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INTRODUCTION |
Integrin-dependent cell adhesion helps to control cell
proliferation and apoptosis, as well as cell spreading, migration, morphogenesis, and differentiation (1-5). Upon cell adhesion, integrin
engagement leads to downstream activation of focal adhesion kinase,
mitogen-activated protein kinase, and many other key signaling molecules (6). At the same time, integrin-dependent
reorganization of cytoskeletal proteins and signaling complexes
facilitates growth factor signaling (7, 8). A distinctive property of
integrins is that they not only deliver "outside-in" signals upon
engagement with ligand but also their function is regulated by
"inside-out" signals (9-12). In this regard, integrin function can
be strongly modulated upon overexpression of various oncogenes (13-15)
or upon engagement of various cell-surface receptors with ligands or
antibodies (10, 16, 17).
The cytoplasmic tails of integrin
and
subunits play critical
roles in two-way signaling through integrins (18-20). For the large
subgroup of integrins containing the
1 subunit, the
1 tail is particularly important. For example, each of
the four naturally occurring alternatively spliced forms of the
1 tail has distinctive functions (21-24). In
experimental systems, perturbing the
1 tail can markedly
alter
1 integrin-mediated function. For example,
exchange of the
1 and
5 tails altered
integrin localization into focal adhesions and support of cell
migration and proliferation (25). Also, overexpression of single chain
tail chimeras severely impaired cell adhesion and spreading mediated by endogenous
1 integrins (26-29). In
addition, mutations within the
1 tail can alter integrin
localization (30-32), conformation, and/or ligand binding (33-35) and
1 integrin-mediated endocytosis (36). Amino acids
particularly important for
1 localization into focal
adhesions have been mapped to three
1 tail subregions called cyto-1, cyto-2, and cyto-3 (32).
In vitro biochemical studies have suggested that the
1 tail may directly interact with focal adhesion kinase
(37) and with cytoskeletal proteins
-actinin (38), talin (39, 40),
paxillin (37, 41), and filamin (40). Additionally, yeast two-hybrid screening has implicated integrin-linked kinase (42), receptor for
activated protein kinase C
(RACK1)1 (43), and integrin
cytoplasmic domain associated protein (ICAP-1) (44) as proteins that
may interact directly with the
1 tail. Many of these
proposed direct interactions still need to be further explored in terms
of biochemistry and functional relevance.
We also have undertaken a yeast two-hybrid screen to identify
1 tail-associated proteins. In the yeast, we initially
identified two candidate
1 tail-interacting proteins.
These were (i) a fragment of RACK1 and (ii) a protein called ICAP-1
(integrin cytoplasmic tail associated protein). Additional yeast
two-hybrid studies suggested that the RACK1 interaction was
nonspecific. However, the ICAP-1 protein did show specific interaction
with the
1 tail, both in yeast and in human cell lines.
Furthermore, the site of ICAP-1 association was mapped to the 14 carboxyl-terminal amino acids of
1 (which includes an
NPXY motif); ICAP-1 phosphorylation was found to be
regulated upon cell spreading on fibronectin, and ICAP-1 appeared to
play a role in
1-dependent cell migration.
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EXPERIMENTAL PROCEDURES |
Cell Lines and Antibodies--
Human embryonic kidney 293 (HEK293), monkey kidney (COS7), and Chinese hamster ovary (CHO) cell
lines were obtained from the American Type Culture Collection
(Bethesda, MD). Anti-hemagglutinin (HA) monoclonal antibody (mAb) 12CA5
was from Dr. J. DeCaprio (Dana-Farber Cancer Institute, Boston), and
rabbit anti-ICAP-1 polyclonal antibodies were either produced using
purified full-length ICAP-1 polypeptide as immunogen or provided by Dr.
D. Chang (UCLA). Other antibodies were as follows: anti-human integrin
1, mAb A-1A5 (45); anti-hamster
1, mAb
7E2 (46); anti-hamster
5 mAb PB1 (47), and negative
control mAb P3 (48).
Yeast Two-hybrid Screening--
Yeast genetic screening for
proteins that bind to the integrin
1 cytoplasmic tail
was carried out essentially as described (49, 50). Integrin
1 subunit cDNA encoding for carboxyl-terminal amino
acid residues 750-798 (see Table I) was amplified from full-length
1 cDNA by polymerase chain reaction (PCR). This PCR product was ligated into plasmid pEG202N to generate the LexA-integrin fusion "bait" plasmid pEG202-
1. Host yeast strain EGY48 (MAT
, his3, trp1, ura3-52, leu2::pLEU 2-LexAop6, constructed by E. Golemis, Massachusetts General Hospital, Boston) was cotransformed with pEG202N bait and pSH18-34 reporter plasmids to verify that the bait
plasmid is itself transcriptionally inert. Also, by using the pJK101
reporter plasmid we confirmed that baits (LexA-integrin cytoplasmic
tail fusions) could be expressed inside the nucleus of EGY48 yeast
cells and bind to LexA operator.
