Molecular and Cellular Biochemistry 262: 187–193, 2004.c© 2004 Kluwer Academic Publishers. Printed in the Netherlands.

Ribosomal protein S18 identifiedas a cofilin-binding protein by using phagedisplay library

Kaoru Kusui,1 Haruyo Sasaki,1 Reiko Adachi,1 Sachiko Matsui,1,3

Kazuo Yamamoto,2 Teruhide Yamaguchi,1 Tadashi Kasahara3 andKazuhiro Suzuki11National Institute of Health Sciences, Kamiyoga, Setagaya-ku, Tokyo, Japan; 2Graduate School of Frontier Sciences,University of Tokyo, Chiba, Japan; 3Kyoritsu College of Pharmacy, Shibakoen, Minato-ku, Tokyo, Japan

Received 16 August 2003; accepted 15 December 2003

Abstract

We previously reported that an actin-binding protein, cofilin, is involved in superoxide production, phagocytosis, and chemotaxisin activated phagocytes through cytoskeletal reorganization. To elucidate the functions of cofilin in greater detail we tried toidentify cofilin-binding proteins by using a phage-displayed cDNA library constructed from human brain mRNAs. Several phageclones capable of binding to cofilin were obtained, and the phage with the strongest binding affinity contained the C-terminalhalf of ribosomal protein S18. To confirm the interaction between the S18 protein and cofilin, we investigated whether cofilinwould bind to His-tagged S18 protein immobilized in Ni-NTA-agarose gel. Cofilin and the S18 protein co-eluted with a low pH(4.5) buffer, suggesting that the proteins interact with each other. Preincubation of cofilin with actin abrogated the binding toprotein S18, indicating that cofilin interacts with S18 protein at the actin-binding site, and cofilin co-immunoprecipitated withFLAG-tagged S18 protein expressed in COS-7 cells. These results suggest that some cofilin molecules bind the ribosomal S18protein under physiological conditions. (Mol Cell Biochem 262: 187–193, 2004)

Key words: actin, cofilin, phage display, ribosomal protein S18

Introduction

In the absence of any stimulation, phagocytic neutrophils arenormally in the resting state. However, once they are activatedby microorganisms, toxins, cytokines, or lipid mediators,they play an important role in self-defense systems throughadhesion, chemotaxis, phagocytosis, superoxide production,degranulation, and release of cytokines [1]. We found alow-molecular-weight phosphoprotein that appeared to beinvolved in activation of phagocytes through dephosphory-lation [2], and the phosphoprotein was subsequently iden-tified as cofilin [3]. Cofilin was first discovered in porcinebrain in 1984 [4], and it was later found to be an actin- and

Address for offprints: K. Suzuki, National Institute of Health Sciences, 1-18-1, Kamiyoga, Stetagaya-ku, Tokyo 158-8501, Japan (E-mail: ksuzuki@nihs.go.jp)

phosphatidylinositol 4,5-bisphosphate (PIP2)-binding pro-tein that is well conserved and ubiquitous [5]. Usually onlythe dephosphorylated form of cofilin is capable of bindingboth filamentous (F-actin) and monomeric (G-actin) actinto depolymerize the former and sequester the latter frompolymerization. The dephosphorylation of cofilin in acti-vated phagocytes has been confirmed by other groups [6–8],and it may be a downstream event of activation of a src-family tyrosine kinase [9] and phospholipase C [10]. In ac-tivated phagocytes cofilin translocates from the cytosol tothe plasma membrane and plays a crucial role in superoxideproduction[3, 11], phagocytosis [9, 12, 13], and chemotaxis[14].

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On the other hand, it has been reported that LIM-kinase 1(LIMK1) and LIM-kinase 2 (LIMK2) phosphorylate cofilinspecifically at Ser-3 [15, 16] and that a cofilin phosphatasenamed Slingshot dephosphorylates it [17]. Very recently wehave observed that cofilin phosphorylation by LIMK1 anddecreased expression of cofilin by antisense oligonucleotidelead to enhancement of respiratory bursts and phagocytosis[12, 13], observations which are apparently inconsistent withthe previous findings. The above findings suggest that cofilinhas complicated roles that depend on its intracellular local-ization and interactions with other functional proteins.

