From the Departments of Cell adhesion kinase The Hic-5 N-terminal domain directly associated in vitro
with the extreme C-terminal region (residue 801 to the end) of CAK We found, by cDNA cloning, the second protein-tyrosine kinase
of the focal adhesion kinase
(FAK)1 subfamily, which we
named cell adhesion kinase CAK CAK In a study to elucidate the upstream and downstream signaling pathways
of CAK Cloning of a cDNA Encoding a CAK Biochemistry and
¶ Pathology,
Department
of Pathology, Sapporo Medical University School of Medicine, South-1,
West-17, Chuo-Ku, Sapporo 060, Japan
ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References
(CAK
/PYK2) is the
second protein-tyrosine kinase of the focal adhesion kinase subfamily.
We identified a cDNA that encodes a CAK
-binding protein. This
cDNA clone encodes the human homologue of Hic-5, the cDNA of
which was cloned in 1994 as transforming growth factor
1- and
hydrogen peroxide-inducible mRNA. We found that Hic-5 exclusively
localized at focal adhesions in a rat fibroblast line, WFB. This
localization of Hic-5 was confirmed in WFB cells expressing Myc-tagged
Hic-5. The amino acid sequence of Hic-5 is highly similar to that of
paxillin in the four LD motifs as well as in the four contiguous LIM
domains.
. CAK
was coimmunoprecipitated with Hic-5 from the WFB cell lysate. The coimmunoprecipitation of CAK
with Hic-5 was markedly inhibited by the addition of the extreme C-terminal region of CAK
.
Coimmunoprecipitation of Hic-5 with CAK
, which was shown in COS-7
cells doubly transfected with cDNA constructs of CAK
and
Myc-tagged Hic-5, was lost when the CAK
amino acid residues 741-903
were deleted. Hic-5 was tyrosine-phosphorylated in Src-transformed 3Y1
cells and in cells treated with pervanadate. Hic-5 associated with
CAK
was selectively tyrosine-phosphorylated in WFB cells exposed to
hypertonic osmotic stress. These results indicate that Hic-5 is a
paxillin-related component of focal adhesions and binds to CAK
,
implying possible involvement of Hic-5 in the downstream signaling of
CAK
.
INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References
(CAK
) (1). The cDNAs of the
protein have also been cloned by other groups of researchers, and the
protein has been named PYK2 (2), RAFTK (3), FAK2 (4), and CADTK (5).
The amino acid sequence of human CAK
is 95.4% identical with that
of rat CAK
(2-4, 6). The human gene of CAK
was mapped to
chromosome 8 at p21.1 (7).
and FAK are closely related in their overall structures and have
sequence similarity over their entire length except for the extreme
N-terminal regions and 10 C-terminal residues. The 88 N-terminal
residues of CAK
are markedly different from the corresponding 81 N-terminal residues of FAK (1). FAK is important as a docking protein.
Four regions of the FAK sequence have been identified as the ligand
sequences (8). All these ligand sequences are at least partly conserved
in CAK
. Tyrosine residue 397 of FAK and the corresponding residue
402 of CAK
are sites of autophosphorylation and also ligand sites to
the SH2 domains of the Src family protein-tyrosine kinases with
conserved ligand sequence, YAEI (9); this binding activates the Src
family kinases (9, 10). The second ligand sequence in FAK for SH2, Y925ENV of the mouse FAK, is known to be the ligand site
for Grb2 (11) and is also functionally conserved in residues 881-884 of CAK
, YHNV of rat CAK
, and YLNV of human CAK
. The third
ligand sequence in FAK, EAPPKPSR, participates in the binding to the SH3 domains of pp130cas and related proteins (12, 13) and is
functionally conserved in CAK
residues 712-719, EPPPKPSR. A weak
coimmunoprecipitation of pp130cas with CAK
was shown (14).
There is one more proline-rich sequence in the C-terminal domain
(C-domain) of FAK, PAAPPKKPPRPGAP (residues 869-882). An
SH3-containing GTPase-activating protein for Rho and Cdc42, named Graf
by Hildebrand et al. (15), was identified as a protein with
specific affinity to this sequence. CAK
also has a proline-rich
sequence at the corresponding region, PPQKPPR (residues 855-861).
in cultured epithelial cells is mainly found in the perinuclear
region and the cytoplasm in addition to the cell-to-cell border. In rat
tissues, CAK
is present in association with microvilli, cilia, and
axons (16). FAK mediates signaling through integrins. A complex
assembly of proteins is formed at focal adhesions in association with
FAK (8, 17). It has been shown that paxillin and talin bind to the
C-domain of FAK (18, 19). Two short stretches of 17 and 9 amino acid
residues were identified as the FAK sequences participating in the
binding to paxillin (18). These sequences are highly conserved in
CAK
. Paxillin binding to CAK
was recently shown (20). FAK is also
activated by stimulation of receptors coupled to phospholipase C
activation such as neuropeptide receptors and the platelet-derived
growth factor receptor (21). This second mode of activation is also
found in CAK
(2). Moreover, the tyrosine phosphorylation of CAK
is markedly enhanced when the cytoplasmic free Ca2+
concentration is increased (2) and when cells are stressed by osmotic
shock (5, 22). The differences in the subcellular and tissue
distributions of CAK
and FAK indicate different functions for these
two protein-tyrosine kinases.
, we used an expression cloning technique to identify binding
partners for the C-domain of CAK
. We report here the identification
of a cDNA that encodes a CAK
-binding protein. The predicted
amino acid sequence of this cDNA indicated that the protein was the
human homologue of Hic-5, the cDNA of which was described by
Shibanuma et al. (23) as a transforming growth factor
1-
and hydrogen peroxide-inducible mRNA. Hic-5 is closely related to
paxillin in its amino acid sequence. Immunocytochemical staining of rat
fibroblast line WFB with a specific anti-Hic-5 antibody revealed that
Hic-5 localized exclusively at focal adhesions as paxillin did, a
result in disagreement with the original identification of Hic-5 as a
nuclear protein (23, 24). We further demonstrated coimmunoprecipitation
as well as direct binding of Hic-5 and CAK
. Hic-5 associated with
CAK
was preferentially tyrosine-phosphorylated, implying a
functional interplay between CAK
and Hic-5.