A yeast expression library with a complexity of 106 was
generated from oligo(dT)-primed cDNA from HeLa (human cervical
carcinoma cell line) mRNA. The cDNA was cloned unidirectionally
into the EcoRI/XhoI sites of galactose-inducible,
TRP+ yeast expression vector pJG4-5 (constructed by J. Gyuris,
Massachusetts General Hospital, Boston). For genetic screening, yeast
strain EGY48 was transformed sequentially with pEG202-
1, pSH18-34,
and pJG4-5 using the lithium acetate method (51). Approximately 2 × 106 yeast transformants were pooled and subjected to
selections as described (49, 50). Positive interaction is defined as
(i) growth on leucine-deficient, galactose-conditioned medium but not
on leucine-deficient, glucose-conditioned medium, and (ii) forming blue
colonies on galactose X-gal plates but not on glucose X-gal plates.
Plasmid DNAs from positive colonies were rescued using
Escherichia coli KC8. Retransformation of EGY48 with prey plasmid DNA, pSH18-34, and pEG202N-
1cyto plasmid DNA was done to
confirm the interaction. As described above for pEG202-
1, other bait
plasmids were constructed to encode for the various integrin
cytoplasmic domains listed in Table I. Also constructed were bait
plasmids containing chimeric
1/
5 integrin
cytoplasmic domains (bottom of Table I).
Cloning of Full-length ICAP-1 cDNA--
A human HeLa S3
5'-stretch plus cDNA library in bacterial phage lambda gt11 vector
(CLONTECH, Palo Alto, CA) was plated and transferred to Colony/Plaque Screen membrane (NEN Life Science Products) according to the manufacturer's protocol. The library was
screened by in situ hybridization (52) with
32P-labeled insert as a probe (obtained from Clone
No. 4, Fig. 1). After an additional two rounds of purification,
eight positive lambda phage clones were obtained, and the cDNA
inserts were sequenced using a double-stranded DNA cycle sequencing kit
(Life Technologies, Inc.).
Construction and Expression of Plasmids--
For prokaryotic
expression of GST fusion proteins, the cytoplasmic domain of
1 was ligated into pGEX vector (Amersham Pharmacia Biotech, Piscataway, New Jersey) and then expressed in E. coli DH5
. The GST-
1cyto fusion proteins were
purified on glutathione-conjugated Sepharose beads (Pharmacia Biotech,
Uppsala, Sweden) as described (53). Vectors pGEX-
2cyto,
pGEX-
3cyto, and pGEX-
5cyto were provided
by Dr. A. Arnaout (Massachusetts General Hospital, Boston), Dr. E. Ruoslahti, and Dr. R. Pasqualini (Burnham Institute, La Jolla, CA),
respectively. Production and purification of GST-
2cyto, GST-
3cyto, and GST-
5cyto fusion proteins
was as described (53). Full-length ICAP-1 cDNA was ligated into
EcoRI/XhoI sites of pGEX-4T-1, and the resulting
pGEX-4T-1-ICAP-1 fusion protein was purified from E. coli
BL21 (Novagen, Madison, WI).
For stable eukaryotic expression, we used the pECE vector containing a
full-length
1 insert (54) which is designated here as
pECE-
1cyto1.1. The pECE-
1cyto5.5
construct codes for wild type
1 extracellular and
transmembrane domains fused to the
5 cytoplasmic domain
(25). Additional
1/
5 cytoplasmic tail
exchange mutants, in the pECE vector, were generated by sequential PCR (52). These contained
1 extracellular and transmembrane
domains, fused to
1,
5, or
1/5 chimeric cytoplasmic tails listed at the bottom of
Table I.
CHO cells negative for dihydrofolate reductase gene (dhfr
) were grown
in MEM
+ medium with 10% FCS, and then switched to MEM 
with
10% dialyzed fetal calf serum (JRH Biosciences, Lenexa, KS) after
transfection. The dhfr+ p901 vector (55) was provided by Dr. M. Rosa
(Biogen Co., Cambridge, MA). For transfection, CHO cells were
electroporated with a mixture of p901 dhfr+ plasmid DNA and
pECE-
1 or -
1/
5 mutant
plasmid DNA at a ratio of 1 to 10, using a gene pulser (Bio-Rad) set at
280 V and 960 microfarads. Growth of transfected CHO cells in MEM 
medium and selection of positive clones by flow cytometry were carried
out as described (25). The CHO-
1 and
-
1/
5 mutants were selected to have
comparable surface expression levels as measured by flow cytometry
using anti-
1 mAb A-1A5.
