From the Department of Microbiology, Showa University School of
Pharmaceutical Sciences, Hatanodai 1-5-8, Shinagawa-ku, Tokyo, Japan
and the Department of Biochemistry, McGill University,
Montreal, Quebec H3G 1Y6, Canada
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ABSTRACT |
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The Hic-5 protein is encoded by a transforming
growth factor- hic-5 was isolated as one of transforming growth
factor- The LIM domain is a unique cysteine-rich motif that defines a double
zinc finger structure with a consensus sequence of
(CXXCX16-23HXXCXXCXXCX16-21CXX(D/H/C)) and is found in a variety of proteins with diverse functions and subcellular distributions, including transcription factors, components of adhesion plaques, and the actin-based cytoskeleton (5). The members
of LIM proteins can be classified into five groups as follows: LIM
homeodomain, LIM only protein, LIM kinase, GTPase-activating protein
family, and zyxin family including enigma, paxillin, and Hic-5 (5).
Accumulating evidence demonstrated that the LIM domains serve as an
interface for protein-protein interactions (6), but the function of the
LIM domains in the Hic-5 protein has not yet been determined.
Recent immunocytochemical studies showed that the Hic-5 protein is
localized in focal adhesions in rat fibroblasts (cell strain WFB) and
associates with cell adhesion kinase- Protein tyrosine phosphorylation plays an important role in the
assembly of focal adhesions followed by transmission of extracellular stimuli (13), and protein-tyrosine kinases such as FAK (16), proline-rich kinase 2 (17), and cell adhesion kinase- Screening of Factors That Associate with Hic-5--
The cDNA
library for expression of fusion proteins in yeast was constructed
using mRNA from differentiated mouse myogenic cells, C2C12 (21).
Total RNA was extracted from cells that were cultured for 4 days in a
differentiation medium (Dulbecco's modified Eagle's minimal essential
medium supplemented with 2% horse serum) by the guanidinium/hot phenol
method (1). Poly(A)+ RNA was isolated by (dT)30
latex (OligotechTM-dT30, Takara Shuzo, Co., Kyoto, Japan).
Double-stranded cDNA was synthesized by using the Two-hybrid
cDNA Library Construction Kit (CLONTECH, Palo
Alto, CA), according to the instruction manuals, and cloned into the EcoRI site of the pGAD10 vector
(CLONTECH). As a bait, a cDNA fragment that
encodes the LIM domain of Hic-5 (amino acids 187-444) was ligated into
BamHI site of pGBT9 to express a fusion protein with
GAL4 DNA binding domain. By using the MATCHMAKER two-hybrid system
(CLONTECH), approximately 1 × 105
independent clones were screened, and cDNA clones that interacted with the Hic-5 LIM domains were selected by growth on His-deficient plates. Positive clones were further screened for LacZ expression, and positive clones were then allowed to test their two plasmid dependencies. From 105 his+
transformants, we obtained 16 interacting cDNA clones. These clones were sequenced using Cy5TM AutoReadTM Sequencing Kit
(Amersham Pharmacia Biotech Inc, Little Chalfont, UK) according to the
manufacturer's instructions.
Cell Culture and Transient Transfection of Plasmids--
Mouse
myoblastic cells, C2C12, were cultured in Dulbecco's modified Eagle's
minimal essential medium supplemented with 15% fetal bovine serum and
50 µg/ml kanamycin under a humidified atmosphere of 5%
CO2 in air. Human immortalized fibroblasts, KMST-6 (42), were cultured in Dulbecco's modified Eagle's minimal essential medium
supplemented with 10% fetal bovine serum. Cells were passaged at 1:8
dilution when they became confluent. Cells were transfected with
plasmids using LipofectAMINE PLUS (Life Technologies, Inc.) according
to manufacturer's manuals. Twenty-four hours post-transfection, cells
were harvested and suspended in lysis buffer (20 mM Hepes, pH 7.4, 150 mM NaCl, 1% Triton X-100, 1 mM
phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, 10 µg/ml
aprotinin, 1 mM sodium orthovanadate) and incubated at
4 °C for 30 min. The insoluble material was then removed by
centrifugation for 15 min at 20,000 × g, and the
resultant lysates were analyzed by Western blotting and immunoprecipitation.
