Interaction of the Integrin beta 1 Cytoplasmic Domain with ICAP-1 Protein*

Xin A. Zhang and Martin E. HemlerDagger

From the Dana-Farber Cancer Institute and Department of Pathology, Harvard Medical School, Boston, Massachusetts 02115

    ABSTRACT
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ABSTRACT
INTRODUCTION
REFERENCES

In a yeast two-hybrid screen, a protein named ICAP-1 (beta 1 integrin cytoplasmic domain associated protein) associated with the integrin beta 1 cytoplasmic tail but not with tails from three other integrin beta  subunits (beta 2, beta 3, and beta 5) or from seven different alpha  subunits. Likewise in human cells, ICAP-1 associated specifically with the beta 1 but not beta 2, beta 3, or beta 5 tails. The carboxyl-terminal 14 amino acids of beta 1 were critical for ICAP-1 interaction. ICAP-1 is a ubiquitously expressed protein of 27 and 31 kDa, with the smaller form being preferentially solubilized by Triton X-100. Phosphorylation of both 27- and 31-kDa forms was constitutive but was increased by 1.5-2-fold upon cell spreading on fibronectin, compared with poly-L-lysine. Also, ICAP-1 contributes to beta 1 integrin-dependent migration because (i) ICAP-1 transfection markedly increased chemotactic migration of COS7 cells through fibronectin-coated but not vitronectin-coated porous filters, and (ii) support of beta 1-dependent cell migration (in Chinese hamster ovary cells transfected with various wild type and mutant beta 1 forms) correlated with ICAP-1 association. In summary, ICAP-1 (i) associates specifically with beta 1 integrins, (ii) is phosphorylated upon beta 1 integrin-mediated adhesion, and (iii) may regulate beta 1-dependent cell migration.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
REFERENCES

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 alpha  and beta  subunits play critical roles in two-way signaling through integrins (18-20). For the large subgroup of integrins containing the beta 1 subunit, the beta 1 tail is particularly important. For example, each of the four naturally occurring alternatively spliced forms of the beta 1 tail has distinctive functions (21-24). In experimental systems, perturbing the beta 1 tail can markedly alter beta 1 integrin-mediated function. For example, exchange of the beta 1 and beta 5 tails altered integrin localization into focal adhesions and support of cell migration and proliferation (25). Also, overexpression of single chain beta  tail chimeras severely impaired cell adhesion and spreading mediated by endogenous beta 1 integrins (26-29). In addition, mutations within the beta 1 tail can alter integrin localization (30-32), conformation, and/or ligand binding (33-35) and beta 1 integrin-mediated endocytosis (36). Amino acids particularly important for beta 1 localization into focal adhesions have been mapped to three beta 1 tail subregions called cyto-1, cyto-2, and cyto-3 (32).

In vitro biochemical studies have suggested that the beta 1 tail may directly interact with focal adhesion kinase (37) and with cytoskeletal proteins alpha -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 beta 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 beta 1 tail-associated proteins. In the yeast, we initially identified two candidate beta 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 beta 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 beta 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 beta 1-dependent cell migration.

    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 beta 1, mAb A-1A5 (45); anti-hamster beta 1, mAb 7E2 (46); anti-hamster alpha 5 mAb PB1 (47), and negative control mAb P3 (48).