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
-binding Protein--
An
oligo(dT)- and random-primed human (normal female, 2 years old)
hippocampus cDNA library constructed in
ZAPII vector
(Stratagene, La Jolla, CA; catalog number 936205) was screened by
affinity binding to a glutathione S-transferase (GST)-CAK
fusion protein, GST-CAK
-Cdom, that had been labeled with
32P. Preparation of 32P-labeled GST-CAK
-Cdom
was as follows. cDNA encoding the C-domain (amino acid residues
670-1009) of rat CAK
was amplified from a cDNA clone 17N (1) by
polymerase chain reaction and inserted into pGEX-2TK vector. The GST
fusion protein was expressed in Escherichia coli strain
BL21(DE3), affinity-purified by using glutathione-agarose, and
phosphorylated in vitro using the catalytic subunit of
cAMP-dependent protein kinase (Sigma) and
[
-32P]ATP (ICN Biochemicals Inc., CA) as described by
Hildebrand et al. (15) and Kaelin et al.
(25).
ZAPII expression libraries was done as described below.
Phage plaques were formed on culture plates at 37 °C, and the
protein expression was induced by overlaying nitrocellulose membranes
(BA85; Schleicher & Schuell) that had been soaked in 10 mM
isopropyl-
-D-thiogalactopyranoside. After overnight
incubation at 37 °C, the membranes were removed and washed twice at
4 °C in Hyb75 buffer (20 mM Hepes (pH 7.5), 75 mM KCl, 0.1 mM EDTA, 2.5 mM
Mg2Cl, 1 mM dithiothreitol, 0.05% Nonidet
P-40) (25). The membranes were soaked twice for 10 min in Hyb75 buffer
supplemented with 6 M guanidine hydrochloride each time in
fresh solution and then for 5 min each in Hyb75 buffers supplemented
with 3 M, 1.5 M, and 0.75 M
guanidine hydrochloride, in this order. After soaking in Hyb75 buffer
containing 5% skim milk at 4 °C for 1 h with constant shaking,
which represents a blocking step, the membranes were then incubated
with 106 cpm/ml of the 32P-labeled
GST-CAK
-Cdom (50 ng/ml) in Hyb75 buffer, which contained 1% skim
milk and 0.6 µg/ml of GST prepared by expression from pGEX-2TK
vector. The membranes were washed seven times for 15 min each in Hyb75
buffer containing 1% skim milk. Positive plaques were made visible by
exposure of the membranes to x-ray films. The positive clone thus
obtained, cbp-1, was subcloned into pBluescript and
subjected to sequencing in both directions after the preparation of
internal deletion mutants.
Epitope-tagged Hic-5 and Fusion Proteins--
The plasmid
construct encoding the N-terminally Myc-tagged Hic-5 was generated as
follows. Using polymerase chain reaction, the cDNA encoding
full-length (according to the open reading frame described by Shibanuma
et al. (23)) human Hic-5 was amplified, and BamHI
and EcoRI restriction sites were created at nucleotide positions 6 (immediate 5
-side of the presumed translational initiation codon ATG) and 1492, respectively. The amplified cDNA was ligated in frame to the BamHI and EcoRI sites
of the pcDNA3Myc vector to obtain pHic5-Myc. The pcDNA3Myc
vector was constructed by ligating the
HindIII-BglII fragment of the pJ3M vector (26) into the HindIII and BamHI sites of pcDNA3
(Invitrogen). The 10 amino acid residues of the epitope tag are
specifically recognized by the anti-Myc monoclonal antibody 9E10.
Deletion Mutants of CAK--
The full-length CAK
cDNA
clone, 17N, and the C-terminally epitope-tagged CAK
cDNA were
subcloned into expression vector pSRE to obtain pCAK
(S) and
pCAK
Tag as described previously (1). pCAK
(S) and pCAK
Tag were
used for the generation of CAK
variants. Deletion (dl) mutations are
designated by the amino acid residues deleted. The base pair (bp)
designation corresponds to the nucleotide sequence of the CAK
cDNA counting from the translational initiation codon (1). Mutation
dl 86-321 was generated by digesting pCAK
Tag with PvuII
(which cleaves at bp 253, 529, 862, 961, and 2698) and isolating the
largest fragment and the fragment of 1737 base pairs followed by
rejoining of the PvuII termini; rejoining of the 1737-bp
fragment in the right direction was confirmed. To construct dl
159-552, pCAK
(S) was digested with BspEI (which cleaves
at bp 473, 680, and 1655) followed by religation of the BspEI termini. This dl 159-552 mutant of pCAK
(S) was
digested with SacI (which cleaves at bp 2854), and then the
SacI fragment of pCAK
Tag containing the Tag cDNA
sequences was ligated into the SacI site of the deletion
mutant of pCAK
(S) to generate the dl 159-552 mutant of pCAK
Tag.
Mutation dl 741-903 was created by digesting pCAK
Tag with
Bsu36I (which cleaves at bp 2218 and 2707) followed by
religation of the Bsu36I termini.
Production of Antiserum to Hic-5 and Affinity Purification of the Antibody-- The anti-Hic-5 antibody was raised in rabbits against a GST fusion protein of full-length human Hic-5, GST-Hic5(fl). Anti-GST antibody was first removed from the serum by the use of a column of covalently bound GST. Anti-Hic-5 was then affinity-purified on a column of the immunogen covalently bound to cyanogen bromide-activated Sepharose 4B, from which the antibody was eluted with 0.5 M ammonium hydroxide containing 3 M sodium thiocyanate (pH 11.0). The anti-Hic-5 antibody immunoprecipitated Hic-5 of human and rat origin and was good for use in immunoblotting and immunocytochemistry.
