As the primary effector cells in the innate immune system,
phagocytes such as neutrophils and macrophages contribute
to host defense by engulfing and destroying invading mi-
crobes.1)Various actin-binding proteins have been implicated
in the regulation of actin–cytoskeleton interactions, essential
for properties integral to phagocytic cell function, including
chemotaxis, phagocytosis, and degranulation.2,3)As de-
scribed previously,4)we have identified an immune cell-spe-
cific actin-binding protein p57 that has significant homology
with coronin, an actin-binding protein of Dictyostelium dis-
coideum. Coronin plays crucial roles in various cell func-
tions, including cell locomotion, phagocytosis, and cytokine-
sis of Dictyostelium.5—7)Homologous proteins to coronin
have been identified in many eukaryotes from yeast to
human,8—17)and are assigned an important role in cell motil-
ity.18)Recent reports have suggested that p57 participates as
well in the formation of phagosome and its maturation in
neutrophils and macrophages.19—21)
Most coronin protein family members, including p57, con-
sist of 450—500 amino acid residues and share common
structural features, five WD (tryptophan-aspartic acid) re-
peats located at the center of the molecule and a coiled-coil
motif containing a leucine zipper4)at the C-terminus.8)These
motifs are known to mediate protein–protein interac-
tions,22,23)but their specific function in the coronin family of
actin-binding proteins has not been elucidated. Since coronin
has no significant homology with other actin-binding pro-
teins, the region(s) responsible for the binding to actin re-
mains undetermined. Recently, two groups have reported pu-
tative actin binding regions in coronin family members of
two distinct species.8,13)In Crn1p, a coronin in budding yeast,
the N-terminal part containing WD repeats, has been shown
to have actin-binding activity.8)In Xcoronin, a coronin homo-
logue in Xenopus oocytes, the deletion of N-terminal 63
amino acids or of C-terminal 65 amino acids reduced its
actin-binding activity, although the WD repeats themselves
were not directly involved in the binding to actin.13)Thus,
there is an argument concerning the region(s) responsible for
actin binding of the coronin family of proteins.
To identify the region(s) responsible for actin binding of
p57, we prepared various mutants of p57 in E. coli and as-
sessed their ability to bind to actin by two approaches. We
evaluated their abilities to co-sediment with F-actin after
actin polymerization, and to co-localize with actin filaments
in cells after transfection with the plasmid constructs for the
mutant. The results obtained suggest that there are at least
two regions responsible for actin binding of p57.
MATERIALS AND METHODS
Expression of Glutathione S-Transferase (GST) Fusion
Proteins of Full-Length, and Deletion Mutants of p57
Full-length and partial coding sequences of p57 were ex-
pressed in E. coli as GST fusion proteins using the vector
pGEX-5X-1 (Pharmacia, Uppsala, Sweden). Expression
plasmids pGEX-p57 (full-length), pGEX-p571—371(Met-1 to
Asp-371), and pGEX-p57372—461(Pro-372 to Lys-461) (Fig.
1), were prepared by restriction endonuclease digestion and
Biol. Pharm. Bull. 26(4) 409—416 (2003)409
* To whom correspondence should be addressed.e-mail: email@example.com© 2003 Pharmaceutical Society of Japan
Two Regions Responsible for the Actin Binding of p57, a Mammalian
Coronin Family Actin-Binding Protein
Teruaki OKU,aSaotomo ITOH,aMasamitsu OKANO,aAkiko SUZUKI,aKensuke SUZUKI,b
Shizuo NAKAJIN,aTsutomu TSUJI,*,aWilliam Michael NAUSEEF,cand Satoshi TOYOSHIMAd
aHoshi University School of Pharmacy and Pharmaceutical Sciences; 2–4–41 Ebara, Shinagawa-ku, Tokyo 142–8501,
Japan: bPharmaceutical Frontier Research Laboratories, Japan Tobacco, Inc.; 1–13–2 Fukuura, Kanazawa-ku, Yokohama
236–0004, Japan: cThe Inflammation Program and Department of Medicine, University of Iowa and Veterans Affairs
Medical Center; Iowa City, Iowa 52242, U.S.A.: and dPharmaceuticals and Medical Devices Evaluation Center, National
Institute of Health Science; 3–8–21 Toranomon, Minato-ku, Tokyo 105–8409, Japan.