For eukaryotic transient expression, full-length ICAP-1 cDNA was
ligated in frame into a modified pMT2HA vector (56) to form
pMT2HA-ICAP-1, which encodes for the influenza hemagglutinin (HA)
antigen epitope just upstream of ICAP-1. Calcium phosphate (52) was
used to transiently transfect pMT2HA-ICAP-1 into HEK293 or COS7 cells,
and cells were analyzed after 48 h. Immunofluorescence analysis
revealed that ICAP was typically expressed in 20-40% of HEK293 cells,
and transfection into COS7 was at least as efficient.
In Vitro Immunoprecipitation and GST Fusion Protein
Assays--
The HEK293 cell line was labeled with ~0.15 mCi
[32P]orthophosphate (10 mCi/ml) in sodium
phosphate-deficient DMEM supplemented with 10% dialyzed FCS and
antibiotics. Labeling was typically begun at 48 h after HEK293
cell transfection and continued for 3 h unless otherwise
indicated. For immunoprecipitation, cells were lysed either in Triton
X-100 buffer (1% Triton X-100, 25 mM HEPES, pH 7.4, 150 mM NaCl, 5 mM MgCl2, 2 mM phenylmethylsulfonyl fluoride, 20 µg/ml aprotinin, and
10 µg/ml leupeptin) or in RIPA buffer (Triton X-100 buffer
supplemented with 0.2% SDS and 1% deoxycholate) at 4 °C for 1 h. After centrifugation at 12,000 rpm for 10 min, soluble material was
precleared by incubation with normal rabbit serum immobilized on
protein A-Sepharose 4B beads (Amersham Pharmacia Biotech) at 4 °C
for 1 h. Next, immune complexes were collected on protein A beads
already pre-bound with rabbit anti-ICAP-1 antibodies and washed four
times with cell lysis buffer. Immunoprecipitated proteins were
separated on SDS-polyacrylamide gel electrophoresis, and then dried
gels were exposed with O-Xar film (Eastman Kodak Co.) for 1-7 days at
70 °C.
For GST fusion protein assays, HEK293 and CHO cell lysates (prepared as
above) were incubated with glutathione-conjugated Sepharose beads
(Amersham Pharmacia Biotech) for 1 h to remove background binding
material. Lysates were then incubated overnight at 4 °C with GST or
GST fusion proteins pre-bound to glutathione-conjugated Sepharose
beads. Beads were then washed three times with lysis buffer, and bound
proteins were eluted in Laemmli sample buffer and subjected to
SDS-polyacrylamide gel electrophoresis under reducing conditions.
Separated proteins were electrophoretically transferred to
nitrocellulose membranes (Schleicher & Schuell) at 4 °C overnight.
Membranes were blocked with 5% fat-free dried milk in PBS/Tween 20 buffer at 25 °C for 1 h and then sequentially blotted with
specific mAb and horseradish peroxidase-conjugated goat anti-mouse IgG
antibody, followed by four washes (15 min each) with PBS/Tween 20 buffer after each blot. Proteins were visualized using Renaissance
chemiluminescent assay (NEN Life Science Products).
Cell Migration Assay--
Migration assays were performed
essentially as described (25), using 96-well chambers and framed
polycarbonate filters with 8-µm pores (Neuroprobe, Cabin John, MD).
Filters were spotted with fibronectin, vitronectin, or
poly-L-lysine diluted in 0.1 M
NaHCO3, allow to dry, rinsed with PBS, and assembled with
matrix-side down in the chamber. Lower wells of the chamber contained
33 µl of MEM
medium (for CHO cells) or DMEM (for COS7 cells) with 10% FCS, unless indicated otherwise. Cells harvested in PBS with 2 mM EDTA were labeled using BCECF-AM
(2',6'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein acetoxymethyl ester;
Molecular Probes, Eugene, OR) for 30 min, pelleted, and resuspended at
3 × 105 cells/ml in 1% FCS (for CHO cells) or 0.1%
FCS (for COS7 cells). After no preincubation (COS7 cells) or with
anti-hamster
5
1 mAb PB1 for 30 min on ice
(CHO cells), cells (suspended in 100 µl) were added to upper wells of
the chamber and allowed to migrate at 37 °C for 4 h. After
migration, cells attached to the upper side of the filter were
mechanically removed by scraping, and cells on the lower side were
quantitated using a Cytofluor 2300 fluorescence measurement system
(Millipore Corp., Bedford, MA). Percent cell migration equals: (cell
fluorescence on filter with matrix coating
control cell
fluorescence on filter without matrix)/(total fluorescence of input
cells) × 100.
