technologies) sup-plemented with 10% FBS, 1% chicken serum, and 50   M 2-ME. Cloning of chicken Caprin-1 cDNA A 2.1-kb cDNA-encoding chicken Caprin-1 was amplified by RT-PCRusing RNA from DT40 cells and the primers 5  -GGGATCCACCATGCCCTCGGCTACCACGGCACC-3   (sense) and 5  -GTTTAATTCACTTGCTGAGCGTTCA-3  (antisense). Targeting constructs The 8.2-kb genomic region of the  caprin-1  locus shown in Fig. 2  A  wasamplified by PCR, using DT40 genomic DNA as a template and the prim-ers S4, 5  -CTTGACGATTACCAGGAACGAATGA-3   and AS5, 5  -TCCTTCTCACTACTGCTGAACTG-3  , and inserted into the TOPO-TAcloning vector (Invitrogen Life Technologies). This plasmid was used as atemplate for PCR to generate the targeting constructs, using the followingprimers: S7, 5  -AGGAATTCCTCTTGCCTAGAAGATCTCA-3  ; AS7,5  -GATGGATCCATCTCTGAACAAACTGCAGCA-3  ; S8, 5  -GATCTCGAGATCCATTGTTTTCTGGCATG-3  ; AS8, 5  -TCTTCTAGAAGAAATAGGCCATTTATGCG-3  ; and S531, 5  -GAGCGGCCGCAGAGACAGTGCGCTGAATTTATGC-3  . The amplified products were clonedinto the TOPO-TA cloning vector, and then subcloned into pPNT-NHS14(11) as the left arm (S7-AS7) and the right arm (S8-AS8 or S531-AS8), The Biomedical Research Centre, University of British Columbia, Vancouver, BritishColumbia, CanadaReceived for publication March 1, 2005. Accepted for publication July 20, 2005.The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby marked  advertisement   in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact. 1 This work was supported by grants from the Canadian Institutes of Health Researchand fellowships to M.D.D. and B.W. from the Canadian Arthritis Network. currently supported by a grant from the Arthritis Society of Canada. 2 B.W. and M.D.D. equally contributed to this study. 3 Address correspondence and reprint requests to Dr. John W. Schrader, BiomedicalResearch Centre, University of British Columbia, 2222 Health Sciences Mall, Van-couver, British Columbia, V6T 1Z3, Canada. E-mail address: 4 Abbreviations used in this paper: Caprin, cytoplasmic activation/proliferation-asso-ciated protein; Dox, doxycycline; G3BP-1, Ras-GTPase-activating protein Src ho-mology 3 domain-binding protein; PI, propidium iodide; PKB, protein kinase B. The Journal of Immunology Copyright © 2005 by The American Association of Immunologists, Inc. 0022-1767/05/$02.00   b  y g u e  s  t   on N o v e m b  e r 1  5  ,2  0 1  5 h  t   t   p :  /   /   w w w . j  i  mm un ol   . or  g /  D o wnl   o a  d  e  d f  r  om   respectively. Finally, the phosphoglycerate kinase promoter- neomycin  cas-sette of these pPNT-derived vectors was replaced with cassettes, orientedin the direction opposite to the arms, containing a chicken  -actin promoterdriving expression of the  puromycin resistance  gene, for the pPNT2-  puro (S7-AS7) construct, or of the  blasticidin resistance  gene, for the pPNT2- bsr   (S8-AS8) and the pPNT3- bsr   (S531-AS8) constructs.  DNA transfection and gene disruption A total of 10 7 DT40 cells was electroporated with 50  g of   Nde I-linearizedpPNT2-  puro  construct at 550 V, 50   F, using a Gene Pulser (Bio-Rad),and selected in presence of 1   g/ml puromycin (Sigma-Aldrich) for 10–14days. Drug-resistant colonies were further selected in 2   M ganciclovir(Sigma-Aldrich) for 3 days. The heterozygous  caprin-1   /   clones wereidentified by PCR amplification of the  caprin-1 -  puromycin  fusion gene,using the primers S4 and Puro (5  -CAGCGCCCGACCGAAAGGAGCGCACGACC-3  ). To generate  caprin-1 -null DT40 cells, a heterozygous  ca- prin-1   /   clone was transfected with the pPNT2- bsr   (first attempt) or thepPNT3- bsr   constructs. Clones were selected in presence of 15   g/ml blas-ticidin-S (Invitrogen Life Technologies) for 10–14 days, and then in pres-ence of 2   M ganciclovir for 3 days. Genomic DNA was analyzed by PCRusing the primers S4 and AS10 (5  -TGTCACTTCCTGGTATTTTGACACTGC-3  ). Disruption of the remaining allele of   caprin-1  was indicatedby absence of the S4-AS10 band. PCR using the primers S7 and Puro wasused as a positive control to verify the quality of the preparation of genomic DNA from each clone. The resulting homozygous  caprin-1 -nullcells were referred to as  caprin-1   /   DT40. Generation of the R- caprin-1   /   cells The pEGFP-hCaprin-1 plasmid (1) was digested by  Bam HI, and the re-sulting 2.2-kb fragment encoding human