Immunoprecipitation and Immunoblotting--
The anti-mouse Hic-5
polyclonal antibody (number 1024) was raised against the recombinant
Hic-5 N-terminal region (amino acids 2-194). The monoclonal
anti-phosphotyrosine antibody (PY20) and the anti-HA antibody (12CA5)
were purchased from Transduction Laboratories (Lexington, KY) and
Boehringer Mannheim, respectively. Immunoprecipitation of the HA-tagged
proteins and the Hic-5 were performed after covalent coupling of
antibodies to protein A- or protein G-beads (Pharmacia Biotech, Inc.,
Uppsala, Sweden) with the chemical cross-linking agent dimethyl
pimelimidate (22). The covalently coupled antibody beads were incubated
for 16 h at 4 °C in the lysates. The immune complexes were
collected by centrifugation, washed with the lysis buffer, and eluted
with SDS sample buffer (50 mM Tris-HCl, pH 6.8, 2% SDS,
0.1% bromphenol blue, 10% glycerol, 0.1 M dithiothreitol).
For immunoblotting, cell lysates and immunoprecipitates were separated
by SDS-PAGE and transferred to nitrocellulose membranes (Hybond C,
Amersham Pharmacia Biotech). The membranes were blocked with bovine
serum albumin and incubated with the anti-Hic-5 or the anti-HA antibody
for 16 h. The primary antibodies were detected with horseradish
peroxidase-conjugated secondary antibodies and a chemiluminescent
detection kit using (ECL, Amersham Pharmacia Biotech).
Plasmid Construction--
To generate Hic-5 deletion mutants
(LIM 1-3, LIM 1-2, LIM 1, and
The Hic-5 cDNA fragment was subcloned into
HindIII/XbaI sites of pKF 19k (Takara Shuzoh,
Co., Kyoto) for site-directed mutagenesis. According to the instruction
manual, the mutagenesis was performed with Takara's Mutan-Super
Express Km Kit. The mutated fragments (mLIM 2 (H293L/C296G) and mLIM 3 (C352G/C355G)) were excised from the pKF 19k vector and subcloned into
the HindIII/XbaI sites of pRc/CMV. An expression
vector for PTP-PEST that was defective in the Pro-2 domain
(HA-
The PEP construct, PEP CL3/pBluescript, was provided by Dr. Kiminori
Hasegawa (Department of Immunology, Tokyo Metropolitan Institute of
Neuroscience, Kodaira, Tokyo, Japan).
To construct a prokaryotic expression vector encoding the GST fusion
protein of PTP-PEST (GST-PEST-185-433) including amino acids 185-433,
the EcoRI fragment cut out from the pGAD10 vector (CLONTECH) containing PTP-PEST cDNA was ligated
into the EcoRI site of pGEX-5X-1 (Amersham Pharmacia
Biotech). Other PTP-PEST GST fusion proteins have been previously
described (24). The GST fusion protein was expressed in
Escherichia coli strain DH5 GST Affinity Matrix Binding Assay--
The fusion proteins were
expressed by incubation of E. coli harboring expression
vectors with 1 mM
isopropyl-D-thiogalactopyranoside for 3 h at
30 °C. The bacteria were collected by centrifugation, lysed by
sonication, and solubilized in phosphate-buffered saline containing 1%
Triton X-100, 1 mM phenylmethylsulfonyl fluoride, 1 µg/ml
aprotinin, and 1 µg/ml leupeptin. The GST fusion proteins were
purified using glutathione-Sepharose beads.
Labeled Hic-5 and paxillin were synthesized in vitro using
rabbit reticulocyte lysate (Promega, Madison, WI) in the presence of
[35S]Met using pRc/S5 (1) and pRc/CMV/paxillin (25) as
templates. In vitro translated products were then incubated
with immobilized GST fusion proteins in the binding buffer (20 mM Tris, pH 8.0, 0.1 M KCl, 0.1 mM
ZnCl2, 10% glycerol, 1% Triton X-100, 5 mM
In Vivo Association of PTP-PEST and Hic-5
Mutants--
Flag-tagged Hic-5 mutants of the LIM domains (mLIM 1, mLIM 2, and mLIM 3) were inserted into pFLAG-CMV-2 vector (Eastman
Kodak Co.) and co-transfected with HA-tagged PTP-PEST into KMST-6 human fibroblasts by calcium phosphate precipitation method. Cells were collected 24 h after transfection, and their association was
analyzed by immunoprecipitation with anti-Flag and Western blotting
with anti-HA as described above.