Received August 22, 2002; accepted December 17, 2002
The actin-binding protein p57, a member of the coronin protein family, is expressed in a variety of immune
cells. It has five WD repeats and a coiled-coil motif containing a leucine zipper, both of which are known to medi-
ate protein–protein interactions. In order to identify the precise actin-binding regions in p57, and to assess the
contribution of these structural motifs, we prepared various truncated p57 as fusion proteins with glutathione
S-transferase (GST) and examined their actin-binding activity. A co-sedimentation assay demonstrated that
p571—371(C-terminal truncated p57) had the ability to bind F-actin, but p57372—461(a fragment containing the
coiled-coil motif) did not. A segment consisting of the N-terminal 34 amino acids of p57 (p571—34) was found to
bind to F-actin in the co-sedimentation assay. Furthermore, fluorescence microscopic observation showed that
p571—34was co-localized with F-actin in COS-1 cells after the transfection with the p571—34construct. Deletion of
10KFRHVF15, a sequence conserved among coronin-related proteins, from p571—34abolished its actin-binding ac-
tivity, suggesting that this sequence with basic and hydrophobic amino acids is crucial for p57 to bind to F-actin.
However, the N-terminal deletion mutant p5763—461retained the binding ability to F-actin. This result suggests
the presence of a second actin-binding region. Further deletion analysis revealed that p57111—204, which includes
the second and third WD repeats, also exhibited weak actin-binding activity in the co-sedimentation assay. Taken
together, these data strongly suggest that at least two regions within Met-1 to Asp-34 and Ile-111 to Glu-204 of
p57 are responsible for its binding to the actin cytoskeleton.
actin-binding protein; coronin; cytoskeleton; WD repeats; leucine zipper
adapter ligation.4)Briefly, the construct for the expression of
p57 was prepared by digestion of the full-length cDNA of
p57 with PmaCI and SacI, and was ligated with both adapter
I and adapter II (adapter I, upper strand 5?-GAT CCG AAT
GAG CCG GCA GGT GGT CCG CTC CAG CAA GTT
CCG CCA C-3? and lower strand 5?-GTG GCG GAA CTT
GCT GGA GCG GAC CAC CTG CCG GCT CAT TCG-3?;
adapter II, upper strand 5?-CCA GAA GCG CTT GGA CAG
GCT GGA GGA GAC AGT CCA GGC CAA GTA GG-3?
and lower strand 5?-TCG ACC TAC TTG GCC TGG ACT
GTC TCC TCC AGC CTG TCC AAG CGC TTC TGG
AGC T-3?). The construct for the expression of p571—371was
prepared by digestion of the full-length cDNA with PmaCI
and BglI, and ligated with adapter I and adapter III (adapter
III, upper strand 5?-CGG CTG AGG AGT GGC TGG CTG
GGG GGT CGG GAT GCT GGG TAG G-3? and lower
strand 5?-AGT GCC GAC TCC TCA CCG ACC CCC CAG
CCC TAC GAC CCA TCC AGC T-3?). The construct for the
expression of p57373—461was prepared by digestion of the
full-length cDNA with BglI and SacI, and ligated with
adapter IV and adapter II (adapter IV, upper strand 5?-GAT
CCC TGC CCT CA-3? and lower strand 5?-GGA CGG G-
3?). The ligated fragments and adapters were subcloned into
pGEX-5X-1, that had been digested with BamHI and SalI.