In this study, we searched for novel proteins that interactwith cofilin (non-muscle type, cofilin 1) by using a humanbrain cDNA library displayed on phage particles. This ap-proach led to the identification of ribosomal protein S18 as aspecific cofilin-binding protein, and further analysis demon-strated that the interaction between ribosomal protein S18and cofilin is inhibited in the presence of actin. Another im-portant finding was the detection of an association betweencofilin and S18 protein in living cells by immunoprecipi-tation. The possible role of cofilin and the cytoskeleton inribosomal protein synthesis is discussed.

Materials and methods

Materials

Expression vectors pGEX-6P-1 and pQE30 were purchasedfrom Amersham Pharmacia Biotech (Piscataway, NJ, USA)and Qiagen (Hilden, Germany), respectively. A PCR-READY cDNA kit (for ribosomal protein S18) was obtainedfrom Toyobo (Tokyo, Japan). Glutathione-Sepharose 4Band PreScission protease were obtained from AmershamPharmacia Biotech. A T7 select human brain cDNA li-brary was purchased from Novagen (Madison, WI, USA).Monoclonal anti-cofilin antibody (MAB22) was donatedby Drs. T. Obinata and H. Abe (Chiba University, Japan),and rabbit anti-cofilin antibody was a gift from Drs. I.Yahara and K. Iida (Tokyo Metropolitan Institute of MedicalScience). Non-muscle actin (APHL95) was purchased fromCytoskeleton (Denver, CO, USA). Ni-NTA agarose andNi-NTA HRP were purchased from Qiagen. p3XFLAG-myc-CMV-26 expression vector and monoclonal anti-FLAGantibody were products of Sigma (St. Louis, MO).

Expression of the GST-cofilin fusion protein

The full-length cDNA of human cofilin (non-muscle type,cofilin 1) was cloned from a cDNA library derived fromU937 cells by PCR using two primers, 5′-CCGGAATTC-ATGGCCTCCGGTGTGGCTGTCTCT-3′ (containing an

EcoRI site) and 5′-CCGCTCGAGTCACAAAGGCTTGCCCTCCAGGGA-3′ (containing an Xho I site). After digestionof the amplified human cofilin cDNA with EcoRI and XhoI, it was inserted between the EcoRI and Xho I sites in themulticloning site of pGEX-6P-1 to yield expression plasmidpGEX-6P-cofilin. Then E. coli strain BL21(DE3) wastransformed with the pGEX-6P-cofilin, and the sequencesof the plasmid DNAs purified from the generated colonieswere confirmed with an ABI PRISM 310 genetic analyzer.The transformed BL21(DE3) was cultured in an LB/Ampmedium at 37 ◦C until the mid-log phase, and expressionof cofilin was induced by adding 1 mM isopropyl-β-D(–)-thiogalactopyranoside (IPTG) to the medium for 4–5 h.After induction, an aliquot of the E. coli cells was collectedby centrifugation and lysed with SDS-sample buffer.Expression of the GST-cofilin fusion protein was confirmedby SDS-PAGE using 12.5% polyacrylamide gel accordingto the method of Laemmli [18].

Preparation of the human recombinant cofilin

E. coli BL21(DE3) expressing the GST-cofilin fusion pro-tein was collected and sonicated in ice-cold PBS containing1% Triton X-100. The cell suspension was then centrifugedat 15,000 rpm for 10 min to remove the insoluble fraction,and the soluble supernatant was collected. The supernatantwas loaded on a Glutathione-Sepharose 4B column equili-brated with PBS. After washing the column with 30 column-volumes of PBS, proteins bound to the column were digestedwith PreScission protease and allowed to stand overnight at4 ◦C. The recombinant cofilin (with the GST portion cutout) was eluted from the column. The purity of the elu-ate prepared in this manner was analyzed by SDS-PAGE asdescribed.