Immunocytochemistry--
Cells were transfected with Flag-tagged
Hic-5 mutants into KMST-6 cells by calcium phosphate method and fixed
in 3.7% formaldehyde in phosphate-buffered saline 24 h after the
transfection. Cells were permeabilized in 0.2% Triton X-100 in
phosphate-buffered saline, treated with monoclonal anti-Flag antibody
(Sigma) at 4 °C for 1 h, and then treated with fluorescein
isothiocyanate-conjugated anti-mouse IgG. After extensive washing,
cells were visualized under a fluorescent microscope.
Isolation of cDNA Clones Encoding Hic-5-binding
Proteins--
To investigate the molecular basis of Hic-5-mediated
cellular functions, we attempted to isolate interacting proteins of
Hic-5 with the yeast two-hybrid system (26). As previously reported, the Hic-5 levels decreased in the most immortalized cell lines (1), but
mouse myoblastic cells, C2C12, express significant amounts of Hic-5.
The cDNA library was constructed with mRNA from C2C12 cells and
screened using the LIM domain of Hic-5 as a bait. Sequences of 5 clones
out of 16 positives were 100% identical to a part of a previously
cloned cDNA, murine protein-tyrosine phosphatase-PEST (PTP-PEST)
(27-30). The longest cDNA contained amino acids 185-433 and the
shortest one 212-420 of PTP-PEST. It is a ubiquitously expressed
protein-tyrosine phosphatase that is composed of an N-terminal
phosphatase domain and a C-terminal noncatalytic region. The cDNA
fragments isolated from the two-hybrid screening contained a part of a
catalytic domain and proline-rich domains.
Hic-5 and PTP-PEST Associate in Vivo--
To confirm that these
two are associated in mammalian cells, we introduced expression vectors
of HA-tagged PTP-PEST into the C2C12 cells, and we examined their
binding by immunoprecipitation and Western blotting. The N-terminal
HA-tagged PTP-PEST (29) or its mutant (C231S) in which a cysteine
residue in the active center was converted to serine residue was
transiently expressed in C2C12 cells, and then endogenous Hic-5 was
immunoprecipitated with anti-Hic-5 antibody (number 1024). HA-PTP-PEST
or HA-C231S was co-immunoprecipitated with Hic-5 (Fig.
1A), showing their in
vivo association. In the immunoprecipitation using anti-HA antibody (12CA5), Hic-5 was co-immunoprecipitated with HA-PTP-PEST or
HA-C231S (Fig. 1B). Surprisingly, the interaction of Hic-5 to wild type PTP-PEST and to mutant C231S were almost at similar levels. HA-C231S has a point mutation in the catalytic domain that can
stably bind the substrates, but more investigation will be required to
eliminate the possibility that Hic-5 is a substrate of PTP-PEST.
Domains of PTP-PEST for Interaction with Hic-5--
One of the
isolated clones of PTP-PEST encoded the amino acid residues 185-433
encompassing the C-terminal portion of the catalytic domain and the
N-terminal region of the noncatalytic domain that includes Pro-1 and
Pro-2 domains. In the noncatalytic domain, PTP-PEST has five
proline-rich domains (Fig.
2A). These domains have been
shown to provide the interface for interacting with Src homology domain
3-containing signal transduction molecules. In order to identify the
domain responsible for association with Hic-5, we performed GST
affinity matrix binding assay using a series of GST fusions of Pro-rich
domains derived from PTP-PEST (Fig. 2A). The fusion proteins
were immobilized on glutathione beads and incubated with
35S-labeled Hic-5 produced by in vitro
translation. The proteins bound to the beads were eluted, separated by
SDS-PAGE, and exposed to x-ray film. The result demonstrated that the
Hic-5 preferentially binds to the GST-Pro-2 domain, whereas Pro-1, and
Pro-3-5 showed essentially no binding activity (Fig. 2, B
and C). A fusion protein of the Pro-2 domain showed unusual
mobility on SDS-PAGE, but it is generally accepted that Pro-rich
peptides migrate unpredictably. Furthermore, all constructs have been
sequenced, and we are sure that they encode for the proper proteins
(36). Paxillin also bound to the Pro-2 domain of PTP-PEST.