Caprin-1 was subcloned into thetetracycline/doxycycline (Dox)-repressible expression vector, pTRE2-hyg(BD Clontech), to generate pTRE2-hCaprin-1. As illustrated in Fig. 4, aclone of heterozygous  caprin-1   /   DT40 cells was transfected with thepTet-Off plasmid (BD Clontech) that contains the  Tet-Off trans activator  and the  neomycin resistance  genes. Stable clones of   caprin-1   /   Tet-Off DT40 cells were then transiently transfected with the pTRE-Luc plasmid(BD Clontech), and tested for their ability to repress expression of the luciferase reporter   gene after treatment for 48 h with 1   g/ml Dox. One of the clones of   caprin-1   /   Tet-Off DT40 cells in which treatment with Doxefficiently suppressed luciferase expression driven by the transiently trans-fected pTRE-Luc plasmid was then stably transfected with the pTRE2-hCaprin-1. These resultant clones were cultured with or without 1   g/mlDox for 4 days, when levels of human Caprin-1 mRNA and chicken hy-poxanthine guanine phosphoribosyltransferase mRNA were assessed byreal-time RT-PCR, and levels of human Caprin-1 protein by immunoblot-ting. We then selected clones that, in medium alone, exhibited levels of human Caprin-1 that were equal to, or exceeded by no more than 2-fold,the levels of Caprin-1 expressed in dividing human tumor cells (Jurkatcells), but that, when cultured with 1   g/ml Dox, exhibited no detectablehuman Caprin-1, and finally transfected these cells with the pPNT2- bsr  gene-targeting construct to disrupt the remaining chicken  caprin-1  allele.Clones in which the remaining endogenous  caprin-1  allele had been dis-rupted were identified by PCR and RT-PCR using the primer pairs S4-AS10 and S53-AS6, respectively. These rescued clones were designatedR- caprin-1   /   DT40 cells.  Immunoblotting Cell lysates were prepared, and immunoblotting using the anti-rGST-Caprin-1 polyclonal Ab was performed, as described previously (1).For analysis of signaling, cells were treated for 4 min with 10   g/ml M4anti-chicken IgM mAb (Southern Biotechnology Associates) to ligate theBCR and lysed in a buffer containing 1% Nonidet P-40, 100 mM NaCl, 50mM Tris, pH 7.5, 2 mM EDTA, 1 mM sodium vanadate, 1 mM PMSF, anda mixture of protease inhibitors (Roche). Lysates were clarified by centrif-ugation at 15,000  g  for 20 min at 4°C, and equivalent amounts of proteinwere resolved by SDS-PAGE. After transfer onto nitrocellulose mem-branes, the presence of specific proteins was assessed by immunoblottingusing the anti-phospho-ERK (9101) and anti-phospho-protein kinase B(PKB) (9271) polyclonal Abs from Cell Signaling Technology.  RT-PCR and real-time PCR A total of 1   g of total RNA was reverse transcribed using random hex-amer primers (Invitrogen Life Technologies) and SuperScript II (Invitro-gen Life Technologies), and used as a template for real-time PCR using aLightCycler (Roche). For amplification of chicken Caprin-1 cDNA, prim-ers S53 (5  -TTGGGTGACGATGAAGTGCGCAG-3  ) and AS6 (5  -CGTTCAACAACCTCCTTCAGG-3  ) were used as forward and reverse prim-ers, respectively. For amplification of chicken Caprin-2 cDNA, we used theforward primer 5  -CCAGCGGAGAGAGAGTCTATTG-3   and the re-verse primer 5  -GGTGCGAGGAGTTGGAATAC-3  . For amplification of human Caprin-1 cDNA, we used the forward primer 5  -ACGGTGTTCAATATGAATGCCCCA-3   and the reverse primer 5  -CCCGTTGATAGCCAGAGTAATCC-3  . After a 3-min denaturation step at 95°C, am-plification reactions of Caprin-1 cDNAs were conducted during 45(chicken) or 40 (human) cycles of successive incubations at 95°C for 5 s,60°C (chicken) or 65°C (human) for 5 s, and 72°C for 18 s. Chickenhypoxanthine guanine phosphoribosyltransferase cDNA was amplified asan internal control using the forward primer 5  -TCAGTGAGACGGGGAAGCGAAG-3   and the reverse primer 5  -GGGCAGCAATAGTCGGTAGAGTC-3  . Statistical analysis of the quantification of relative levels of mRNA expression by real-time PCR was conducted using the softwareREST-XL-version 2 and the Pair-Wise Fixed Reallocation RandomizationTest (12). Cell cycle analysis Cells were synchronized at metaphase by treatment with 0.5   g/ml no-codazole for 10 h, and then washed thoroughly. At the indicated times,cells were resuspended at 10 6 cells/ml in PBS containing 50   g/ml pro-pidium iodide (PI) and 0.1% sodium azide, mixed with an equal volume of