The expression plasmids of pGEX-p571—71(Met-1 to Asp-
71) and pGEX-p571—34(Met-1 to Thr-34) (Fig. 1), were gen-
erated from pGEX-p571—371by introducing stop codons
using a QuikChangeTMSite-directed mutagenesis kit (Strata-
gene, La Jolla, CA, U.S.A.). The sequences of mutagenic
primers are as follows (the substituted bases are underlined):
K72Z, sense primer 5?-GGC AAG ACT GGA CGT GTG
GAC TAG AAT GCG CCC ACG GTC TGT GG-3? and anti-
sense primer 5?-CCA CAG ACC GTG GGC GCA TTC
TAG TCC ACA CGT CCA GTC TTG CC-3?; and W35Z,
sense primer 5?-CGT CTC ACA GAC CAC CTA GGA
CAG TGG CTT CTG TGC-3? and anti-sense primer 5?-GCA
CAG AAG CCA CTG TCC TAG GTG GTC TGT GAG
ACG-3?. Deletion of amino acid residues 10 to 15 from
p571—34was also performed by a QuikChange Site-directed
mutagenesis kit. The primers used were: D(10—15), sense
primer 5?-G GTG GTC CGC TCC AGC GGA CAG CCG
GCC AAG G-3? and anti-sense primer 5?-C CTT GGC CGG
CTG TCC GCT GGA GCG GAC CAC C-3?.
The other deletions of p57, pGEX-p5763—461(Pro-63 to
Lys-461), pGEX-p5763—127(Pro-63 to Leu-127), pGEX-
p57111—204(Ile-111 to Glu-204), pGEX-p57205—296(Pro-205
to Glu-296), and pGEX-p57297—429(Ile-297 to Ala-429) (Fig.
1), were obtained by polymerase chain reaction (PCR), fol-
lowed by digestion and subcloning in expression vector
pGEX-5X-1. The amplified DNA fragments were digested
with BamHI and EcoRI, then cloned into pGEX-5X-1. The
primers used are as follows (restriction enzyme sites are un-
derlined): BamHI-p5763—, 5?-GG GGG ATC CCC CTG
GGC AAG ACT GGA CGT GTG G-3? and EcoRI-HindIII-
p57461—reverse, 5?-GGG GAA TTC AAG CTT GGG GCT
CTA CTT GGC CTG G-3? for p5763—461; BamHI-hp5763—
and EcoRI-p57127—reverse, 5?-GGG GAA TTC CAG GGT
GAC GAC GGG CTC CCG CAG G-3? for p5763—127;
BamHI-p57111—, 5?-GGG GGG ATC CCC ATC CCG GAT
GGG GGC CTG AT-3? and EcoRI-p57204—reverse, 5?-GGG
GAA TTC CTC GAT GAT GCG CAC GCG CT-3? for
p57111—204; BamHI-p57205—, 5?-GGG GGG ATC CCC CCC
CGC AAA GGC ACT GTC GT-3? and EcoRI-p57296—re-
verse, GGG GAA TTC CTC AAA GTA CCG GAT TGA GC
for p57205—296; and BamHI-p57297—, GGG GGG ATC CCC
ATC ACT TCC GAG GCC CCT TT and EcoRI-p57429—re-
verse, GGG GAA TTC CCT CCT CCA GCC GAG ACA
CG for p57297—429.
The pGEX-p5763—299was generated from pGEX-p5763—461
by introducing stop codons. The primers used were: E300Z,
sense primer 5?-GGT ACT TTG AGA TCA CTT CCT AGG
CCC CTT TCC TGC ACT ATC-3? and antisense primer 5?-
GAT AGT GCA GGA AAG GGG CCT AGG AAG TGA
TCT CAA AGT ACC-3?.
Expression and Purification of GST Fusion Proteins
E. coli JM109 cells, transformed with each plasmid de-
scribed above, were cultured overnight in 6ml of Luria-
Bertani (LB) medium (10g/l of tryptone, 5g/l of yeast ex-
tract, 10g/l of NaCl, pH 7.0) at 37°C, diluted to 500ml with
fresh Terrific Broth (TB; 12g/l of tryptone, 24g/l of yeast ex-
tract, 0.8% of glycerol and 1.25mM of potassium phosphate
buffer, pH 6.5), and grown for 48h at 20°C. Fusion proteins
410Vol. 26, No. 4
The upper panel indicates the schematic representation of p57. Hatched boxes indicate WD repeats and a shaded box indicates a coiled-coil motif.