Antibodies and Other Materials--
The first anti-CAK rabbit
antibody used in this study, anti-CAK
(C-a), was raised against a GST
fusion protein of residues 670-716 of rat CAK
and was
affinity-purified on a column of the immunogen covalently bound to
cyanogen bromide-activated Sepharose 4B. Anti-CAK
(C-a) was found to
be specific to CAK
; the antibody did not immunoprecipitate or
immunoblot FAK. The second anti-CAK
rabbit antibody, anti-CAK
(N),
was raised against a GST fusion protein of rat CAK
residues from
5
to 416, GST-CAK
-Ndom; this antiserum was used either without
purification or after removal of anti-GST followed by affinity
purification on a column of immobilized immunogen. Anti-CAK
(N) was
also found to be specific to CAK
. Anti-CAK
(N)-mAb and
anti-CAK
(C)-mAb are mouse IgG1 monoclonal antibodies
raised against GST-CAK
-Ndom and GST-CAK
-Cdom, respectively.
Cells--
A rat fibroblast line, WFB (30), was obtained from
the establisher of the line, Dr. N. Sato (Sapporo Medical University, Sapporo, Japan). A rat fibroblast line transformed with Rous sarcoma virus, SR-3Y1-1 (SR-3Y1, RCB0353) (31), and its parent line, 3Y1-B
clone 1-6 (3Y1, RCB0488) (32), were obtained from Riken Cell Bank
(Tsukuba, Japan). COS-7 (ATCC CRL 1651) and HEK 293 (CRL 1573) were
obtained from the American Type Culture Collection (Rockville, MD).
These cells were cultured in Iscove's modified Dulbecco's medium
(Iscove's medium) supplemented with 10% heat-inactivated (56 °C
for 30 min) fetal calf serum, 2 mM glutamine, 1 mM sodium pyruvate, 50 units/ml penicillin, and 50 µg/ml
streptomycin. When WFB cells were stimulated, the medium of confluent
monolayer cultures of the cells in 10-cm dishes was removed and
replaced with 2 ml of warm Iscove's medium without serum. After 1 h of incubation at 37 °C, the cells were stimulated at 37 °C for
3-10 min with either 2 µM oleoyl
L--lysophosphatidic acid (Sigma) or 0.2 µM endothelin 1 (Sigma) or exposed to hypertonic medium at 37 °C for
5-20 min by replacing the medium with 2 ml of warm Iscove's medium
containing 0.3 M sorbitol.
Immunofluorescence Microscopy and Confocal Laser-scanning
Microscopy--
Cells grown on glass coverslips coated with rat tail
collagen (33) were fixed with cold absolute ethanol unless otherwise stated and kept at 20 °C until use. After being rinsed with
phosphate-buffered saline (PBS), the cells were incubated with Block
Ace (Dainippon Pharmaceutical Co., Tokyo, Japan) at room temperature
for 30 min. Then a primary antibody was applied. The incubation with
anti-Hic-5 antibodies was done for 1 h at room temperature, and
the incubation with other antibodies was done for 30 min at room
temperature. Fluorescein isothiocyanate- or rhodamine-conjugated
antibodies were then applied for 30 min at room temperature. The cells
were thoroughly washed in PBS and incubated with the secondary
antibody. After being rinsed with PBS, the coverslips were mounted in a solution of 10% PBS and 90% glycerol containing 1 mg/ml
p-phenylenediamine (Kanto Chemical Co., Tokyo, Japan). For
double staining, the following combinations of primary antibodies or
stains were used; anti-Hic-5 and anti-FAK antibodies, anti-Hic-5
antibody and rhodamine-conjugated phalloidin, anti-Myc antibody and
rhodamine-conjugated phalloidin, anti-Hic-5 and anti-vinculin
antibodies, and anti-CAK
and anti-vinculin antibodies. The samples
were examined with an Olympus epifluorescence photomicroscope (Olympus,
Tokyo, Japan). Some samples were imaged with a confocal laser-scanning
microscope (model TCS NT, Leica, Heebrugg, Switzerland).
Immunoprecipitation of Hic-5, CAK, and Other
Proteins--
Confluent monolayer cultures of cells in 10-cm dishes
were washed twice with PBS and then lysed on ice in 0.5 ml per dish of
a lysis buffer (20 mM Tris-HCl (pH 7.4), 150 mM
NaCl, 2.5 mM EDTA, 1% Nonidet P-40, 10% glycerol, 10 µg/ml each leupeptin and aprotinin, 1 mM
phenylmethylsulfonyl fluoride, 50 mM NaF, 1 mM Na3VO4, 20 mM
Na4P2O7). The lysates were
subjected to centrifugation at 12,000 × g for 10 min
at 4 °C to obtain clarified lysates. Portions of the lysates were
precleared by mixing for 2 h at 4 °C with either normal rabbit
IgG bound to protein A-Sepharose or mouse IgG bound to anti-mouse IgG
agarose, depending on the antibody to be used in the
immunoprecipitation. The cell lysates thus precleared were then
incubated at 4 °C for 4 h or overnight with antibody beads. The
anti-Hic-5 beads and the anti-CAK
beads were prepared for each assay
by mixing either 1 µg of protein of affinity-purified anti-Hic-5, 3 µg of protein of affinity-purified anti-CAK
, or 4 µl of
anti-CAK
serum with 10 µl (packed volume) of protein A-Sepharose
and washing the Sepharose beads with the lysis buffer. The mouse
antibody beads were prepared for each assay by mixing 1 µg of protein
of either anti-paxillin or anti-FAK monoclonal antibody or 3 µg of
protein of an anti-CAK
monoclonal antibody with 10 µl (packed
volume) of anti-mouse IgG-agarose and washing the agarose beads with
the lysis buffer. Each immunoprecipitation was done from 1 mg of
protein of the clarified lysates. As a control, rabbit immunoglobulin
beads, mouse IgG beads, or preimmune rabbit serum beads were prepared
and used in each assay. Immunoprecipitates were washed three times with
the lysis buffer, and proteins were separated by SDS-PAGE according to
the method of Laemmli and Favre (34). The separated proteins were
blotted onto polyvinylidene difluoride (PVDF) membranes (Immobilon-P,
Millipore Corp., Bedford, MA). The membranes were blocked with 3%
bovine serum albumin in TBST (25 mM Tris-HCl (pH 7.5), 150 mM NaCl, and 0.05% Tween 20) for 20 min at 60 °C and
then probed with a primary antibody in TBST containing 1% bovine serum
albumin for 1 h at room temperature. For immunoblotting,
affinity-purified anti-Hic-5 and affinity-purified anti-CAK
(C-a)
antibodies were used at 1 µg of protein/ml, and anti-CAK
serum was
used at a 200-fold dilution. The membranes were washed with TBST three
times and probed again in TBST for 1 h with a second antibody
conjugated with alkaline phosphatase or, for enzyme-linked
chemiluminescence, with a second antibody conjugated with horseradish
peroxidase, followed by washing three times in TBST. Positive bands
were detected either by enzyme-linked chemiluminescence according to
the manufacturer's (Amersham) protocol or by incubation in nitro blue
tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate.