Biopanning with a pre-made T7 select human brain phagedisplay library

Recombinant cofilin (10 µg/ml) obtained by the above pro-cedure was coated onto a 96-well plate, and the T7 humanbrain cDNA library (2 × 107 pfu) was added to the well andallowed to react at 4 ◦C overnight. The well was then washedwith TBS containing 0.1% Tween 20, the recombinant cofilin(10 µg/ml) was added, and the solution was allowed to standat room temperature for 1 h. The phages that bound to thecoated recombinant cofilin were specifically eluted, and afteramplifying the eluted phages with transforming E. coli strainBLT 5615 and concentrating them by the polyethylene glycol6000 (PEG) precipitation method, the amplified phages (1010

pfu) were used for the next biopanning. The biopanning cyclewas repeated four times.

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Cloning and identification of the proteins that bound to therecombinant cofilin

Phages eluted after the final (fourth) biopanning cycle wereplated on an LB/carbenicillin plate. Several single plaqueswere isolated at random, and their inserted cDNAs wereamplified by the PCR method. The sequences of the ampli-fied DNAs were analyzed, and a homology was conducted byusing the BLAST computer program to identify the protein.Phage clones whose inserted cDNAs coded the open readingframe of the proteins expressed in the correct reading frameswere selected, and we confirmed that these recombinantphages specifically bound to the coated recombinant cofilinby comparing them to the negative control phage clone.

Expression of the His-tagged human ribosomal protein S18

Human ribosomal protein S18 cDNA was cloned fromhuman leukemia promyelocytic HL-60 PCR-READY cDNA(Toyobo, Tokyo) by the PCR method using two primers,5′-CGG-CAGGATCCATGTCTCTAGTGATCCCTGAAA-AG-3′ (containing a BamHI site) and 5′-CGGCAGTCGAC-TTATTTCTTCTTGGACACACCCAC-3′ (containing a Sal Isite). After digestion of the amplified human ribosomal pro-tein S18 cDNA at the BamHI and Sal I sites, it was insertedinto the multicloning site of pQE30 to yield recombinantplasmid pQE-S18-2. E. coli strain M15 [pREP4] was thentransformed by pQE-S18-2, and expression was inducedwith 1 mM IPTG as described. Expression of the ribosomalprotein S18 and its solubility was confirmed by SDS-PAGE.

Co-elution of cofilin with ribosomal protein S18

The E. coli M15 [pREP4] cells expressing human riboso-mal protein S18 were solubilized for 1 h at room temper-ature with 100 mM NaH2PO4, 10 mM Tris·HCl, and 6 Mguanidine-hydrochloride (GuHCl) pH 8.0. After collectingthe supernatant, the His-tagged ribosomal protein S18 wasimmobilized on Ni-NTA agarose beads by rotating. The beadswere sequentially washed with 100 mM NaH2PO4, 10 mMTris·HCl, 8 M urea pH 8.0, 4 M urea pH 8.0, 100 mMNaH2PO4, 10 mM Tris·HCl, and 2 M urea pH 8.0 to re-move the GuHCl and reduce the concentration of urea. Thebeads thus prepared were reacted with the recombinant cofilin(pre-incubated or not pre-incubated with non-muscle actin)in 100 mM NaH2PO4, 10 mM Tris·HCl, 2 M urea pH 8.0 byrotating for 3 h at room temperature. The beads were thenwashed with 100 mM NaH2PO4, 10 mM Tris·HCl, and 2 Murea, pH 6.3, three times, and eluted with 100 mM NaH2PO4,10 mM Tris·HCl, and 2 M urea pH 4.5 twice. The eluateswere analyzed by immunoblotting with anti-cofilin antibody(MAB22) and Ni-NTA HRP. Immunoblot analysis was car-ried out as described previously [3].