Structures of p57 and its Deletions
were induced by overnight culture of the bacteria with
0.5mM isopropylthio-b-D-galactoside. After the culture, the
bacteria were pelleted, suspended in a sonication buffer
(50mM Tris–HCl, pH 8.0, 150mM NaCl, 1mM EDTA), and
incubated with 1.0mg/ml of lysozyme for 20min on ice. The
suspensions were sonicated and centrifuged at 10000?g for
30min. Then, the supernatants were incubated with 0.25ml
of glutathione-Sepharose beads (Pharmacia) overnight at
4°C. The beads were washed three times with a sonication
buffer. The bound GST fusion proteins were eluted with
50mM reduced glutathione in 50mM Tris–HCl buffer, pH
Actin Co-sedimentation Assay
tion assay was performed to investigate the ability of fusion
proteins to bind actin. Each fusion protein, with or without
G-actin (30mg, Sigma, St. Louis, MO, U.S.A.) was incubated
in F-actin buffer (20mM Tris–HCl, pH 8.0, 160mM KCl, and
0.2mM ATP) for 1.5h at 25°C. After the incubation, the re-
action mixtures were ultracentrifuged at 198000?g for 1.5h
at 4°C. The reaction mixture before ultracentrifugation is re-
ferred to as the total reaction mixture in the text. The super-
natants, pellets, and total reaction mixtures were resolved by
sodium dodecyl sulfate (SDS)-polyacrylamide gel elec-
trophoresis (PAGE) (10% gel), and gels were stained with
Coomassie brilliant blue (CBB). To confirm the presence of
fusion proteins, immunoblot analysis using anti-p57 poly-
clonal antibody4)or anti-p57 monoclonal antibody9)was also
Expression Plasmids of p57 and Its Deletions
struct plasmids for the expression of p57 in mammalian
cells, the DNA fragment encoding full-length p57 was ampli-
fied by PCR. The primers used were (restriction enzyme sites
are underlined): 5?-GGG GAA TTC AGA ATG AGC CGG
CAG GTG G-3? and 5?-GGG GAA TTC AAG CTT GGG
GCT CTA CTT GGC CTG G-3?. The amplified DNA frag-
ments were digested with EcoRI and cloned into pEF1/Myc-
His A (Invitrogen, Carlsbad, CA, U.S.A.). Then, TAG stop
codon was replaced by GCG to generate a fusion protein
with Myc-His tag using a QuikChange Site-directed mutage-
nesis kit (Stratagene). The primers used were: 5?-GGA GAC
AGT CCA GGC CAA GGC GAG CCC CAA GCT TGA
ATT CTG C-3? and 5?-GCA GAA TTC AAG CTT GGG
GCT CGC CTT GGC CTG GAC TGT CTC C-3?. To gener-
ate the expression plasmid pcDNA3.1/p57-V5-His for full-
length p57, which expresses p57 fused with viral epitope V5
and 6xHis tag, the DNA fragments encoding p57 were sub-
cloned into pcDNA3.1/V5-His A (Invitrogen).
For expression of p571—371and p57372—461, corresponding
DNA fragments were subcloned into pcDNA3.1/V5His A
(pcDNA3.1/p571—371and pcDNA3.1/p57372—461). Expression
plasmids encoding p571—34and p571—71were generated
from pcDNA3.1/p57-V5-His by introducing stop codons
(pcDNA3.1/p571—34and pcDNA3.1/p571—71, respectively).
The p57111—204was expressed as a fusion protein with an
N-terminal XpressTMtag (Invitrogen) in order to facilitate de-
tection of the expressed protein. The cDNA fragment of
p57111—204was prepared by digestion of pGEX-p57111—204
with BamHI and EcoRI, and subcloned into pcDNA3.1/His
Cell Culture and Transfection
cell line) cells (American Type Culture Collection) were
An actin co-sedimenta-
COS-1 (monkey kidney
grown in Iscove’s Modified Dulbecco’s Medium (IMDM,
GIBCO) supplemented with 10% heat inactivated FCS
(Sanko Junyaku Co., Ltd., Tokyo), penicillin (50U/ml), and
streptomycin (50mg/ml) under the standard cell culture con-
dition (37°C, humidified 5% CO2in air).