Determination of the LIM Domains Essential for Interaction with
PTP-PEST--
The Hic-5 C-terminal region used as a bait contains four
LIM domains. Because a single LIM domain can act as a protein
association interface (5), we tried to map the LIM domain(s) required
for binding to PTP-PEST. The binding assay was carried out using GST fusion protein of PTP-PEST isolated from two-hybrid screening and
35S-labeled Hic-5 deletion mutants. Since both LIM 1-3 and
LIM 3-4 could bind to PTP-PEST, LIM 1, LIM 2, and LIM 4 are not
necessary for the association (Fig. 3,
A and B). These data indicate that LIM 3 of Hic-5
is important for interaction with PTP-PEST. However, the Hic-5 mutant
containing only LIM 3 could not bind to PTP-PEST (Fig. 3B),
suggesting that LIM 3 alone is not sufficient for the binding. It is
most likely that each LIM may affect the structural stability of
adjacent LIMs or functions cooperatively.
To ascertain the requirement of LIM 3 for binding to PTP-PEST, we
introduced two point mutations in LIM 2 or LIM 3 by site-directed mutagenesis. A single LIM motif is composed of two zinc fingers with
the following consensus sequence:
CX2CX16-23HX2CX2CX2CX16-21CX2(C/H/D). Each zinc finger is folded into its ternary structure chelating a zinc
ion via conserved cysteine, histidine, or aspartate residues, and the
introduction of point mutations in zinc-chelating residues can disrupt
the ternary folding of LIM structure (31). The mutants used here, mLIM
2(H293L/C296G) and mLIM 3(H352G/C355G), contained point mutations at
zinc-chelating residues within both the first and second zinc fingers,
disrupting the structure of an individual LIM domain. We performed the
binding assay using these mutants and a GST fusion Pro-2 domain of
PTP-PEST as a GST matrix (Fig. 3C). In contrast to the mLIM
2 protein that bound to PTP-PEST efficiently, mLIM 3 did not show any
binding activity. This indicates that the LIM 3 domain of Hic-5 is
essential to associate with PTP-PEST. Taken together with the above
results, we concluded that Hic-5 and PTP-PEST associate through LIM 3 domain and Pro-2 domain, respectively, in vitro.
We also examined the specificity of interaction between Hic-5 and
PTP-PEST by comparing to another protein-tyrosine phosphatase, PEP
(32). PEP is a nonreceptor type PTP with similar structure to PTP-PEST,
and we performed in vitro binding assay using a GST fusion
protein of the LIM domains of Hic-5 and 35S-labeled PEP.
LIM domains of Hic-5 failed to bind to PEP (data not shown), suggesting
that these LIM domains have binding selectivity to a certain type of PTPs.
In Vivo Association between Mutants of Hic-5 and PTP-PEST--
The
above results show that LIM 3 of Hic-5 and Pro-2 of PTP-PEST
specifically bind each other in vitro, but their in
vivo association was examined by transfection of each expression
vectors. In Fig. 4A, plasmids
that express HA-tagged full-length PTP-PEST or its Pro-2 deletion
mutant ( Subcellular Localization of the LIM Mutants--
It is reported
that the LIM 3 of paxillin determines its localization to focal
adhesion (38), and subcellular localization of Hic-5 with mutations at
the LIM domains was examined using Flag-tagged Hic-5 expressing either
wild type or mutated LIMs. KMST-6 cells were transfected with
pRc/CMV-based expression vectors, and cells were fixed 24 h later
for immunostaining with anti-Flag antibody (Fig.
5, A-D). The results show
that wild type Hic-5 localized in focal adhesion, but the mutant in LIM
3 completely lost this phenotype (Fig. 5D). Localization of
the mutants at LIM 1 and LIM 2 in focal adhesion seemed weakened (Fig.
5, B and C) compared with that of wild type
Hic-5.
Involvement of PTP-PEST for subcellular localization of Hic-5 was
tested using fibroblasts from PTP-PEST(+/ Hic-5, a member of LIM proteins, has been shown to be involved in
cellular senescence and differentiation processes (2, 3). However, the
molecular mechanisms for its biological effects have been remained
unsolved, whereas its localization in focal adhesion (7) suggested to
us that Hic-5 has some roles in modulating integrin-mediated signal. In
this study, we isolated the Hic-5 interacting factors using a yeast
two-hybrid system and identified PTP-PEST/P19-PTP, which is a member of
the intracellular PTP family, as one of such factors.