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RESULTS |
Yeast 2-Hybrid Selection and Cloning of
1 Integrin Tail-binding Proteins--
A
HeLa cell library was expressed in 2 × 106 yeast
transformants, selection for interaction with the integrin
1 cytoplasmic tail protein was carried out, and 25 positive clones were obtained. Among these clones, 9 coded for protein
fragments that included the carboxyl-terminal half of the receptor of
activated protein kinase C (RACK1) protein. The carboxyl-terminal half
of RACK1 interacted strongly with
1, weakly with
5, and not at all with
2 or
3
integrin tail bait proteins. However, yeast 2-hybrid analyses also
revealed interactions between the RACK1 carboxyl-terminal fragment and
integrin
V and
4 cytoplasmic tail bait
proteins (Table II). Because the
V and
4
tail sequences show no obvious similarity to the
1 tail
(Table I), the RACK1 interactions
appeared to be nonspecific and were not pursued further.
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Table I
Amino acid sequences of integrin cytoplasmic domains and integrin
1/ 5 cytoplasmic tail mutants studied in the yeast
two-hybrid system
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Another 7 of the initial 25 positive clones coded for a related group
of polypeptides, with identical carboxyl termini but variable amino
termini (Fig. 1). These results suggest
that regions essential for interaction with the integrin
1 tail reside within the 162 residues present in the
shortest clones (clones 4 and 5). Two of the polypeptide sequences
contained divergent amino termini (clones 6 and 7), which did not
appear in full-length clones (as obtained below), and thus may be
cloning artifacts.

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Fig. 1.
Schematic representation of related
"ICAP-1" polypeptides interacting with integrin 1
cytoplasmic tail in yeast.
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Open reading frames coding for the longest polypeptides did not include
a methionine start site. Thus, to obtain a full-length sequence for the
ICAP-1 protein, we used a cDNA probe corresponding to clone 4 to
screen a bacteriophage lambda gt11-HeLa cell cDNA library. The
resulting sequence contained a putative ATG start codon, just upstream
of the sequence represented in clone 1. This methionine is present in a
near consensus translation initiation sequence (57) and is located
downstream of stop codons in all three frames, suggestive of an
authentic start codon (Fig. 2).
The full-length ICAP-1 consists of 200 amino acids and is rich in
serine (16%), with the amino-terminal 50 amino acids containing 19 serine residues. There are three possible protein kinase C phosphorylation sites at Ser-20 and Ser-46 and Ser-197 (58), and one
cAMP or cGMP-dependent protein kinase phosphorylation site
at Ser-10 (59). No signaling motifs or domains, such as SH2 or SH3,
were found in ICAP-1. Subsequent to our isolation of ICAP-1, an
identical protein was described and named "ICAP-1" (44). Also, an
unpublished sequence coding for the mouse homologue of ICAP-1 has
appeared in GenBankTM (accession number AJ001373).
Interaction of ICAP-1 with the Integrin
1
Cytoplasmic Tail Is Highly Specific--
In the context of the yeast
two-hybrid system, ICAP-1 (as a pJG4.5-ICAP-1 prey construct)
interacted strongly with the
1 tail but failed to
interact with the integrin
2,
3, or
5 tails (Table II). Also
ICAP-1 did not associate with 7 different integrin
chain tails
(Table II). All of the pEG202-encoded bait proteins containing integrin
or
chain cytoplasmic domains were able to bind to LexA but by
themselves were transcriptionally inert, thus they meet the criteria
for bait constructs suitable for study in the two-hybrid system. In
other yeast two-hybrid experiments, ICAP-1 failed to interact with
additional bait proteins including phosphatidylinositol 3-kinase 85-kDa
subunit, Max, v-Myc, p300 CH3 domain, CD2 cytoplasmic domain, and the
LAR phosphatase cytoplasmic domain.
To determine the subregion of the
1 cytoplasmic domain
that is critical for ICAP-1 association, we utilized bait plasmid pEG202 to synthesize chimeric
1/
5
cytoplasmic tail mutants (listed in Table I, bottom). In yeast, both
the wild type
1 tail (cyto.11) and the cyto.51 chimera
showed strong interaction with ICAP-1, whereas cyto.15 and cyto.55 did not.
Tissue Expression and Biochemical Features of ICAP-1--
Northern
blotting showed that ICAP-1 mRNA is present in nearly all human
tissues (Fig. 3). It was highly expressed
in heart, colon (mucosal lining), skeletal muscle, and small intestine, barely detectable in liver, and present at intermediate levels in all
other tissues. The major ICAP-1 transcript was 1.2 kilobase pairs, with
variable amounts of another form at ~1.8 kilobase pairs.

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Fig. 3.
Tissue expression of ICAP-1 mRNA.
Filters containing mRNA from multiple human tissues
(CLONTECH) were used for Northern blotting
according to manufacturer's instructions. ICAP-1 cDNA probe was
prepared by EcoRI/XhoI digestion from pJG4.5
vector and labeling with [ -32P]dCTP using RadPrime DNA
kit (Life Technologies, Inc.). After stripping of the ICAP-1 probe,
filters were rehybridized with 32P-labeled human actin
cDNA. kb, kilobase pair; PBL, peripheral
blood lymphocyte.