Plasmids encoding p57 and its deletions were introduced
into COS-1 cells by electroporation. Briefly, COS-1 cells
(4?106cells/300ml) in phosphate buffered saline (PBS) were
mixed with 10mg plasmid DNA and placed in a 0.4cm elec-
trode gap electroporation cuvette (Bio-Rad, Hercules, CA,
U.S.A.). Cells were subjected to a single pulse of 750V/cm
at a capacitance setting of 975 microfarads, transferred to
10ml of IMDM containing 10% FCS, and maintained for
48h under standard cell culture conditions.
were cultured on a Lab-Tek II Chamber Slide (Nalge Nunc,
Rochester, NY, U.S.A.) for 10min, fixed with 4% neutral
buffered formaldehyde (Kanto Chemicals Co., Ltd., Tokyo,
Japan), and then permeabilized by treatment with 0.2% Tri-
ton X-100 in PBS for 10min at room temperature. Permeabi-
lized cells were rinsed with PBS and incubated with a mono-
clonal antibody to p57, N7,20)which recognizes the C-termi-
nal region of p57, at 5mg/ml in PBS containing 3% BSA for
1h. For the detection of N-terminal fragments of p57, a rab-
bit polyclonal antibody, which recognizes N-terminal 20
amino acids, was used. For the detection of Xpress-tagged
fusion protein, an anti-Xpress antibody (1:5000, Invitrogen)
was used. After being washed with PBS, the cells were incu-
bated with FITC-labeled goat anti-mouse IgG (KPL,
Gaithersburg, MD, U.S.A.) or FITC-labeled goat anti-rabbit
IgG (KPL) for 30min. F-actin was detected by using rho-
damine-conjugated phalloidin (Sigma). Fluorescently labeled
cells were washed three times with PBS and then mounted
with 2.3% 1,4-diazabicycle-2,2,2-octane (Sigma) containing
glycerol on slide glass. Samples were analyzed by using a
confocal laser microscope (Radiance 2100 laser scanning
system, Bio-Rad, Hercules, CA, U.S.A.).
The transfected cells
Identification of Region(s) in p57 Responsible for Actin
To identify the region(s) of p57 responsible for
actin binding, we examined the abilities of GST-fusion pro-
teins with full-length and several truncated forms of p57 to
co-sediment with F-actin in vitro. GST-p57 (a fusion protein
of full-length p57 with GST) and GST-p571—371(a fusion
protein containing five WD repeats but lacking a C-terminal
coiled-coil motif) (Fig. 1) co-precipitated with F-actin (Figs.
2A, B). Although some impurities were present in the GST-
p57 preparation, the identity of GST-p57 was confirmed by
immunoblotting using an anti-p57 antibody, and the impuri-
ties remained in the supernatant after ultracentrifugation. In
contrast, GST-p57372—461(a fusion protein containing a C-ter-
minal fragment with a coiled-coil motif) (Fig. 1) did not co-
sediment with F-actin (Fig. 2C). These results suggested that
the region responsible for p57 binding to actin was located in
the N-terminal part of p57 that includes WD repeats, but
does not include the coiled-coil motif at the C-terminus. We
then prepared two additional truncated proteins containing
the N-terminus of p57 but lacking the WD repeats, GST-
p571—71and GST-p571—34(Fig. 1). These deletion mutants
co-precipitated with F-actin (Figs. 3B, C), suggesting that the
N-terminal 34 amino acids of p57 possessed the capacity to
bind F-actin. Moreover, the deletion of six amino acid
residues, 10KFRHVF15, from p571—34(GST-p571—34;D10—15)
abolished the actin-binding ability (Fig. 3D), implicating a
sequence rich in basic and hydrophobic amino acids as es-
sential for actin binding of p571—34.
Further Analysis of Region(s) in p57 Responsible for
p57 molecules containing WD repeats in actin binding, we
analyzed the F-actin binding capacity of six truncated fusion
proteins of p57 that lack the putative N-terminal actin-bind-
ing sequence, namely GST-p5763—461, GST-p5763—299, GST-
p57297—429, GST-p5763—127, GST-p57111—204and GST-p57205—
296(Fig. 1). GST-p5763—461, which lacks the N-terminal 62
amino acids, co-precipitated with F-actin (Fig. 4A), suggest-
To evaluate the role of the central region of
412 Vol. 26, No. 4
The supernanant (S) and the precipitate (P) after ultracentrifugation, as well as the total reaction mixture (T), were separated on SDS-polyacrylamide gel electrophoresis. Two
sets of samples in the presence (?) or absence (?) of G-actin were assayed.