Expression of ribosomal S18 protein in COS-7 cells, andimmunoprecipitation

Cloned cDNA of ribosomal S18 protein was inserted intop3XFLAG-myc-CMV-26 vector and transfected into COS-7cells with Effectene (Qiagen). The cells were then washedwith Tris-buffered saline and solubilized with RIPA as pre-viously described [14]. The supernatant was incubated withanti-FLAG antibody-coated protein A/G resin (CytoSignalCo., Irvine, CA) at 4 ◦C for 15 h, and the resin was washedwith RIPA. The materials adsorbed onto the resin were elutedat 80 ◦C for 1 min with SDS-sample buffer containing 20 mMdithiothreitol and subjected to SDS-PAGE followed by im-munoblotting using rabbit anti-cofilin antibody.

Results

Expression of the GST-cofilin fusion protein andpreparation of the recombinant cofilin

We first prepared the GST-cofilin fusion protein as a meansof obtaining recombinant cofilin to biopan the phage displaylibrary. Approximately half of the GST-cofilin fusion proteinexpressed was recovered into a soluble fraction by the sonica-tion method. The soluble fraction was applied to Glutathione-Sepharose 4B affinity column chromatography and then di-gested with PreScission protease to yield the recombinantcofilin. Figure 1A shows the results of SDS-PAGE of thehighly purified recombinant cofilin corresponding to the MW19 kDa, which was confirmed by immunoblotting analysiswith anti-cofilin antibody (MAB22) (data not shown).

Screening for cofilin-binding proteins with pre-made T7select human brain phage display library

We chose the phage display method [19], which has the ad-vantage of less non-specific binding than other methods, toidentify the molecules that interact with cofilin. We also con-sidered the phage-library-inserted cDNA to be more use-ful than library-inserted random primed peptides, becausein preliminary experiments using the latter we cloned nu-merous phage clones whose displayed peptide sequences didnot match those of native proteins. In the present experimentwe used phage display library inserted human brain cDNAs(about 300–800 bp) and repeated the biopanning cycle fourtimes. The purified cofilin was employed as a specific lig-and for elution instead of conventional protein-denaturingagents. After the fourth biopanning, we randomly selected45 independent phage clones and sequenced the inserted cD-NAs to identify the coding protein by means of the BLASTcomputer program. The inserted proteins of 11 of these 45clones were expressed in the correct reading frames andcoded seven different proteins in Table 1. The seven phage

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Fig. 1. SDS-polyacrylamide gel electrophoresis of purified recombinant cofilin and the eluate from ribosomal protein S18-conjugated gel beads. (A) Purifiedrecombinant cofilin that was used for specific elution in the biopanning experiment. (B) Co-elution of ribosomal protein S18 and cofilin. S18 protein-immobilizedNi-NTA-agarose was incubated with cofilin at pH 8.0, extensively washed with buffer (pH 6.3), and eluted with low pH buffer (pH 4.5) twice. Lanes 1 and2 show the first and second eluate, respectively. The recombinant cofilin (left panel) and His-tagged ribosomal protein S18 (right panel) were detected withanti-cofilin antibody and Ni-NTA-horseradish peroxidase (HRP). The positions of the molecular weight markers are indicated on the left.

clones displaying these proteins were amplified, and the samenumbers of phages (1 × 108 pfu) were used to confirm thebinding to cofilin in comparison with the control phage clone.The numbers of the bound and eluted phages are summarizedin Table 2, and the results show that only three clones (CL-6,CL-8, CL-24) could bind to cofilin. CL-6 displayed thestrongest binding among these coding clones (Table 2), andit contained the cDNA of the human ribosomal protein S18

Table 1. Identified inserted proteins expressed on bound phage clones

Name of the clone Protein inserted

CL-6 Ribosomal protein S18

CL-8 Protein 1a

CL-15 Protein 1a

CL-20 Protein 1a

CL-24 Protein 2a

CL-28 Protein 1a

CL-34 Protein 2a

CL-35 Filamin

CL-38 TF II B-related factor

CL-41 80 kDa Golgi protein

CL-42 Peanut-like 2 (PNUTL2)