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Anti-ICAP-1 antiserum immunoprecipitated a protein of ~27 kDa from
Triton X-100 lysate of 35S-labeled ICAP-1-transfected
HEK293 cells that was not obtained from mock-transfected cells and was
not seen using preimmune rabbit serum (not shown). A protein of ~27
kDa was also obtained upon anti-HA Western blotting of HA-tagged ICAP-1
from Triton X-100 lysate of ICAP-1-transfected COS7 cells (data not
shown). From A431 cells, HeLa cells, Jurkat cells, human endothelial
cells, and human fibroblasts lysed in RIPA buffer, anti-ICAP-1
antiserum blotted endogenously expressed ~27- and ~31-kDa proteins,
with the latter being particularly prominent in endothelial cells and fibroblasts (not shown). Whereas the ~27-kDa protein was routinely observed when using mild detergent extraction (e.g. Fig.
4, lanes 3 and 4),
visualization of the ~31-kDa protein was enhanced by use of stringent
detergent lysis conditions (Fig. 4, lanes 7 and 8). Analysis of the Triton X-100 pellet revealed that the
~31-kDa protein was indeed retained in the Triton-insoluble fraction
(compare lanes 9 and 10). In contrast, analysis
of the RIPA-insoluble fraction (lane 12) indicated that the
majority of both 27- and 31-kDa proteins had already been extracted
(lane 11). Adhesion to fibronectin (in comparison to cell
suspension) did not alter the appearance of either form of ICAP-1
protein (Fig. 4, compare lanes 3 and 4 and
7 and 8).

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Fig. 4.
Characterization of ICAP-1 protein.
Unlabeled HA-ICAP-1-HEK293 and mock-HEK293 cells were either allowed to
adhere to fibronectin for 2 h at 37 °C (Adhe.) or
held in suspension for 2 h (Susp.) prior to lysis with
1% Triton X-100 or RIPA buffer (lanes 1-8). Alternatively,
HA-ICAP-1-HEK293 cells were lysed (with Triton or RIPA) in suspension
at 4 °C for 30 min (lanes 9 and 11), and
insoluble materials were further solubilized in Laemmli sample buffer
(lanes 10 and 12). After separation by
SDS-polyacrylamide gel electrophoresis, Western blotting was carried
out using anti-HA mAb 12CA5.
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Reciprocal Demonstration of
1 Tail-ICAP-1
Interaction in Mammalian Cells--
To investigate the interaction of
the
1 tail with mammalian cell ICAP-1, immobilized
tail GST fusion proteins were incubated with soluble HA-ICAP-1 from
lysates of transfected HEK293 cells. As indicated by Western blotting
with anti-HA mAb (Fig. 5A),
HA-tagged ICAP-1 bound selectively to GST-
1 (lane
2) but not to GST itself, GST-
2,
GST-
3, or GST-
5 fusion proteins
(lanes 1 and 3-5). We estimate that ~1-2% of
the total ICAP-1 in the cell lysate bound to the GST-
1
beads. In control experiments, ICAP-1 was readily visualized from a
whole cell lysate of ICAP-1-transfected HEK293 cells (lane
11) but not from mock-transfected HEK293 cells (Fig. 5A,
lanes 6-10 and 12).

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Fig. 5.
Reciprocal interactions between ICAP-1 and
1 integrin from mammalian cell lysates.
A, lysates from ICAP-1-transfected HEK293 cells (lanes
1-5) or mock-transfected cells (lanes 6-10) were
incubated with GST fusion proteins immobilized on beads, and then bound
proteins were eluted and detected by Western blotting with anti-ICAP-1
antiserum. Also shown are whole cell lysates from ICAP-1 (lane
11) and Mock (lane 12)-transfected HEK293 cells.
B, lysates from the indicated CHO cell transfectants were
incubated with immobilized GST-ICAP-1 fusion protein, and bound
proteins were detected by Western blotting with anti-
mAb A-1A5.
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In a reciprocal experiment we next analyzed binding of solubilized
1 integrin to immobilized GST-ICAP-1. First, CHO cells were transfected to stably express wild type or mutant human
1 subunits. In each case, the
1
extracellular and transmembrane domains were present, whereas the
cytoplasmic tail was either unaltered or fully or partly exchanged with
regions of the
5 tail (See Table I, bottom, for
sequences). Upon incubation with GST-ICAP-1 fusion protein, selective
binding of wild type
1 (
1cyto.11) and
1cyto.51, but not
1cyto.55 or
1cyto.15, was observed (Fig. 5B), as detected
by Western blotting with anti-human
1 mAb A-1A5. No
1 was found to associate with immobilized GST control
protein (not shown). Wild type human
1 and various tail
mutants were present in CHO cells at comparable levels as seen by
cell-surface flow cytometry (Fig. 6) and
also as indicated by blotting with anti-human
1 mAb
A-1A5 (not shown).