Co-sedimentation Assay of GST-p57 (A), GST-p571—371(B), and GST-p57372—461(C) Fusion Proteins with F-Actin
GST-p571—34;D10—15(D) Fusion Proteins with F-Actin
After F-actin was sedimented by ultracentrifugation, each fraction was subjected to SDS-polyacrylamide gel electrophoresis; T, total reaction mixture; S, ultracentrifuged super-
natant; P, ultracentrifuged precipitate. Each fusion protein, with (?) or without (?) G-actin, was incubated in F-actin buffer for co-sedimentation assay.
Amino Acid Sequences of GST-p571—34and GST-p571—34;D10—15(A) and the Co-sedimentation Assay of GST-p571—34(B), GST-p571—71(C), and
ing the presence of a second region responsible for actin-
binding of p57 in addition to the N-terminal sequence
(p571—34). Furthermore, GST-p5763—299and GST-p57111—204
also co-precipitated with F-actin (Figs. 4B, C), whereas
GST-p5763—127, GST-p57205—296, and GST-p57297—429did not
(Figs. 4D—F). Based on these results on the differential rep-
resentation of the WD repeats among these mutant forms of
p57 (Fig. 1), it is strongly suggested that p57 contains at least
two regions that mediate actin binding; i.e., the N-terminal
region (p571—34) and the central region containing the second
and third WD repeats (p57111—204).
Binding of p57 Deletions to Actin in COS-1 Cells
extend our analysis of the putative actin-binding regions of
p57 to intact cells, we expressed in COS-1 cells full-length
and deletion mutants of p57 (p571—371, p57372—461, p571—71,
p571—34and p57111—204). Expressed forms of p57 constructs
were visualized in transiently transfected cells using im-
munofluorescence microscopy (Fig. 5). Full-length p57 and
its deletions that exhibited actin-binding activity in vitro,
including p571—371, p571—71, p571—34and p57111—204, were
localized in cortical F-actin rich regions. In contrast,
p57372—461, which did not exhibit actin-binding activity in
vitro, was diffusely distributed in cytosol and did not show
cortical localization. These results are in good agreement
with those of the co-sedimentation assay in vitro.
p57297—429(F) Fusion Proteins with F-Actin
After F-actin was sedimented by ultracentrifutgation, each fraction was subjected to SDS-polyacrylamide gel electrophoresis; T, total reaction mixture; S, ultracentrifuged super-
natant, P, ultracentrifuged precipitate. Each fusion protein, with (?) or without (?) G-actin was incubated in F-actin buffer for co-sedimentation assay.
Co-sedimentation Assay of GST-p5763—461(A), GST-p5763—299(B), GST-p57111—204(C), GST-p5763—127(D), GST-p57205—296(E), and GST-
Our previous study demonstrated that the transient pe-
riphagosomal association of p57 plays an essential role in the
maturation of phagolysosome in phagocytes.24)Considering
that reorganization of the actin cytoskeleton is required for
phagocytosis, the interaction between p57 and F-actin is one
of the key steps during phagocytic processes. In this study,
we have demonstrated that a p57 molecule possesses two re-
gions responsible for its binding to F-actin. One is located in
a short stretch of N-terminal 34 amino acids, and the other is
located in the central region of the molecule containing WD
repeats. Our results are partly consistent with a recent report
414Vol. 26, No. 4
Expression plasmids pcDNA3.1/p57V5His (p57), pcDNA3.1/p571—371(p571—371), cDNA3.1/p57372—461(p57372—461), pcDNA3.1/p571—71(p571—71), pcDNA3.1/p571—34(p571—34)
and pcDNA3.1/p57111—204(p57111—204) were transiently expressed in COS-1 cells. Deletion mutants of p57 and F-actin were stained by anti-p57 antibodies or anti-Xpress antibody,
followed by secondary antibody conjugated with FITC and by rhodamine-labelled phalloidin, respectively, and they were observed by fluorescence microscopy. Experiments were
performed more than three times, and representative data are shown. Scale bars, 20mm.