Blot Overlay Assay--
Four to five µg each of GST-Hic5(fl),
GST-Hic5-Ndom, GST-Hic5-Cdom, and GST was subjected to SDS-PAGE and
blotted to a PVDF membrane. The membranes were soaked in solutions of
guanidine hydrochloride for denaturation and renaturation of the bound
proteins as described above under "Cloning of a cDNA Encoding a
CAK-binding Protein." Then the proteins on membranes were probed
with 32P-labeled probes as also described in that section.
Probes used were GST fusion proteins of the N- and C-domains of CAK
and FAK and portions of them. The GST fusion proteins were expressed
from pGEX-2TK vector as described above and thus contained a site of phosphorylation by cAMP-dependent protein kinase.
32P labeling of these probes was as described above. One
µg of each probe was labeled at about 3 × 107 cpm.
The probes were added to each assay at a concentration of 3 × 106 cpm/ml.
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RESULTS |
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Isolation of a cDNA Clone Encoding a Binding Protein to the
CAK C-domain--
In an effort to identify proteins that bind
CAK
, we used the N- and C-domains of CAK
, which are the regions
contiguous to the kinase domain at the N- and C-terminal sides, to
screen a
ZAPII expression library derived from human hippocampus
cDNA. One clone (clone cbp-1) was positively identified
by screening of 2 × 106 plaques with the C-domain,
GST-CAK
-Cdom (consisting of amino acid residues 670-1009 of rat
CAK
), as a probe. A comparison of 1759 base pairs of the
cbp-1 cDNA sequence with those in the GenBankTM data base by the BLASTx program (35) of NCBI
revealed high similarity of the amino acid sequence translated from
cbp-1 in one reading frame and the Hic-5 amino acid sequence
(GenBankTM L22482) over their entire length. This result
indicated that cbp-1 is a cDNA clone encoding the
full-length, human homologue of Hic-5, which had been cloned from a
mouse cDNA library as a transforming growth factor
1- and
hydrogen peroxide-inducible mRNA by Shibanuma et al.
(23). Human Hic-5 has the same number of amino acid residues as mouse
Hic-5, and their amino acid sequences are 97.0% identical in their
double zinc finger LIM domains (amino acid residues 211-444 (23)) (36,
37) and 84.3% identical in their N-domain (amino acid residues 1-210
(23)). The clone cbp-1 contained flanking 5
- and
3
-untranslated sequences of 49 and 376 base pairs, respectively. In
the fusion protein encoded by clone cbp-1, the 49 base pairs
at the presumed 5
-untranslated region (23) predicted 16-amino acid
residues contiguous to
-galactosidase encoded by the phage vector
used in the construction of the cDNA library.
|
Hic-5 Localizes at Focal Adhesions-- An anti-Hic-5 antibody was raised in rabbits against the GST fusion protein of human Hic-5 (GST-Hic5(fl)) and affinity-purified on a column of immobilized immunogen. The anti-Hic-5 antibody was found to be specific to Hic-5 as shown by immunoblotting and immunoprecipitation from the rat fibroblast WFB cell lysate, where a band of about 55 kDa was detected by the antibody (Fig. 2). The anti-paxillin monoclonal antibody obtained from Transduction Laboratories immunoprecipitated and immunoblotted not only paxillin but also Hic-5 (Fig. 2). The anti-Hic-5 antibody neither immunoprecipitated paxillin nor bound to paxillin blotted from gel after immunoprecipitation with anti-paxillin and separation by SDS-PAGE (Fig. 2).
|
|
|
Direct Binding of the Hic-5 N-domain to the C-terminal Region of
CAK--
Direct binding of Hic-5 to CAK
was shown by blot
overlay assays, and the specificity of this binding was examined. GST
fusion proteins of almost full-length Hic-5 (amino acid residues
16 to 444; GST-Hic5(fl)), the Hic-5 N-domain (amino acid residues 1-223;
GST-Hic5-Ndom), the Hic-5 C-domain (amino acid residues 203-444
containing the LIM domains; GST-Hic5-Cdom), and, as a control, GST were
subjected to SDS-PAGE and immobilized on a PVDF membrane. Hic-5 in the
WFB cell lysate was immunoprecipitated with anti-Hic-5 and was also
immobilized on the membrane after the electrophoretic separation. After
procedures for denaturation and renaturation of the proteins on the
membrane, the fusion proteins and Hic-5 were probed for binding to
CAK
and FAK. The probes used for the binding assay were
32P-labeled GST fusion proteins of the N- and C-domains of
CAK
, GST-CAK
-Ndom and GST-CAK
-Cdom; fragments of the C-domain,
GST-CAK
-CdomA and GST-CAK
-CdomB; and the C-domain of FAK,
GST-FAK-Cdom, and its fragment, GST-FAK-CdomB. The CAK
C-domain,
GST-CAK
-Cdom, was bound by GST-Hic5(fl), GST-Hic5-Ndom, and Hic-5
from WFB cells but not by GST-Hic5-Cdom (Fig.