Protein tyrosine phosphorylation is one of the most important entities
constituting signal transduction pathways from cell surface to the
nucleus in response to various extracellular stimuli (33). The level of
protein tyrosine phosphorylation increases not only upon stimuli with
soluble factors, such as growth factors, but also upon cell adhesion to
extracellular substrate (34). Many molecules are now known to be
tyrosine-phosphorylated upon cell adhesion events and thought to
function in transmitting the signal emanating from integrins, which
have no intrinsic tyrosine-phosphorylating activity, to the nucleus
(reviewed in Refs. 12-15). For example, paxillin and FAK are
tyrosine-phosphorylated following stimulation of integrin signals (19,
34). Protein tyrosine phosphorylation is so critical that its level
should be tightly regulated by the balanced activities of
protein-tyrosine kinases and phosphatases. Thus, like protein-tyrosine
kinases, PTPs are important molecules in controlling the level of
protein tyrosine phosphorylation.
PTP-PEST (also known as P19-PTP) is a member of the intracellular PTP
family and is characterized by the presence of several PEST motifs in
the carboxyl segment of the protein adjacent to the catalytic domain
(26-29). In addition, its C terminus contains five proline-rich
segments (29). At least two of these domains represent type 2 proline-rich consensus segments known to interact with Src homology
domain 3 motif. To date, several molecules have been identified to
interact with PTP-PEST, including adaptor proteins such as SHC (35) and
Grb2 (24). The recent study using fibroblasts derived from PTP-PEST
knock-out mice has identified p130Cas as one of the
downstream targets of PTP-PEST (36). Cas is localized in focal
adhesions (37), as well as PTP-PEST following cell adhesion to
fibronectin and concomitant engagement of integrins (2). It is
plausible that the PTP-PEST interacting factors including Hic-5
modulate such PTP-PEST activity or substrate specificity, thereby
contributing to signal transduction in focal adhesions.
From the series of experiments using various types of mutant proteins,
we concluded that Hic-5 interacts with the Pro-2 domain of PTP-PEST by
LIM 3 in vitro as summarized in
Fig. 6. On the other hand, the results
obtained from in vivo binding assay shown in Fig.
4B indicate that Hic-5 with point mutation either in LIM 1, LIM 2, or LIM 3 formed a complex with HA-tagged PTP-PEST. This may
result from the presence of another factor that bridges between Hic-5
and PTP-PEST through other domains.
1- and hydrogen peroxide-inducible gene,
hic-5, and has striking similarity to paxillin, especially
in their C-terminal LIM domains. Like paxillin, Hic-5 is localized in
focal adhesion plaques in association with focal adhesion kinase in
cultured fibroblasts. We carried out yeast two-hybrid screening to
identify cellular factors that form a complex with Hic-5 using its LIM
domains as a bait, and we identified a cytoplasmic tyrosine phosphatase
(PTP-PEST) as one of the partners of Hic-5. These two proteins are
associated in mammalian cells. From in vitro binding
experiments using deletion and point mutations, it was demonstrated
that the essential domain in Hic-5 for the binding was LIM 3. As for
PTP-PEST, one of the five proline-rich sequences found on PTP-PEST,
Pro-2, was identified as the binding site for Hic-5 in in
vitro binding assays. Paxillin also binds to the Pro-2 domain of
PTP-PEST. In conclusion, Hic-5 may participate in the regulation of
signaling cascade through its interaction with distinct tyrosine
kinases and phosphatases.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1- and hydrogen peroxide-inducible clones from mouse
osteoblastic cells by a differential hybridization method and encodes a
protein of 55 kDa (1). Its overexpression induced senescence-like
phenotypes in immortalized fibroblasts (2) and promoted cellular
differentiation process in cell lines such as osteoblasts (3). One of
the unique features of the Hic-5 protein is its similarity to paxillin,
especially in their C-terminal four LIM domains. Paxillin is a
vinculin-binding protein co-localized with
FAK1 and integrins in focal
contacts (4). It is a phosphoprotein that interacts with tyrosine
kinases of the Src family as well as with focal adhesion kinase and
vinculin at focal adhesions (4).
(7). Focal adhesions are
specialized sites of cell adhesion to the extracellular matrix,
consisting of the integrins, cytoskeletal proteins, and signaling
molecules such as protein-tyrosine kinases and small G proteins
(8-10). Integrins are the transmembrane extracellular matrix receptors
(11), and the interaction of extracellular matrix and integrins elicits
numerous cellular responses such as proliferation, migration, and
differentiation (12-14). Formation of focal adhesions thus plays a
central role in these biological responses (15).