Intracellular Localization of p57 and Its Deletions Expressed in COS-1 Cells
by Goode et al.8)They demonstrated that the actin-binding
region of a yeast homologue of coronin, Crn1p, was located
in the N-terminal part containing WD repeats, but not in its
C-terminal region with a coiled-coil motif. By contrast,
Mishima and Nishida13)reported that the deletion of N-ter-
minal 63 amino acids or of C-terminal 65 amino acids from
Xcoronin, a Xenopus homologue of coronin, reduces, but
does not eliminate, its actin-binding activity. The reduction
of actin-binding activity in Xcoronin lacking its C-terminal
coiled-coil motif is not consistent with the results from p57
and Crn1p. This apparent conflict may be reflected by the
differences among coronin species, and coronin homologues
do not necessarily share common characteristics in the actin-
A co-sedimentation assay and fluorescent microscopic ob-
servation strongly suggest that the N-terminal fragment of
p57 with 34 amino acids (p571—34) was bound to F-actin
(Figs. 3, 5). Deletion of the sequence 10KFRHVF15abolished
the actin-binding activity of p571—34, suggesting that the
stretch of these six amino acids is essential for actin binding
of the N-terminal fragment. Several known actin-binding
proteins possess regions rich in basic amino acid residues
that contribute to actin binding through their interaction with
acidic amino acid clusters contained in actin.25—28)This clus-
ter of basic amino acid residues may be responsible not only
for actin binding by p57, but may also represent a motif com-
mon to related proteins, as the KXRHXX (where X repre-
sents a hydrophobic amino acid) is conserved in many other
coronin-family actin-binding proteins (Fig. 6).
In addition to p571—34, we identified a second region of
p57 that exhibited actin-binding activity with Ile-111 to Glu-
204 (Fig. 4). p57111—204includes two out of the five WD re-
peats present in p57. The WD motifs were found in a variety
of proteins and are known to participate in various critical
cell functions, including signal transduction, gene regulation,
vesicular trafficking, regulation of the cytoskeleton and the
cell cycle.29)Regions in proteins with WD repeats are
thought to organize into a b-propeller structure and to pro-
mote protein–protein interactions.30,31)However, the WD re-
peats in p57 do not seem to mediate its actin-binding, but
more likely serve another function, perhaps providing a scaf-
fold for the responsible regions of p57 to bind actin and/or
link actin to another protein. In the case of p57111—204there
are WD repeats sufficient for only one blade of a b-propeller,
thus it is inadequate to fold properly into a functional b-pro-
peller structure.30)Therefore, the actin binding of p57111—204
more likely depends on a structure other than the WD re-
peats. The determination of the second sequence responsible
for the binding of p57111—204to F-actin awaits further investi-
The presence of two actin-binding regions in a p57 mole-
cule would increase its affinity for F-actin, as multiple actin-
binding regions in several actin-binding proteins are believed
to augment their interaction with actin. This hypothesis is
supported by the result that the interaction of intact p57 with
F-actin seems to be stronger than those of deletions contain-
ing only one of two actin-binding regions (Figs. 2, 4).
Ministry of Education, Culture, Sports, Science and Technol-
ogy of Japan, the Ministry of Health, Labour and Welfare of
Japan, and the U.S. Public Health Service (RO1AI34879).
This work was supported by the
1) Nauseef W. M., Clark R. A., “Principles and Practice of Infectious
Diseases,” 5th ed., Chap. 8, eds. by Mandell G. L., Bennett J. E., Dolin
R., Churchill Livingstone, Philadelphia, 2000, pp. 89—111.
May R. C., Machesky L. M., J. Cell Sci., 114, 1061—1077 (2001).
Noegel A. A., Schleicher M., J. Cell Sci., 113, 759—766 (2000).
Suzuki K., Nishihata J., Arai Y., Honma N., Yamamoto K., Irimura T.,
Toyoshima S., FEBS Lett., 364, 283—288 (1995).
de Hostos E. L., Bradtke B., Lottspeich F., Guggenheim R., Gerisch
G., EMBO J., 10, 4097—4104 (1991).
de Hostos E. L, Rehfuess C., Bradtke B., Waddell D., R., Albrecht R.,
Murphy J., Gerisch G., J. Cell Biol., 120, 163—173 (1993).