5B). The CAK
N-domain was
not bound by these GST fusion proteins or by Hic-5 from WFB cells (data
not shown). The FAK C-domain, GST-FAK-Cdom, was also bound by
GST-Hic5(fl) and GST-Hic5-Ndom (data not shown). When the two regions
of the divided CAK
C-domain were tested for the binding, only the
extreme C-terminal region, GST-CAK
-CdomB, was bound by GST-Hic5(fl)
and GST-Hic5-Ndom (Fig. 5, C and E). These
results are consistent with the results obtained by dot blots, in which the
ZAPII phage plaques of the original cbp-1 clone
induced to produce the fusion protein were probed with
32P-labeled GST-CAK
-CdomA and GST-CAK
-CdomB; the
positive signal was obtained only with GST-CAK
-CdomB (data not
shown). The same region of FAK contained the binding site; only the
extreme C-terminal region of the FAK C-domain, GST-FAK-CdomB, was bound
by GST-Hic5(fl) and GST-Hic5-Ndom (Fig. 5F). It was noted
that the extreme C-terminal halves of the CAK
and FAK C-domains gave
better signals than the whole C-domains in the blot overlay assays.
These results indicate that a common structure in the extreme
C-terminal halves of the CAK
and FAK C-domains has specific affinity
to the N-domain of Hic-5.
|
GST Fusion Protein of the CAK C-terminal Region Bound in Vitro
Hic-5 from the WFB Cell Lysate--
Hic-5 in the WFB cell lysate was
bound to the GST fusion protein of full-length CAK
, GST-CAK
(fl),
and to the fusion protein of the C-terminal portion of CAK
,
GST-CAK
-CdomB (Fig. 6, lanes 4 and 10). Paxillin was also bound to GST-CAK
-CdomB
(Fig. 6, lane 10) but was not significantly bound to
GST-CAK
(fl) (Fig. 6, lane 4). The anti-paxillin
monoclonal antibody used in this study immunostained Hic-5 in addition
to paxillin as shown in Fig. 2 (Fig. 6, middle). The
C-terminal portion of FAK, GST-FAK-CdomB, also bound Hic-5 and paxillin
(Fig. 6, lane 12). The GST fusion proteins of the CAK
N-domain, GST-CAK
-Ndom, and the region of the CAK
C-domain
proximal to the kinase domain, GST-CAK
-CdomA, did not bind Hic-5 or
paxillin (Fig. 6, lanes 6 and 8). These results
further prove that Hic-5 and paxillin bind to the extreme C-terminal
halves of CAK
and FAK C-domains.
|
Coimmunoprecipitation of CAK with Hic-5 from WFB Cell
Lysate--
When Hic-5 was immunoprecipitated with anti-Hic-5 from the
lysate of WFB cells, CAK
was coimmunoprecipitated with Hic-5 (Fig. 7, lane 4 of top).
This association of CAK
with Hic-5 was found when the lysate was
prepared in a lysis buffer containing 1% Nonidet P-40 as the
detergent. The addition of sodium deoxycholate at 0.5% to the lysis
buffer prevented successful demonstration of the coimmunoprecipitation.
As shown in Fig. 2, the anti-paxillin monoclonal antibody
immunoprecipitated Hic-5 (Fig. 7, lane 12 of
middle) in addition to paxillin, which migrated as a broad band behind Hic-5; thus, CAK
was also found in the immunoprecipitate with this anti-paxillin (Fig. 7, lane 12 of top).
Hic-5 was resolved by SDS-PAGE into double bands just above
immunoglobulin heavy chains. When the association of Hic-5 and CAK
was examined in the reverse direction by immunoprecipitating CAK
from the WFB cell lysate, it was hard to find Hic-5 in the anti-CAK
immunoprecipitate by blotting with anti-Hic-5 (Fig. 6, lane
2 and 3 of middle). In an attempt to show
coimmunoprecipitation of Hic-5 with CAK
, we immunoprecipitated
CAK
with different anti-CAK
antibodies: anti-CAK
(C-a),
anti-CAK
(N), anti-CAK
(N)-mAb, and anti-CAK
(C)-mAb. In the
immunoprecipitates from the WFB cell lysate with any of these
anti-CAK
antibodies, no significant amount of Hic-5 was demonstrated
by blotting with anti-Hic-5 (Fig. 7). However, as shown in Fig. 11,
coimmunoprecipitation with CAK
of a tyrosine-phosphorylated protein
migrating at the position of Hic-5 was found by blotting with
anti-phosphotyrosine. Moreover, when the A-431 cell lysate was used, a
small amount of Hic-5 was found by blotting with anti-Hic-5 in the
immunoprecipitates with anti-CAK
(C-a) and anti-CAK
(C)-mAb (data
not shown). A small amount of paxillin, but no Hic-5, was found
coimmunoprecipitated with FAK from the WFB cell lysate (Fig. 7,
lane 8) (the blotting with anti-paxillin is not shown).
Paxillin was faintly detected in the immunoprecipitates with
anti-CAK
(N), anti-CAK
(N)-mAb, and anti-CAK
(C)-mAb by extended
blotting with anti-paxillin (data not shown). As shown below,
coimmunoprecipitation of Hic-5 with CAK
was clearly demonstrated in
COS-7 cells expressing these proteins from transfected cDNA
constructs.
|
Inhibition of CAK Coimmunoprecipitation with Hic-5 by GST Fusion
Proteins of the Extreme C-terminal Portions of CAK
and FAK--
The
association of the C-terminal region of CAK
with Hic-5, shown in
Fig. 5 by blot overlay assay and in Fig. 6 by pull-down assay, was
confirmed by showing an inhibition of CAK
coimmunoprecipitation with
Hic-5 from the WFB lysate. The addition of the extreme C-terminal region of CAK
fused to GST, GST-CAK
-CdomB, to the WFB cell lysate prior to the immunoprecipitation with anti-Hic-5 markedly interfered with the CAK
coimmunoprecipitation with Hic-5 (Fig. 7, lane
5 of top). This inhibition of the CAK
association
with Hic-5 was also found when the corresponding C-terminal region of
FAK, GST-FAK-CdomB, was added to the lysate, but the inhibition was not
found when the N-domain of CAK
, GST-CAK
-Ndom, was added (Fig. 7,
lanes 6 and 7 of top). These results
are consistent with the in vitro binding data shown in Figs.