(18) are
involved in the formation of focal adhesion complexes (19). Recently,
it has been shown that protein-tyrosine phosphatases such as PTEN (20)
and PTP-PEST2 are also
localized in the focal adhesion plaques. In the present communication,
we describe evidence that PTP-PEST is associated with Hic-5 through its
distinct LIM domains.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
LIM), Hic-5 cDNA on the CMV/S5
vector (1) was truncated with restriction enzymes at the
PmaCI site (nucleotide 1365), the BssHII site
(nucleotide 1230), the BstPI site (nucleotide 1039), or the
StuI site (nucleotide 822), respectively. To obtain other
deletion constructs, a HindIII/XbaI-digested
Hic-5 cDNA fragment from CMV/S5 was cloned into the
HindIII/XbaI site of pVZ-1 vector (23). This
construct was named pVZ-1/S5, digested with StuI and
XbaI, and used as a primary vector. Desired fragments were
generated by polymerase chain reaction amplification of selected regions using primers incorporating 5'-StuI and
3'-XbaI restriction site. After restriction digestion,
polymerase chain reaction products were introduced into the
StuI/XbaI-digested primary vector. The mutant
fragments were excised from pVZ-1/S5 vector with HindIII and
XbaI and ligated into the HindIII/XbaI
sites of pRC/CMV (Invitrogen). These deletion mutants lack the
following nucleotide sequences of the Hic-5 cDNA; LIM 1-3
(nucleotides 165-1365), LIM 1-2 (nucleotides 165-1230), LIM 1 (nucleotides 165-1039), LIM 2-4 (nucleotides 165-822/982-1531), LIM
3-4 (nucleotides 165-822/1157-1531), LIM 3 (nucleotides
165-822/1157-1352), and LIM (nucleotides 165-822).
Pro-2) was generated by polymerase chain reaction.
.
-mercaptoethanol, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin) for 10 min at 4 °C. The beads were washed four
times with the wash buffer (20 mM Tris, pH 8.0, 0.1 M KCl, 0.1 mM ZnCl2, 10% glycerol,
0.1% Triton X-100, 5 mM
-mercaptoethanol, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin).
The protein complexes were eluted with elution buffer (50 mM Tris-HCl, pH 8.0, 0.2 M KCl, 20 mM glutathione) and subjected to SDS-PAGE. After drying,
the gels were exposed to x-ray films.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Hic-5 associates with PTP-PEST in the
mammalian cell. A, C2C12 cells were transfected with
eukaryotic expression constructs engineered to express HA-tagged wild
type PTP-PEST (lanes 2 and 5) or HA-tagged C231S
mutant PTP-PEST (lanes 3 and 6). 24 h after
transfection, the cells were lysed, and the lysates were incubated with
preimmune serum (lanes 1-3) or anti-Hic-5 polyclonal
antibody, number 1024 (lanes 4-6), conjugated to protein A
beads. The immunocomplexes were collected and analyzed by Western
blotting (WB) using anti-HA monoclonal antibody, 12CA5,
(upper panel) or anti-HIC-5 antibody, number 1024, (lower panel). B, C2C12 cell lysates transfected
with the wild type (lane 2) or C231S mutant (lane
3) PTP-PEST expression vectors were subjected to
immunoprecipitation (IP) with the anti-HA antibody. The
immunocomplexes were analyzed by Western blotting using the anti-HIC-5
antibody (upper panel) or anti-HA antibody (lower
panel).
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Fig. 2.
Pro-2 domain of PTP-PEST binds directly to
HIC-5. A, a schematic representation of the different
GST-PTP-PEST fusion proteins. A striped region indicates a phosphatase
catalytic domain. Pro-1-5 indicate five proline-rich domains: Pro-1,
332-338 amino acid residues; Pro-2, 355-374 amino acid residues;
Pro-3, 519-527 amino acid residues; Pro-4, 672-682 amino acid
residues; Pro-5, 764-771 amino acid residues (40). B,
Coomassie Blue-stained gel after SDS-PAGE of the GST-PTP-PEST fusion
proteins used in this study. Equivalent amounts of protein were used in
the binding assay. C, Hic-5 protein and paxillin were
in vitro translated in the presence of
[35S]methionine and incubated with glutathione-Sepharose
beads coupled with GST (lane 1) or GST fusion proteins
containing the Pro-1-5 domains (residues 276-775, lane 2),
Pro-1 domain (residues 316-346, lane 3), Pro-2 domain
(residues 344-437, lane 4), Pro-3 domain (residues
500-540, lane 5), Pro-4 domain (residues 666-689,
lane 6), or Pro-5 domain (residues 730-775, lane
7). After washing, bound proteins were eluted with elution buffer
and analyzed by autoradiography after SDS-PAGE.