Yeast Two-hybrid Screening-- Yeast genetic screening for proteins that bind to the integrin beta 1 cytoplasmic tail was carried out essentially as described (49, 50). Integrin beta 1 subunit cDNA encoding for carboxyl-terminal amino acid residues 750-798 (see Table I) was amplified from full-length beta 1 cDNA by polymerase chain reaction (PCR). This PCR product was ligated into plasmid pEG202N to generate the LexA-integrin fusion "bait" plasmid pEG202-beta 1. Host yeast strain EGY48 (MATalpha , 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-beta 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-beta 1cyto plasmid DNA was done to confirm the interaction. As described above for pEG202-beta 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 beta 1/beta 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 beta 1 was ligated into pGEX vector (Amersham Pharmacia Biotech, Piscataway, New Jersey) and then expressed in E. coli DH5alpha . The GST-beta 1cyto fusion proteins were purified on glutathione-conjugated Sepharose beads (Pharmacia Biotech, Uppsala, Sweden) as described (53). Vectors pGEX-beta 2cyto, pGEX-beta 3cyto, and pGEX-beta 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-beta 2cyto, GST-beta 3cyto, and GST-beta 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 beta 1 insert (54) which is designated here as pECE-beta 1cyto1.1. The pECE-beta 1cyto5.5 construct codes for wild type beta 1 extracellular and transmembrane domains fused to the beta 5 cytoplasmic domain (25). Additional beta 1/beta 5 cytoplasmic tail exchange mutants, in the pECE vector, were generated by sequential PCR (52). These contained beta 1 extracellular and transmembrane domains, fused to beta 1, beta 5, or beta 1/5 chimeric cytoplasmic tails listed at the bottom of Table I.

CHO cells negative for dihydrofolate reductase gene (dhfr-) were grown in MEM alpha + medium with 10% FCS, and then switched to MEM alpha - 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-beta 1 or -beta 1/beta 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 alpha - medium and selection of positive clones by flow cytometry were carried out as described (25). The CHO-beta 1 and -beta 1/beta 5 mutants were selected to have comparable surface expression levels as measured by flow cytometry using anti-beta 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 alpha  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 alpha 5beta 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.

    RESULTS

Yeast 2-Hybrid Selection and Cloning of beta 1 Integrin Tail-binding Proteins-- A HeLa cell library was expressed in 2 × 106 yeast transformants, selection for interaction with the integrin beta 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 beta 1, weakly with beta 5, and not at all with beta 2 or beta 3 integrin tail bait proteins. However, yeast 2-hybrid analyses also revealed interactions between the RACK1 carboxyl-terminal fragment and integrin alpha V and alpha 4 cytoplasmic tail bait proteins (Table II). Because the alpha V and alpha 4 tail sequences show no obvious similarity to the beta 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 beta 1/beta 5 cytoplasmic tail mutants studied in the yeast two-hybrid system

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 beta 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 beta 1 cytoplasmic tail in yeast.

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).


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Fig. 2.   Sequences of ICAP-1 cDNA and protein.

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 beta 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 beta 1 tail but failed to interact with the integrin beta 2, beta 3, or beta 5 tails (Table II). Also ICAP-1 did not associate with 7 different integrin alpha  chain tails (Table II). All of the pEG202-encoded bait proteins containing integrin alpha  or beta  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.

                              
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Table II
Interaction of ICAP-1, RACK1, and integrin cytoplasmic tails in yeast

To determine the subregion of the beta 1 cytoplasmic domain that is critical for ICAP-1 association, we utilized bait plasmid pEG202 to synthesize chimeric beta 1/beta 5 cytoplasmic tail mutants (listed in Table I, bottom). In yeast, both the wild type beta 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 [alpha -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.

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.

Reciprocal Demonstration of beta 1 Tail-ICAP-1 Interaction in Mammalian Cells-- To investigate the interaction of the beta 1 tail with mammalian cell ICAP-1, immobilized beta  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-beta 1 (lane 2) but not to GST itself, GST-beta 2, GST-beta 3, or GST-beta 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-beta 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 beta 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-beta mAb A-1A5.