5 and 6.
Analysis of the Association of CAK and Hic-5 by the Use of
Deletion Mutants of CAK
Expressed in COS-7 Cells from Transfected
cDNA Constructs--
COS-7 cells endogenously express a small
amount of Hic-5 but almost no CAK
. In the experiments shown in Fig.
8, CAK
and Hic-5 were expressed
in COS-7 cells from transfected cDNA constructs. In this
analysis of the CAK
association with Hic-5 by immunoprecipitation with anti-Hic-5 and anti-CAK
from the lysates of transfected COS-7 cells, we were able to show the coimmunoprecipitation of CAK
with Hic-5 and Hic-5 with CAK
(Fig. 8, lanes 7 and
8). In these experiments, CAK
was expressed as
C-terminally HSV-tagged CAK
and Hic-5 was expressed as N-terminally
Myc-tagged Hic-5. There may be at least two reasons for this successful
demonstration of Hic-5 coimmunoprecipitation with CAK
. We found that
the anti-Myc monoclonal antibody detected Hic-5 at a sensitivity
significantly higher than that with anti-Hic-5 (the data of
immunoblotting with anti-Hic-5 are not shown in Fig. 8). It was also
noted that Hic-5 with an N-terminal Myc-tag was expressed at a
significantly high level in COS-7 cells.
|
Tyrosine Phosphorylation of Hic-5--
The results shown above
indicated that Hic-5 was a protein highly related to paxillin in its
structure and function. It is known that paxillin is markedly
tyrosine-phosphorylated upon activation of FAK (41). Therefore, we
examined whether Hic-5 was tyrosine-phosphorylated upon activation of
CAK and in Src-transformed cells. Hic-5 was strongly
tyrosine-phosphorylated in 3Y1 and WFB cells treated with pervanadate
(Fig. 9). Mobility retardation was
observed in Hic-5 when the protein was heavily phosphorylated. Hic-5 in
Src-transformed 3Y1 cells, SR-3Y1, was also significantly
tyrosine-phosphorylated as compared with the protein in 3Y1 cells (Fig.
9). In accordance with the data reported by Shibanuma et
al. (23, 24), SR-3Y1 cells, a transformed cell line, contained
much smaller amounts of Hic-5 than the untransformed counterpart, 3Y1
cells (Fig. 9A, lanes 1 and 3).
|
|
CAK and Hic-5 Were Tyrosine-phosphorylated in Parallel in WFB
Cells either Exposed to Hypertonic Osmotic Stress or Stimulated with
Lysophosphatidic Acid--
The association of Hic-5 with CAK
was
examined under conditions where the tyrosine phosphorylation of CAK
was enhanced. The level of CAK
tyrosine phosphorylation decreased
upon detachment of WFB cells from culture dishes by trypsinization
(Fig. 11, bottom, lane
3 as compared with lane 5). The tyrosine
phosphorylation of CAK
was enhanced by stimulation of WFB cells with
lysophosphatidic acid (Fig. 11, lane 7) and by exposing the
cells to hypertonic osmotic stress (Fig. 11, lane 9). The
amounts of CAK
coimmunoprecipitated with Hic-5 did not significantly
change under these various conditions of WFB cells where the levels of
CAK
tyrosine phosphorylation varied (Fig. 11). However, blotting
with anti-phosphotyrosine revealed that the tyrosine-phosphorylated
CAK
present in anti-Hic-5 immunoprecipitates decreased upon
detachment of WFB cells from culture dishes by trypsinization (Fig. 11,
lane 4) and increased upon stimulation of the cells with
lysophosphatidic acid and exposure of the cells to osmotic stress (Fig.
11, lanes 8 and 10). A tyrosine-phosphorylated band was found above CAK
in the anti-Hic-5 immunoprecipitates; this
band was most prominent when cells were stimulated with
lysophosphatidic acid (Fig. 11, lane 8) but was also found
in cells adhering on dishes and when cells were exposed to osmotic
stress (Fig. 11, lanes 6 and 10). In another
experiment (data not shown), this band above CAK
revealed with
anti-phosphotyrosine was resolved into double bands, an upper major
band and a lower minor band. The upper major band had a mobility slower
than FAK; possible candidates for these bands are phosphorylated FAK,
vinculin, and pp130cas.
|
A Small Portion of CAK Is Present in WFB Cells at the Site of
Focal Adhesions--
It was shown that the localization of FAK at
focal adhesions is dependent on the association of FAK with paxillin
(18), which is targeted to focal adhesions by itself (38). Since CAK
bound Hic-5 and paxillin and a fraction of CAK
coimmunoprecipitated with Hic-5 from the WFB cell lysate, we examined WFB cells by immunostaining to find CAK
at focal adhesions. The major portion of
CAK
in WFB cells was found in the perinuclear region and in the
cytoplasm (Fig. 12A). In the
same cell line, FAK localized at focal adhesions (Fig. 3a).