Maniak M., Rauchenberger R., Albrecht R., Murphy J., Gerisch G.,
Cell, 83, 915—924 (1995).
Goode B. L., Wong J. J., Butty A. C., Peter M., McCormack A. L.,
Yates J. R., Drubin D. G., Barnes G., J. Cell Biol., 144, 83—98 (1999).
Tardieux I., Liu X., Poupel O., Parzy D., Dehoux P., Langsley G.,
FEBS Lett., 441, 251—256 (1998).
Bricheux G., Coffe G., Bayle D., Brugerolle G., Eur. J. Cell Biol., 79,
Rappleye C. A., Paredez A. R., Smith C. W., McDonald K. L., Aroian
R. V., Genes Dev., 13, 2838—2851 (1999).
Terasaki A. G., Ohnuma M., Mabuchi I., J. Biochem. (Tokyo), 122,
Mishima M., Nishida E., J. Cell Sci., 112, 2833—2842 (1999).
Iizaka M., Han H. J., Akashi H., Furukawa Y., Nakajima Y., Sugano
S., Ogawa M., Nakamura Y., Cytogenet. Cell Genet., 88, 221—224
Nakamura T., Takeuchi K., Muraoka S., Takezoe H., Takahashi N.,
Mori N., J. Biol. Chem., 274, 13322—13327 (1999).
Parente J. A., Jr., Chen X., Zhou C., Petropoulos A. C., Chew C. S., J.
Biol. Chem., 274, 3017—3025 (1999).
Okumura M., Kung C., Wong S., Rodgers M., Thomas M. L., DNA
Cell Biol., 17, 779—787 (1998).
April 2003 415
Asterisks indicate basic amino acid residues conserved in the coronin family of actin-binding proteins.
Alignment of the Putative Actin-binding Region at the N-terminus of p57 and the Corresponding Regions of Other Coronin Family Actin-Binding
de Hostos E. L., Trends Cell Biol., 9, 345—350 (1999).
Grogan A., Reeves E., Keep N., Wientjes F., Totty N. F., Burlingame
A. L., Hsuan J. J., Segal A. W., J. Cell Sci., 110, 3071—3081 (1997).
Allen L. A., DeLeo F. R., Gallois A., Toyoshima S., Suzuki K., Nau-
seef W. M., Blood, 93, 3521—3530 (1999).
Ferrari G., Langen H., Naito M., Pieters J., Cell, 97, 435—447 (1999).
Cohen D. R., Curran T., Mol. Cell. Biol., 8, 2063—2069 (1988).
Vinson C. R., Hai T. W., Boyd S., Genes. Dev., 7, 1047—1058 (1993).
Itoh S., Suzuki K., Nishihata J., Iwasa M., Oku T., Nakajin S., Nauseef
W. N., Toyoshima S., Biol. Pharm. Bull., 25, 837—844 (2002).
Lappalainen P., Fedorov E. V, Fedorov A. A, Almo S. C., Drubin D. G.,
EMBO J., 16, 5520—5530 (1997).
Yao L., Janmey P., Frigeri L. G., Han W., Fujita J., Kawakami Y.,
Apgar J. R., Kawakami T., J. Biol. Chem., 274, 19752—19761 (1999).
de Arruda M. V., Bazari H., Wallek M., Matsudaira P., J. Biol. Chem.,
267, 13079—13085 (1992).
Jongstra-Bilen J., Janmey P. A., Hartwig J. H, Galea S., Jongstra J., J.
Cell Biol., 118, 1443—14453 (1992).
Neer E. J., Schmidt C. J., Nambudripad R., Smith T. F., Nature (Lon-
don), 371, 297—300 (1994).
Sondek J., Bohm A., Lambright D. G., Hamm H. E., Sigler P. B., Na-
ture (London), 379, 369—374 (1996).
Garcia-Higuera I., Fenoglio J., Li Y., Lewis C., Panchenko M. P.,
Reiner O., Smith T. F., Neer E. J., Biochemistry, 35, 13985—13994
416 Vol. 26, No. 4