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Fig. 6.
Surface expression of human integrin
1 and 1/5 mutants in CHO cells.
Transfected CHO cells were incubated with negative control mAb P3,
anti-hamster 1 mAb 7E2, or anti-human 1
mAb A-1A5. Then cells were washed, incubated with fluorescein-coupled
goat anti-mouse antibodies (Calbiochem), and analyzed by flow cytometry
as described previously (66).
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Regulation of ICAP-1 Phosphorylation--
Because of the high
serine composition and putative protein kinase C phosphorylation sites,
we tested whether ICAP-1 might be phosphorylated. First,
ICAP-1-transfected HEK293 cells were incubated with
[32P]orthophosphate for 2 h while in suspension, and
for another 1 h while spreading, prior to lysis using RIPA
buffer. Then, anti-ICAP-1 antibody was used to immunoprecipitate
phosphorylated proteins of ~27 and 31 kDa from
32P-labeled HEK293 cells (Fig.
7A, lanes 3 and 4).
These proteins were not precipitated using preimmune serum or from
mock-transfected cells (lanes 1, 2, and 5-8).
Notably, phosphorylation was enhanced by ~2-fold for the 27-kDa
protein, and 1.5-fold for the 31-kDa protein when ICAP-1-transfected
HEK293 cells were spread on FN (lane 4) compared with
poly-L-lysine (lane 2). In contrast, the level
of phosphorylation of background proteins was unchanged as determined
by comparison of protein band densities (FN/PLL ratios = 1.0). A
long exposure of Fig. 7A confirmed that none of the many
phosphorylated non-ICAP-1 proteins (including Control Band 1 and
Control Band 2) were altered. In a separate experiment (not shown),
phosphorylation of 27- and 31-kDa ICAP-1 proteins was again increased
(by 1.8- and 2.0-fold), respectively, upon adhesion to fibronectin
compared with PLL. Again, phosphorylation of all other (non-ICAP-1)
proteins was unchanged.

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Fig. 7.
Effects of cell adhesion on ICAP-1
phosphorylation. A, ICAP-1-HEK293 and mock-HEK293 cells
were incubated with [32P]orthophosphate while in
suspension for 2 h. Then these cells were plated on plastic
surfaces that had been pre-coated with either 10 µg/ml fibronectin
(Life Technologies, Inc.) or 10 µg/ml PLL (Sigma) and blocked with
0.1% heat-inactivated bovine serum albumin (Sigma). An additional
incubation with [32P]orthophosphate (in phosphate-free
DMEM) was then carried out for 1 h at 37 °C while cells were
adhering and spreading. Cells were then washed, lysed in RIPA buffer,
and immunoprecipitated as described under "Experimental
Procedures," using the indicated antibodies. The indicated ratios
(FN/PLL) were determined using integrated density values
(AlphaImager 2000 Documentation & Analysis System, Alpha Innotech Co.,
San Leandro, CA) for phosphorylated protein bands obtained from cells
on fibronectin and poly-L-lysine. B,
ICAP-1-transfected HEK293 cells were incubated as in A,
except that adhesion to fibronectin was carried out for various
periods.
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A report elsewhere (44) has suggested that the more slowly migrating
form of ICAP-1 may represent a phosphorylated form of the protein that
may appear at elevated levels upon cell adhesion and spreading on
fibronectin for 15 or 30 min (44). Thus, to supplement our results
obtained upon adhesion to fibronectin for 1 h (Fig.
7A), we analyzed additional time points (Fig.
7B). At no time point from 15 to 120 min did we observe that
the slowly migrating form of ICAP-1 (~31 kDa) was highly
phosphorylated relative to the 27-kDa protein, even though the 31-kDa
protein was well represented (e.g. see Fig. 4). Indeed,
under identical extraction conditions, the 31/27-kDa ratio was 0.42 in
terms of total protein but only 0.13 in terms of phosphorylated protein.
ICAP-1 May Contribute to Cell Migration--
In further
experiments, COS7 cells transiently transfected with ICAP-1 were found
to undergo increased transwell migration, when the FCS chemoattractant
was held constant at 10% and different FN levels were coated onto the
underside of the filter (Fig. 8A, left panel). Also, ICAP-COS7 cells showed preferential migration compared with Mock-COS7 cells when FN coating was held constant at 10 µg/ml and different FCS chemoattractant levels were used (Fig.
8A, right panel). Although ICAP-1 caused an elevation of
1-dependent migration on fibronectin, it did
not alter
1-independent migration on vitronectin, as
seen in two separate experiments (Fig. 8B, right and
left panels). Because COS cells express moderate to high
amounts of
V and
5, but little
3, we suspect that vitronectin-dependent migration is largely mediated by
V
5.

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Fig. 8.
ICAP-1 effects on cell migration.