The inserted DNAs were sequenced, and a search was conducted by usingthe BLAST program. Only the proteins expressed in the correct readingframes are listed.aProtein 1 and protein 2 are under investigation.

coding from the amino acid residue 61 to the final residue,amino acid residue 153. We therefore considered these threeproteins to be the candidates for cofilin-binding proteins andconcluded that ribosomal protein S18 had the highest affinityfor cofilin.

Co-elution of recombinant human cofilin and His-taggedhuman ribosomal protein S18

To confirm the direct interaction between cofilin and riboso-mal protein S18, we co-eluted the recombinant cofilin and

Table 2. Results of further biopanning to determine whether specific bindingoccurred

Name of the clone Number of bound phages listed (pfu)

CL-6 5 × 106 (strong binding)

CL-8 1.2 × 106 (moderate binding)

CL-24 5 × 105 (weak binding)

CL-35 2 × 104 (not bound)

CL-38 5 × 104 (not bound)

CL-41 3 × 104 (not bound)

CL-42 2 × 104 (not bound)

Control 2 × 104 (not bound)

The same numbers of single-cloned phages (1 × 108 pfu) were used forfurther biopanning with a negative control phage. The numbers of boundand eluted phages are summarized.

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Fig. 2. Inhibitory effect of actin on binding between recombinant cofilin and His-tagged ribosomal protein S18. The experimental conditions were the same asin Fig. 1B. Agarose beads were incubated in the absence (lanes 1 and 2) or presence (lanes 3 and 4) of non-muscle actin. Lanes 1 and 3 show the first eluates,and lanes 2 and 4 show the second eluates.

recombinant ribosomal protein S18. To do so, we preparedHis-tagged ribosomal protein S18, which had a molecularweight of 18 kDa on the SDS-PAGE gel. Most of the proteinformed insoluble aggregates in inclusion bodies, and we usedurea to solubilize the aggregates as described in the Materialsand Methods section. The recombinant cofilin bound to theHis-tagged ribosomal protein S18 immobilized to Ni-NTAagarose in the presence of 2 M urea at pH 8.0, and the boundcofilin was eluted with His-tagged ribosomal protein S18by lowering the pH to 4.5, which reduces the interaction be-tween Ni and the His-tag (Fig. 1B). The results confirmed thatthe recombinant cofilin did not bind to the Ni-NTA-agarosedirectly. In addition, to assess the effect of actin on the bind-ing between cofilin and ribosomal protein S18, the recombi-nant cofilin was preincubated for 30 min at 30 ◦C with non-muscle actin equivalent to 10 times the molar concentrationof cofilin and then reacted with the His-tagged ribosomal pro-tein S18-immobilized gel beads under the same conditionsas above. Actin clearly inhibited the binding between the re-combinant cofilin and the recombinant ribosomal protein S18(Fig. 2).

Co-immunoprecipitation of cofilin and ribosomal S18protein

To investigate the possibility of association between cofilinand ribosomal S18 protein occurring in living cells, we con-structed a vector that expresses FLAG-tagged ribosomal S18

protein. The vector was transfected into COS-7 cells, and thecell lysate containing FLAG-tagged S18 protein and cellu-lar cofilin was subjected to immunoprecipitation with anti-FLAG antibody. Both FLAG-S18 protein and cofilin weredetected in the immunoprecipitate by anti-FLAG antibody(Fig. 3, lane 1), whereas no cofilin was detected in a sham ex-periment (lane 2). The amount of cofilin that co-precipitatedwith ribosomal S18 protein was limited, probably becausethe cell lysate contained actin derived from COS-7 cells.Based on the ratio of FLAG-S18 protein to cofilin in thelysate (lane 3) and the immunoprecipitate, 3–5% of the cofilin