At the cell periphery of well spread WFB cells, where focal adhesions
were seen by staining with anti-paxillin and anti-vinculin, CAK
was
faintly immunostained at focal adhesions (Fig. 12). Thus, a small
amount of CAK
was found at the sites of focal adhesions in WFB
cells. However, CAK
was immunostained diffusely at the focal
adhesions, not in the well confined, rodlike patchy structure of focal
adhesions, an image obtained by immunostaining vinculin, paxillin, and
Hic-5. When WFB cells were doubly stained for Hic-5 and vinculin, the
localization of Hic-5 at the cell periphery overlapped with that of
vinculin (data not shown).
|
Analysis of Tyrosine Phosphorylation Levels of CAK and Hic-5 in
WFB Cells: Effects of Trypsinization and Replating the Cells onto
Dishes Coated with Fibronectin or Poly-L-lysine--
The
changes in the tyrosine phosphorylation levels of FAK, CAK
, and
Hic-5 were compared by trypsinization of WFB cells and replating the
cells on dishes coated with either fibronectin or poly-L-lysine. The tyrosine phosphorylation levels of FAK
decreased upon detachment of well spread WFB cells from culture dishes
by trypsinization, and the levels were recovered by incubating the cells at 37 °C for 45 and 90 min after plating them onto dishes coated with fibronectin (data not shown). Plating the cells on poly-L-lysine dishes was not effective to recover the
tyrosine phosphorylation level of FAK. These results on FAK obtained in WFB cells are consistent with those reported on FAK in BALB3T3 cells
and NIH3T3 cells (11, 27). In parallel experiments, it was found that
the tyrosine phosphorylation levels of CAK
in WFB cells decreased on
trypsinization and recovered on incubation at 37 °C for 45 min after
plating the cells, but this recovery was observed on plating the cells
not only onto fibronectin dishes but also onto
poly-L-lysine dishes, although the effect of fibronectin was somewhat stronger than the effect of poly-L-lysine
(data not shown). Moreover, a longer incubation of the cells for 90 min after replating did not enhance the tyrosine phosphorylation levels of
CAK
.
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DISCUSSION |
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We identified Hic-5 in a search for CAK-binding proteins by
cDNA expression cloning. Hic-5 had an amino acid sequence highly similar to paxillin. The results shown in this paper demonstrated that
Hic-5 was a component of focal adhesions. This finding disagrees with
the original identification by Shibanuma et al. (23, 24) that Hic-5 is localized in nuclei. A binding of the Hic-5 LIM domain to
certain DNA sequences was postulated (24). We consider that their
assumption that Hic-5 is a nuclear protein was drawn from weak evidence
obtained by the use of a less specific anti-Hic-5 antibody (23),
although a possible function of Hic-5 in the nucleus cannot be excluded
at present. In this paper, we have confirmed the Hic-5 localization at
focal adhesions by immunostaining with anti-Myc in WFB cells expressing
the Myc-tagged Hic-5 from a cDNA construct. The cell biological
functions of Hic-5 should be reevaluated from the standpoint that Hic-5
is a component of focal adhesions. Recently, Brown et al.
(38) showed that LIM domains are responsible for targeting paxillin to
focal adhesion. They identified the LIM3 domain as the principal
determinant of paxillin localization at focal adhesions. The four
contiguous LIM domains of Hic-5 are most similar to those of paxillin
among the known LIM domains (42); the highest similarity is found in
each corresponding domain. Among the corresponding four LIM domains of
Hic-5 and paxillin, the LIM3 domains of these proteins are most highly
conserved with an identity of 70.6% in the amino acid sequences. In
addition to a novel role of the LIM domain as protein dimerization
motifs (36), it has been proposed that the LIM domain recognizes a
tyrosine-containing tight turn (37). The identity of the protein
recognized by the LIM3 domains of paxillin and Hic-5 is most
interesting because Hic-5 may also be targeted to focal adhesions
through its LIM3 domain.
We found that the Hic-5 N-domain directly associated in
vitro with the C-terminal regions of CAK and FAK (Figs. 5 and
6). Moreover, the association of Hic-5 with CAK
was shown in the lysates of WFB cells and of those COS-7 cells that were doubly transfected with CAK
and Hic-5 cDNA constructs. Brown et
al. (38) localized on paxillin the binding sites of FAK and
vinculin to small stretches of amino acid sequences, which they named
LD motifs. They pointed out high sequence similarity between paxillin and Hic-5 in the LD motifs. The paxillin-binding sites on FAK and
vinculin have also been identified by Tachibana et al. (18) and named paxillin-binding subdomains 1 and 2 (PBS1 and PBS2). These
subdomains are also found in CAK
at amino acid residues 875-891
(PBS1) and 990-998 (PBS2). The PBS1 and PBS2 of FAK and CAK
are
highly conserved, being 58.8% identical in PBS1 and 77.8% identical
in PBS2. The Hic-5 N-domain bound to the CAK
C-domain and its
fragment containing both PBS1 and PBS2 (Figs. 5 and 6).
The presence of Hic-5 in association with CAK in the WFB and COS-7
cell lysates (Figs. 7, 8, and 11) suggested that Hic-5 was a component
in signaling pathways downstream of CAK
. The results shown in Figs.
7 and 8 are consistent with the prediction that the CAK
amino acid
residues containing both PBS1 and PBS2 are essential for the binding of
CAK
to Hic-5. In the deletion mutant of CAK
, dl 741-903, PBS2
was present, but PBS1 was deleted. In addition to the association of
Hic-5 with CAK
, the association of paxillin with CAK
and that of
Hic-5 with FAK were also found. It is possible that there are
differences in affinities of the associations between FAK/CAK
and
paxillin/Hic-5. In this relation, we noted that the GST fusion protein
of the full-length CAK
, GST-CAK
(fl), had much less affinity to
paxillin than to Hic-5 (Fig. 6, lane 4). Moreover, the
results in Fig. 6 suggest less binding of Hic-5 to GST-FAK-CdomB than
to GST-CAK
-CdomB.