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Fig. 3.
The interaction between Hic-5 and PTP-PEST
requires LIM 3 of HIC-5. A, a series of mutants deleted
from the C or N terminus of LIM domain of Hic-5 were translated in a
rabbit reticulocyte lysate system in the presence of
[35S]methionine. The products were separated by SDS-PAGE
and autoradiographed. B, GST-PTP-PEST 185-433 fusion
protein was coupled with glutathione-Sepharose beads and incubated with
the 35S-labeled deletion proteins (lanes 2, 4, 6, 8, 10, 12, 14, and 16). Bound protein were eluted,
separated by SDS-PAGE, and autoradiographed. C, expression
vectors for mLIM 2 (H293L/C296G) and mLIM 3 (H352G/C355G) were used as
templates for in vitro translated 35S-labeled
proteins, and products were analyzed by autoradiography after SDS-PAGE
(left panel). The proteins were incubated with
glutathione-Sepharose beads coupled with GST-PTP-PEST 185-433
(lanes 2, 5, and 8), GST-Pro-2 (lanes 3, 6, and 9), and GST (lanes 1, 4, and
7) as a control. Bound fractions were eluted, resolved by
SDS-PAGE, and autoradiographed (right panel).
Pro-2) were introduced into C2C12 cells. Cell lysates were
treated with anti-Hic-5 to precipitate endogenous Hic-5, and the
precipitates were Western-blotted with anti-HA antibody. The results
clearly indicate that Pro-2 deletion mutant did not associate with
Hic-5. Next we transfected human fibroblastic KMST-6 cells with
Flag-tagged Hic-5 mutants together with HA-PTP-PEST. This cell line was
chosen, because of its low level of endogenous Hic-5 that would
interfere with interaction of exogenously expressed products. Cellular
lysates were immunoprecipitated with anti-HA and Western-blotted with
anti-Hic-5 antibody. In contrast to the results obtained from in
vitro binding assay, the results shown in Fig. 4B
indicate that Hic-5 with point mutation either in LIM 1, LIM 2, or LIM
3 formed a complex with HA-tagged PTP-PEST. This may result from the
presence of other factors that bridge between Hic-5 and PTP-PEST
through other domains.
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Fig. 4.
In vivo association between
mutants of Hic-5 and PTP-PEST. A, plasmid that express
HA-tagged full-length PTP-PEST or its Pro-2 deletion mutant ( Pro-2)
was introduced into C2C12 cells. Cell lysates were treated with
anti-Hic-5 to precipitate endogenous Hic-5, and the precipitates were
Western-blotted (WB) and probed either with anti-HA or
anti-Hic-5 antibodies. B, KMST-6 cells were transfected with
Flag-tagged Hic-5 mutants together with HA-PTP-PEST. Twenty four hours
after transfection, cellular lysates were immunoprecipitated
(IP) with anti-HA and Western blotted with anti-HA or
anti-Hic-5 antibodies.
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Fig. 5.
Subcellular localization of the Hic-5 and its
LIM mutants. A-D, flag-tagged Hic-5 expressing either
wild type (A), point mutants of LIM 1 (B), LIM 2 (C), or LIM 3 (D) were transfected into KMST-6
cells, and cells were fixed 24 h later for immunostaining with
anti-Flag antibody (A-D). E and F, an
expression vector of HA-tagged Hic-5 was introduced into embryo
fibroblasts from PTP-PEST(+/ ) (E) and (
/
) mice
(F), and cells were stained with anti-HA antibody.
) and (
/
) mice. An
expression vector of HA-tagged Hic-5 was introduced into embryo fibroblasts, and localization of Hic-5 was observed by staining with
anti-HA antibody. Comparison of Fig. 5, E and F,
indicates that Hic-5 localized in focal adhesion both in PTP-PEST(+/
)
and (
/
) cells, but Hic-5 seems mainly localize in stress fibers in (
/
) cells.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 6.
Delineation of the HIC-5 LIM domain required
for PTP-PEST binding.