Migration that was both chemotactic and haptotactic was carried out as
described under "Experimental Procedures." A, migration
of ICAP-transfected COS7 cells was determined using porous filters
coated on the underside with various doses of fibronectin with 10% FCS
in the lower well (left panel), or using 10 µg/ml
fibronectin with different doses of FCS in the lower well (right
panel). B, filters were coated with either 10 µg/ml
fibronectin or 10 µg/ml vitronectin, with 10% FCS in the lower well.
Two separate experiments were carried out on different days, using
cells derived from different transfections. In each experiment, a
common pool of transiently transfected COS7 cells was divided for
testing on the two different substrates. In each of the seven
experiments on fibronectin (A and B), ICAP-COS7
migration was significantly greater than Mock-COS7 migration
(two-tailed p value < 0.008). Migration on vitronectin
was not significantly different. C, migration of
CHO- 1 and - 1/5 transfectants was
determined using 10 µg/ml fibronectin coating and 10% FCS in the
lower well, with 1% FCS in the upper well. For all migration
experiments (A-C) each data point represents the mean ± S.D. from six replicates.
|
|
CHO transfectants stably expressing comparable surface levels of human
wild type or chimeric
1 (see Fig. 6) were also tested for migration. The assay was performed in the presence of anti-hamster
5
1 mAb PB1 to block the contribution of
endogenous hamster
5
1 (Fig.
8C). The CHO-
1.cyto11 and
-
1.cyto51 transfectants showed substantially more
migration than either the CHO-
1.cyto55 or -
1cyto1.5
transfectants. This differential migratory behavior precisely coincides
with the differential abilities of these mutants to bind to ICAP-1
(e.g. as seen in Table I and Fig. 5B). In the absence of 10% FCS as a chemoattractant, none of the cells showed very
much migration (not shown).
 |
DISCUSSION |
Specific Association of ICAP-1 with Integrin
1
Tail--
Here we have identified and characterized ICAP-1, a 200 amino acid phosphoprotein specifically associating with the
1 integrin tail. Interaction seen in a yeast two-hybrid
assay was confirmed in reciprocal experiments using ICAP-1- and
1 integrin-transfected human and hamster cell lysates.
In both systems the association was highly specific. In both mammalian
cell lysates and in yeast, replacement of the carboxyl-terminal 14 amino acids of
1 with the carboxyl-terminal 24 amino
acids of
5 resulted in loss of ICAP-1 association.
Conversely, the reciprocal exchange (
5 tail with
terminal 14 residues from
1) allowed strong association. Thus the carboxyl-terminal "SAVTTVVNPKYEGK" sequence in
1 is required for ICAP-1 interaction. While this work
was in progress, another group described ICAP-1 as a protein that
associated selectively with the integrin
1 tail (44).
Consistent with results shown here, residues critical for ICAP-1
association resided within the carboxyl-terminal 13 residues of the
1 tail (44).
Association of
1 Tail with RACK1?--
In another
report, the carboxyl-terminal portion of RACK1 was isolated by a yeast
two-hybrid approach and suggested to interact specifically with
integrin
1,
2, and
5 tails
(43). We also obtained a carboxyl-terminal fragment of RACK1 upon yeast
two-hybrid screening but did not study it further due to an apparent
lack of interaction specificity. Although it is still possible that RACK1 could specifically participate in integrin functions, future studies will need to explain its ability to bind to multiple peptide sequences that are seemingly unrelated.
Distribution and Size of ICAP-1--
Northern blotting showed that
the ICAP-1 protein is widely expressed in many human tissues, as
previously shown for the
1 integrin subunit. Also by
Western blotting, ICAP-1 was ubiquitously expressed in most cultured
cell lines. It is not yet clear whether the appearance of RNA of two
different sizes (1.8 and 1.2 kilobase pairs) represents alternative
splicing or different polyadenylation sites as previously suggested
(44). Chang et al. (44) described an apparent alternatively
spliced 16-kDa form of ICAP-1 (ICAP-1
) that lacks amino acids
128-177 and does not interact with integrin
1
cytoplasmic domain. We did not observe such a form while screening for
full-length ICAP-1, possibly because we screened a different cDNA library.
In analyses of several cell lines, and cells transfected with ICAP-1
cDNA, we detected major (~27 kDa) and minor (~31 kDa) ICAP-1
proteins, with the latter only being seen using stringent detergent
conditions. Both the ~27 and ~31-kDa proteins incorporated 32P label, with phosphorylation of the more rapidly
migrating ~27-kDa form being particularly prominent. Elsewhere, it
was suggested that the more slowly migrating form of ICAP-1 may be
preferentially phosphorylated, because it disappeared upon incubation
of lysate in the absence of phosphatase inhibitors (44). Our direct
phosphorylation results contradict that conclusion. We cannot explain
why phosphatase inhibitors may have facilitated the maintenance of the
more slowly migrating form, except to suggest that this effect may be
indirect and possibly involve other components in the cell lysate. At
present, the biochemical basis for the larger size of the 31-kDa ICAP-1 protein and its relative resistance to detergent extraction (compared with the 27-kDa protein) are not clear.