A high sequence similarity was found between the first LD motif of
paxillin and the Hic-5 amino acid sequence translated from the
"5-untranslated region," which was presumed on mouse Hic-5 cDNA by Shibanuma et al. (23), of human Hic-5 cDNA,
cbp-1 (Fig. 1). The published mouse Hic-5 amino acid
sequence (23) contains only three LD motifs lacking the one
corresponding to the first LD motif of paxillin. The translational
initiation site of the Hic-5 cDNA presumed by Shibanuma et
al. (23) does not fit to the sequence context for translational
initiation (39) and may not be the true translational initiation site
of the Hic-5 mRNA. The level of Hic-5 protein expression in the
cDNA transfected cells has not been shown by these authors (23,
24). The expression of Hic-5 from constructs of the original human
Hic-5 cDNA, cbp-1, was quite low in the transfected
cells (data not shown). We will continue our efforts to identify the
N-terminal methionine and to sequence further 5
-upstream region of
Hic-5 cDNA by isolating other Hic-5 cDNA clones.
Although Hic-5 is almost exclusively localized at focal adhesions in
WFB cells (Fig. 3) and CAK has specific affinity to Hic-5 and
paxillin, only a limited, small fraction of CAK
localized at focal
adhesions in WFB cells (Fig. 12). This subcellular localization of
CAK
was different from that of FAK, a large portion of which was
found at focal adhesions in WFB cells (Fig. 3). The major portion of
CAK
in WFB cells is present in the perinuclear region and cytoplasm
(Fig. 12). In cultured epithelial cells, CAK
was also found at the
cell-to-cell borders in addition to the perinuclear regions and
cytoplasm. A small amount of CAK
present at focal adhesions in WFB
cells was found only by careful examination and was not immunostained
in images of typical components of focal adhesions such as paxillin,
vinculin, Hic-5, and FAK (Fig. 12). This intracellular localization of
CAK
was compatible with our finding that CAK
is present in
association with microvilli, cilia, and axons in rat tissues (16).
Tachibana et al. (18) showed that FAK localizes at focal
adhesions through its binding to paxillin. It was shown that the
C-domain of CAK
exclusively localized at focal adhesions when this
domain was singly expressed in chicken embryo fibroblasts from a
designed recombinant cDNA construct (43). It is obvious that CAK
has an intrinsic property of localizing at focal adhesions via its
C-domain. In most cells, however, the major portion of CAK
does not
localize at focal adhesions. The reason for this subcellular
localization of CAK
at sites other than focal adhesions is a focus
of our current study. The regulatory mechanism of CAK
localization
might be important in understanding the unique functions of CAK
different from FAK. We (1) previously showed that the tyrosine
phosphorylation of CAK
is not enhanced in response to plating rat
3Y1 fibroblasts onto fibronectin. Thereafter, different results have
been reported on the changes of the CAK
tyrosine phosphorylation in
response to integrin signaling (14, 44, 45). The subcellular
localization of CAK
shown in Fig. 12 may explain our finding that
the tyrosine-phosphorylation was not regulated by
cell-to-substratum adhesion at focal adhesions.
Although the amino acid sequences of Hic-5 and paxillin are similar at
the LIM domains and also at the LD motifs, the other portions of the
Hic-5 N-domain are not similar to those of the paxillin N-domain.
Paxillin has a role as a signal-integrating protein or a docking
protein. It was shown that the Src family protein kinases associate
with paxillin by their SH3 domains (46). It was also shown that the
transforming protein v-Crk binds with its SH2 domain to the
phosphorylated sequences at tyrosine residues 31 and 118 of the
paxillin N-domain (47-49). Although Hic-5 does not share these ligand
sequences, the proline-rich sequence found at amino acid residues
14-20 and some tyrosine-phosphorylated sequences of Hic-5 may also
have ligand specificities to some SH3 and SH2 domains. A tyrosine
residue at position 43 of Hic-5, which is not found in paxillin, is in
a sequence context indicative of a possible phosphorylation site. The
Hic-5 coimmunoprecipitated with CAK from the lysate of WFB cells
stimulated by osmotic stress was selectively tyrosine-phosphorylated as
compared with the total cellular Hic-5 (Fig. 11). This result indicates
functional coupling of CAK
and Hic-5. However, it remains unanswered
which tyrosine kinase, CAK
, Src, or another one, is responsible for
the phosphorylation of Hic-5. Thus, the portion of the Hic-5 N-domain
outside the LD motifs is probably the site important in signal
transduction. It seems important to study whether the cell biological
effects of Hic-5 reported by Shibanuma et al. (24) are
related to unique downstream signals, which are possibly generated from
Hic-5 but not from paxillin. Because Hic-5 and paxillin have several
properties in common, related but different roles are expected for
their functions.
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ACKNOWLEDGEMENTS |
---|
We thank Dr. Steven K. Hanks (Department of Cell Biology, Vanderbilt University School of Medicine) for providing mouse FAK cDNA, pBSFAK; Dr. J. Chernoff (Fox Chase Cancer Center) for providing pJ3M vector; and Dr. Bruce J. Mayer (Howard Hughes Medical Institute, Children's Hospital, Harvard Medical School) for providing pEBG vector.
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FOOTNOTES |
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* This work was supported in part by grants-in-aid from the Ministry of Education, Science, Sports and Culture, 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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AB007836.
§ The first two authors contributed equally to this work.
** To whom correspondence should be addressed: Dept. of Biochemistry, Cancer Research Institute, Sapporo Medical University School of Medicine, South-1, West-17, Chuo-Ku, Sapporo 060, Japan. Tel.: 81-11-611-2111 (ext. 2380); Fax: 81-11-612-5861; E-mail: sasaki{at}cc.sapmed.ac.jp.
1
The abbreviations used are: FAK, focal adhesion
kinase; CAK, cell adhesion kinase
; N-domain, N-terminal domain;
C-domain, C-terminal domain; SH2, Src homology domain 2; SH3, Src
homology domain 3; PAGE, polyacrylamide gel electrophoresis; PVDF,
polyvinylidene difluoride; ECL, enzyme-linked chemiluminescence; PBS,
phosphate-buffered saline; GST, glutathione S-transferase;
PBS1 and PBS2, paxillin-binding subdomains 1 and 2; bp, base pair(s);
HSV, herpes simplex virus.
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REFERENCES |
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