Vindelov’s PI staining solution (0.01 M Tris base, 0.01 M NaCl, 700 U of RNase, 7.5  10  5 M PI, 0.1% Nonidet P-40), and incubated for 30 minon ice. Flow cytometry was performed using a BD Biosciences FACS-Calibur. Asynchronous DT40 cells were pulsed for 10 min with 10   MBrdU, as previously described (13). A BrdU Flow kit (BD Pharmingen)was used to determine the cell cycle distribution of BrdU-labeled cells byflow cytometry. Cell counting Cell suspensions were mixed with a predetermined number of 10-  m mi-crospheres (Polysciences). The ratio of viable cells to microspheres wasassessed by flow cytometry. Results  Isolation of chicken full-length Caprin-1 cDNA To isolate chicken Caprin-1 cDNA, we searched the BursalExpressed Sequence Tag Database (   ) and found several expressed sequence tag sequences withhigh homology to parts of the mouse and human Caprin-1 cDNAsdescribed previously (1). RNA from DT40 cells was used as atemplate for RT-PCR using primers designed in these conservedsequences. The PCR product was cloned and sequenced (GenBank accession AY745194). It exhibited 81 and 79% identity with hu-man and mouse Caprin-1 cDNAs, respectively, and the predictedprotein of 702 aa that it encoded exhibited 92% identity with bothhuman (709 aa) and mouse (707 aa) Caprin-1 (Fig. 1). These dataindicated that this cDNA encoded the chicken ortholog of the hu-man and mouse Caprin-1 described previously (1), and revealed ahigh level of conservation of Caprin-1 between species. Generation of   caprin-1 -deficient DT40 cells We exploited the unusually high rate of homologous recombina-tion in the DT40 line of chicken B lymphocytes to generate  caprin-1 -deficient cells and investigate its cellular functions. To cloneparts of the chicken  caprin-1  gene, we used genomic DNA fromDT40 cells as a template for PCR. The amplified fragments weresubcloned in a series of targeting constructs based on pPNT. In thepPNT2-  puro  construct, an actin-promoter-  puromycin  cassette wasflanked by fragments corresponding to introns 3 and 7 of the  ca- prin-1  gene. The pPNT2- bsr   construct was identical, except thatthe  puromycin resistance  gene was replaced by a  blasticidin re-sistance  gene. Homologous recombination of the  caprin-1  genewith the pPNT2-  puro  or the pPNT2- bsr   constructs would result indeletion of the region encompassing exons 4–7 of the chicken caprin-1  gene (encoding 80% of the HR-1 region of Caprin-1,4275The Journal of Immunology   b  y g u e  s  t   on N o v e m b  e r 1  5  ,2  0 1  5 h  t   t   p :  /   /   w w w . j  i  mm un ol   . or  g /  D o wnl   o a  d  e  d f  r  om   from aa 87 to 268), and would render the cells resistant to puro-mycin or blasticidin, respectively (Fig. 2  A ). Nonhomologous re-combination events (i.e., random integration of these constructs)would result in sensitivity of transfected cells to ganciclovir, due toexpression of the  thymidine kinase  gene present in all of the pPNT-derived targeting constructs.Wild-type DT40 cells were transfected with the pPNT2-  puro construct, and selected with both puromycin and ganciclovir. Dou-ble drug-resistant clones were examined by PCR using the forwardprimer S4, specific for exon 3 of the  caprin-1  gene, and the reverseprimer Puro, specific for the  puromycin resistance  gene. Success-ful disruptions of a  caprin-1  allele were detected by the presenceof a 2.2-kb band, corresponding to a chimeric fragment containingelements of both the  caprin-1  (exon 3 and intron 3) and the  pu-romycin resistance  genes (Fig. 2  B ). Several such clones wereobtained. FIGURE 1.  Conservation of the sequences of chicken, human, and mouse Caprin-1 proteins. Shown are alignments of the predicted amino acidsequences of chicken Caprin-1 (GenBank accession AY745194), human Caprin-1 (GenBank accession DAA01121.1), and mouse Caprin-1 (GenBank accession DAA01122.1), made using the Clustal method with the Macvector software (Accelrys). Identical residues are darkly shaded, and similar residueslightly shaded. 