Elsewhere it was also shown that appearance of the larger ICAP protein
form was favored upon cell adhesion to fibronectin, whereas it was
greatly diminished when cell matrix interaction was disrupted (44). We
did not observe an adhesion-dependent change in levels of
either the 27- or 31-kDa form of ICAP-1 (e.g. see Fig. 4).
This disparity possibly could be explained by our use of a human
embryonic kidney cell line (HEK293) instead of the osteosarcoma cell
line (UTA-6) used in the other study.
Functional Relevance of
1 Tail Association with
ICAP-1--
Association of the
1 tail with ICAP-1 may
be relevant for multiple reasons. First, phosphorylation of both 27- and 31-kDa forms of ICAP-1 was selectively promoted upon cell adhesion
and spreading on fibronectin but not on poly-L-lysine.
Thus, ICAP-1 phosphorylation appears to be regulated during the
outside-in signaling that occurs upon integrin engagement with ligand.
In future studies, it will be important to place ICAP-1 phosphorylation into the context of established integrin-dependent
signaling events, such as the phosphorylation of focal adhesion kinase,
paxillin, and other downstream targets (6). A previous report suggested that constitutively activated RhoA might down-regulate ICAP-1 phosphorylation (44). In contrast, we found that RhoA.V14 transfection into NIH3T3 cells caused no elevation in ICAP-1 phosphorylation (not
shown). This discrepancy is perhaps easily explained, considering (as
discussed above) that Chang et al. (44) appear not to have been actually measuring ICAP-1 phosphorylation.
Second, ICAP-1 interactions with the integrin
1 tail may
support cell migration. In one set of experiments, expression of ICAP-1
in COS7 cells was associated with increased
1-dependent cell migration on fibronectin
but not
1-independent migration on vitronectin. In
another set of experiments, the carboxyl-terminal amino acids within
the
1 tail that were needed for ICAP-1 association were
also required for enhanced cell migration. The carboxyl terminus of
5 could not substitute for
1 to support
migration. These results may help to explain previously noted
differences between the integrin
1 and
5
tails in terms of supporting cell migration (25).
Other functions known to require the carboxyl-terminal 14 amino acids
of
1 could potentially also involve ICAP-1. For example, the carboxyl-terminal "SAVTTVVNPKYEGK" sequence in the
1 tail includes amino acids (Thr-788, Thr-789, Asn-792,
and Tyr-795 in human
1) that help to regulate integrin
affinity for ligand (33) and amino acids (Asn-792 and Tyr-795) required
for cytoskeletal association (32). Deletion of the carboxyl-terminal 13 amino acids results in loss of integrin co-localization with talin,
-actinin, and focal adhesion kinase (60). Furthermore, alternatively spliced
1B (21) and
1C (61) tails lack
the critical carboxyl-terminal residues present in
1A
and thus should not bind to ICAP-1. This could at least partially
explain why functions of
1A are markedly different from
the functions of
1B or
1C (21, 23). In
this regard, expression of
1B in CHO cells resulted in a
severe reduction of cell motility on fibronectin (62), analogous to the
diminished motility of our non-ICAP-1 binding
1cyto.15 mutant.
Other
1 Tail-associated Proteins--
At present,
the integrin
1 tail has been suggested to interact
directly with cytoskeletal proteins (
-actinin (38), talin (39, 40),
filamin (40), and paxillin (37, 41)), protein kinases (focal adhesion
kinase (37), integrin-linked kinase (42)), and other proteins (RACK1
(43) and ICAP-1 (44)). Interestingly, the ICAP-1 protein shows no
similarity to any of these other proteins, and as far as we are aware,
the ICAP-1 interaction is the only one to map to the carboxyl-terminal
14 amino acids of the
1 tail. In addition, the ICAP-1
protein is completely distinct from proteins that may interact with
other integrin
tails, such as cytohesin-1 (63) and
3-endonexin (64). Despite the growing list of integrin
cytoplasmic tail-associated proteins detected by yeast two-hybrid
screening (65), few if any of these associations have yet been
independently established by more than one laboratory. A strength of
the current report is that now the ICAP-1 interaction with the
1 tail has been independently determined, by at least
two distinct laboratories, upon screening of two different cDNA libraries.
In conclusion, we have demonstrated a direct and specific interaction
between the widely expressed ICAP-1 protein and the widely expressed
integrin
1A cytoplasmic tail. Furthermore, we provide
evidence that the integrin-ICAP-1 interaction may be relevant to cell
migration and adhesion and spreading functions carried out by
1 integrins, and we suggest that phosphorylation of
ICAP-1 could play a role in these events.