Fig. 3. Co-immunoprecipitation of cofilin with ribosomal S18 protein.FLAG-tagged ribosomal protein S18 was expressed in COS-7 cells, andimmunoprecipitation was performed using anti-FLAG antibody (lane 1) ornormal mouse IgG (sham control, lane 2). Cell lysate before immunoprecip-itation contained both cofilin and the FLAG-ribosomal protein S18 (lane 3).The electrophoretic positions of the proteins are indicated. In this pattern,ribosomal S18 protein yielded a band at 23 kDa because it had FLAG(X3)and myc tags. The large amount of IgG light chain stained non-specifically.

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associated with the ribosomal protein. These results suggestsignificant association between cofilin and ribosomal S18protein under physiological conditions.

Discussion

In this study we used a phage display library to search formolecules that interact with cofilin (non-muscle type, cofilin1). The results revealed that ribosomal protein S18 stronglybinds to cofilin, and that the binding is inhibited by actin.The interaction between cofilin and ribosomal S18 proteinwas confirmed in living cells by immunoprecipitation.

Ribosomes are protein-synthesizing organelles that consistof 40S and 60S subunits in eukaryotes, and ribosomal proteinS18 is one of the components of the 40S ribosomal subunit.It has been reported that the protein synthesizing machinerycontaining mRNAs, ribosomes, initiation factors, and elonga-tion factors is associated with the actin cytoskeleton [20, 21].For example, (1) it has been found that some of the elonga-tion factor 2 (EF-2), ribosomes, and initiation factor 2 (IF-2)are co-localized with some of the actin microfilament bun-dles in mouse embryo fibroblasts [22, 23], (2) that elongationfactor 1α (EF-1α), which is also involved in protein synthe-sis, is associated with the actin cytoskeleton in Dictyosteliumand adenocarcinoma cells [24–26]; and (3) that ribosomalproteins L4 and S6 are associated with cytoskeleton-boundpolysomes in HepG2 cells [27]. In addition, evidence has re-cently been found for linking of signaling between the phos-phorylation of a component of protein synthesis and cofilin.The above-mentioned EF-1α is a novel substrate of Rho-associated kinase (Rho-kinase)/ROK/ROCK, a downstreameffector of Rho [28], and phosphorylation of EF-1α by ROCKhas been shown to decrease F-actin-bundling activity. ROCKalso phosphorylates and activates LIM-kinases, resulting inthe phosphorylation of cofilin [29], and thus the Rho/ROCKsignaling pathway may not only be involved in cytoskele-tal organization but in protein synthesis associated with theactin cytoskeleton as well. In this regard, the results in thisstudy that showing a component of the small subunit of ri-bosomes, ribosomal protein S18, associates with the actin-binding protein cofilin provides a clue to the complicatedmechanisms of cytoskeletal organization and protein synthe-sis. Cofilin tends to form oligomers [30], and oligomers ofcofilin may be more suitable for interaction with the riboso-mal protein in the translation complex. One end of the cofilinoligomer might interact with actin and the other with S18 pro-tein to link protein synthesis machinery with the cytoskele-ton. There are three types of ribosomes, microsome-boundribosomes, cytoskeleton-bound polysomes, and cytosolicfree ribosomes, and an electron microscopic study should beperformed to determine the precise intracellular distributionof cofilin.

On the other hand, very recently cofilin has been reportedto interact directly with various proteins including the α sub-unit of Na,K-ATPase [31], 14-3-3 protein ζ [32], and triose-phosphate isomerase [33]. All the above and the fact thatcofilin can bind a signaling phospholipid, phosphatidylinos-itol 4,5-bisphosphate [5], indicate that cofilin has multiplefunctions, and further studies should be conducted to clarifythe roles of cofilin in greater detail.

Acknowledgements

This study was supported in part by research grants providedby the Japan Health Sciences Foundation, the Ministry ofEducation, Science, Sports, and Culture, and the Ministry ofEnvironment of Japan.

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