4276 ROLE OF CAPRIN-1 IN CELLULAR PROLIFERATION   b  y g u e  s  t   on N o v e m b  e r 1  5  ,2  0 1  5 h  t   t   p :  /   /   w w w . j  i  mm un ol   . or  g /  D o wnl   o a  d  e  d f  r  om   To generate homozygous ( caprin-1   /   ) cells, one of the clonesthat were heterozygous for  caprin-1  ( caprin-1   /   ) was transfectedwith the pPNT2- bsr   targeting construct. Subclones were selectedfor resistance to blasticidin and ganciclovir, and disruption of theremaining  caprin-1  allele was identified by PCR using the primersS4 and AS10, specific for the exons 3 and 4 of the  caprin-1  gene,respectively. Thus, PCR of genomic DNA from  caprin-1   /   , butnot  caprin-1   /   DT40 cells would yield a fragment of 1.9 kb,corresponding to the region encompassing exons 3–4 of the wild-type  caprin-1  gene.However, we analyzed 130 clones without finding a single clonein which the remaining  caprin-1  allele had been disrupted. Thisresult suggested that either the pPNT2- bsr   plasmid yielded an un-expectedly lower efficiency of targeting than the pPNT2-  puro  con-struct, or, more likely, that cells in which both  caprin-1  alleles hadbeen disrupted had an impaired ability to proliferate and/orsurvive.To obtain a higher frequency of homologous recombination (4),we lengthened one of the regions in the pPNT2- bsr   targeting con-struct that were homologous to the  caprin-1  gene. Thus, in the newtargeting construct, pPNT3- bsr  , we replaced the 3   arm of thepPNT2- bsr   plasmid that corresponded to the 1.3-kb intron 7 of the caprin-1  gene, with a 3.3-kb fragment encompassing intron 6,exon 7, and intron 7. We transfected this construct into  caprin-1   /   cells, selected the stable transfectants in presence of blasti-cidin and ganciclovir, and screened for disruption of the remainingwild-type  caprin-1  allele by PCR using the primers S4 and AS10.Using this strategy, we obtained a total of four clones in the caseof which PCR amplification of genomic DNA using these primersfailed to yield the 1.9-kb fragment corresponding to the regionencompassing exons 3–4 of the wild-type  caprin-1  gene (Fig. 2 C  ).PCR with the primers S7 and Puro was used as an internal positivecontrol, to verify the quality of genomic DNA derived from eachclone (data not shown). We also used RT-PCR with the primersS53 and AS6, specific for exons 5 and 7, respectively, to confirmthat these clones did not express Caprin-1 mRNA (Fig. 2  D ). Thesedata indicated that, in these clones, we had successfully disruptedboth alleles of the  caprin-1  gene.  Low frequency of recovery of   caprin-1   /   clones As summarized in Table I, homologous recombination in wild-type DT40 cells of one or the other of the  caprin-1  alleles with thepPNT2-  puro  construct occurred in 2 of the 28 recovered clonesthat we analyzed (7% of the total number of clones). However, insimilar experiments using  caprin-1   /   cells and the very similarpPNT2- bsr   construct, we analyzed 130 clones without finding asingle clone in which the remaining  caprin-1  allele had been dis-rupted. With the pPNT3- bsr   construct, we obtained a much higherfrequency of homologous recombination events than with thepPNT2- bsr   construct. Thus, after transfection of pPNT3- bsr   intowild-type DT40 cells and selection in presence of blasticidin and FIGURE 2.  Generation of   caprin-1 -deficient DT40 clones.  A , Sche-matic representation of the partial chicken  caprin-1  locus, the three target-ing constructs (pPNT2-  puro , pPNT2- bsr  , and pPNT3- bsr  ), and the struc-ture of the targeted loci. The open boxes and the numbers indicate exonsof Caprin-1. Arrowheads indicate localization of primer sites for PCR.Genomic DNA from wild-type (   /   ), heterozygous (   /   ), and homozy-gous mutant (   /   ) clones was isolated and examined by PCR using theprimers S4 and Puro (  B ), and S4 and AS10 ( C  ).  D , RT-PCR analysis of mRNA from  caprin-1   /   ,  caprin-1   /   , and  caprin-1   /   clones. TotalRNA was isolated from the cells and amplified by RT-PCR using theprimers S53 and AS6.Table I.  Reduced frequency of recovering  caprin-1 -null clones ConstructTargetCellIntegrations at:ClonesAnalyzedAllele 1 Allele 2 Rd a pPNT2-  puro    /    2 (7.3%) 0 26 28pPNT2- bsr     /    ND 0 130 130pPNT3- bsr     /    10 (31.3%) 0 22 32pPNT3- bsr     /    15 (16.3%) b 3 (3.2%) 74 92 a Random integrations. b Replaces pPNT2-  puro  cassette. 4277The Journal of Immunology   b  y g u e  s  t   on N o v e m b  e r 1  5  ,2  0 1  5 h  t   t   p :  /   /   w w w . j  i  mm un ol   . or  g /  D o wnl   o a  d  e  d f  r  om 
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