The LIM domains of Hic-5 are very similar to those of paxillin, and overall sequence similarity of amino acids in the LIM domains of Hic-5 and paxillin is 68%. In particular, similarity is as high as 74% in LIM 3. Therefore LIM 3 domains of both proteins possibly bear the similar function. We have shown that the LIM 3 of Hic-5 is the binding site for PTP-PEST, and the LIM 3 of paxillin is known to participate in localization of the protein to focal adhesion (38). Besides, both Hic-5 and paxillin are actually localized at focal adhesions and bind to PTP-PEST. Taken together, an interesting possibility is raised that LIM 3 is the determinant of subcellular localization of Hic-5 and paxillin and that PTP-PEST participates in recruiting them to focal adhesions by binding to LIM 3 of both proteins. The results of Fig. 5, A-D, however, suggest that the LIM domains other than LIM 3 may be involved in localization in focal adhesion. These possibilities are an open question for future study.
N-terminal regions of Hic-5 and paxillin are also very similar in structure, although their similarity is less than in LIM domains. The regions contain Pro-rich and LD domains, which are thought to serve as an interface for protein-protein interaction. In fact FAK, proline-rich kinase 2, and vinculin have been shown to bind to the N-terminal regions of paxillin (39), and Fujita et al. (40) recently reported that Hic-5 associates with FAK at the N-terminal domain. Collectively, Hic-5 and paxillin are closely related proteins in many aspects, such as structures, interacting factors, and subcellular localization. However, they are expected to have different functions within cells, since their expression patterns are different; paxillin is ubiquitously expressed, whereas Hic-5 is expressed higher in spleen and lung (1). At molecular levels, Fujita et al. (40) have clearly demonstrated the differences between Hic-5 and paxillin. They have shown that Hic-5 is marginally tyrosine-phosphorylated either by FAK kinase activity or by integrin stimulation, which forms sharp contrast to paxillin that is heavily phosphorylated by either pathway. This result is consistent with the fact that Hic-5 does not have several tyrosine residues that are present in paxillin and are supposed to be binding sites for the Crk SH2 domain and potentially phosphorylated (40). Of interest is that fibronectin-induced tyrosine phosphorylation of paxillin was inhibited by co-expression of Hic-5 (40). From these observations, they proposed that one of putative Hic-5 functions is to inhibit tyrosine phosphorylation of paxillin by binding to protein-tyrosine kinases such as FAK in a competitive manner to paxillin. Recent findings by Shen et al. (41) indicated that paxillin associates with PTP-PEST through the region of 297-494 amino acids encompassing Pro-1 and Pro-2. Although the precise mapping of the interacting domain of PTP-PEST with paxillin has not been yet performed, it is likely that Hic-5 binding to Pro-2 competes out paxillin, resulting in inhibition of the signaling directed by the complex of PTP-PEST and paxillin as is the case of protein-tyrosine kinases.
In conclusion Hic-5 shares structural features, subcellular
localization, and interacting factors with paxillin. FAK and PYK2 have
been reported to bind to both Hic-5 and paxillin. In this study we
added PTP-PEST to the list. All of these molecules are implicated in
protein tyrosine phosphorylation and are believed to have important
functions in integrin-mediated signal transduction pathway. Hic-5 is
thought to compete with paxillin in the binding to these molecules and
thus control tyrosine phosphorylation signaling mediated by paxillin.
Further study on Hic-5 functions is expected to clarify the nature of
integrin-mediated signaling in more detail and the way it is involved
in various types of cellular processes.
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FOOTNOTES |
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* This study was supported in part by a Grant-in-Aid for Cancer Research from the Ministry of Education, Science, Sports, and Culture of Japan.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Recipient of a Cancer Research Society studentship.
¶ Chercheur boursier from Les Fonds De La Recherche En Santé Du Québec.
To whom correspondence should be addressed: Dept. of
Microbiology, Showa University School of Pharmaceutical Sciences,
Hatanodai 1-5-8, Shinagawa-ku, Tokyo, Japan. Tel.: 81-3-3784-8208; Fax: 81-3-3784-6850; E-mail: knose{at}pharm.showa-u.ac.jp.
2 A. Angers-Loustau, J. F. Côté, A. Charest, D. Dowbenko, S. Spencer, L. A. Lasky, and M. K. Tremblay, manuscript submitted for publication.
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ABBREVIATIONS |
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The abbreviations used are: FAK, focal adhesion kinase; PTP, protein-tyrosine phosphatase; GST, glutathione S-transferase; PAGE, polyacrylamide gel electrophoresis; HA, hemagglutinin; CMV, cytomegalovirus.
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