In a reciprocal experiment we next analyzed binding of solubilized beta 1 integrin to immobilized GST-ICAP-1. First, CHO cells were transfected to stably express wild type or mutant human beta 1 subunits. In each case, the beta 1 extracellular and transmembrane domains were present, whereas the cytoplasmic tail was either unaltered or fully or partly exchanged with regions of the beta 5 tail (See Table I, bottom, for sequences). Upon incubation with GST-ICAP-1 fusion protein, selective binding of wild type beta 1 (beta 1cyto.11) and beta 1cyto.51, but not beta 1cyto.55 or beta 1cyto.15, was observed (Fig. 5B), as detected by Western blotting with anti-human beta 1 mAb A-1A5. No beta 1 was found to associate with immobilized GST control protein (not shown). Wild type human beta 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 beta 1 mAb A-1A5 (not shown).


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Fig. 6.   Surface expression of human integrin beta 1 and beta 1/5 mutants in CHO cells. Transfected CHO cells were incubated with negative control mAb P3, anti-hamster beta 1 mAb 7E2, or anti-human beta 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).

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.

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 beta 1-dependent migration on fibronectin, it did not alter beta 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 alpha V and beta 5, but little beta 3, we suspect that vitronectin-dependent migration is largely mediated by alpha Vbeta 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-beta 1 and -beta 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 beta 1 (see Fig. 6) were also tested for migration. The assay was performed in the presence of anti-hamster alpha 5beta 1 mAb PB1 to block the contribution of endogenous hamster alpha 5beta 1 (Fig. 8C). The CHO-beta 1.cyto11 and -beta 1.cyto51 transfectants showed substantially more migration than either the CHO-beta 1.cyto55 or -beta 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 beta 1 Tail-- Here we have identified and characterized ICAP-1, a 200 amino acid phosphoprotein specifically associating with the beta 1 integrin tail. Interaction seen in a yeast two-hybrid assay was confirmed in reciprocal experiments using ICAP-1- and beta 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 beta 1 with the carboxyl-terminal 24 amino acids of beta 5 resulted in loss of ICAP-1 association. Conversely, the reciprocal exchange (beta 5 tail with terminal 14 residues from beta 1) allowed strong association. Thus the carboxyl-terminal "SAVTTVVNPKYEGK" sequence in beta 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 beta 1 tail (44). Consistent with results shown here, residues critical for ICAP-1 association resided within the carboxyl-terminal 13 residues of the beta 1 tail (44).

Association of beta 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 beta 1, beta 2, and beta 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 beta 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-1beta ) that lacks amino acids 128-177 and does not interact with integrin beta 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 beta 1 Tail Association with ICAP-1-- Association of the beta 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 beta 1 tail may support cell migration. In one set of experiments, expression of ICAP-1 in COS7 cells was associated with increased beta 1-dependent cell migration on fibronectin but not beta 1-independent migration on vitronectin. In another set of experiments, the carboxyl-terminal amino acids within the beta 1 tail that were needed for ICAP-1 association were also required for enhanced cell migration. The carboxyl terminus of beta 5 could not substitute for beta 1 to support migration. These results may help to explain previously noted differences between the integrin beta 1 and beta 5 tails in terms of supporting cell migration (25).

Other functions known to require the carboxyl-terminal 14 amino acids of beta 1 could potentially also involve ICAP-1. For example, the carboxyl-terminal "SAVTTVVNPKYEGK" sequence in the beta 1 tail includes amino acids (Thr-788, Thr-789, Asn-792, and Tyr-795 in human beta 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, alpha -actinin, and focal adhesion kinase (60). Furthermore, alternatively spliced beta 1B (21) and beta 1C (61) tails lack the critical carboxyl-terminal residues present in beta 1A and thus should not bind to ICAP-1. This could at least partially explain why functions of beta 1A are markedly different from the functions of beta 1B or beta 1C (21, 23). In this regard, expression of beta 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 beta 1cyto.15 mutant.

Other beta 1 Tail-associated Proteins-- At present, the integrin beta 1 tail has been suggested to interact directly with cytoskeletal proteins (alpha -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 beta 1 tail. In addition, the ICAP-1 protein is completely distinct from proteins that may interact with other integrin beta  tails, such as cytohesin-1 (63) and beta 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 beta 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 beta 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 beta 1 integrins, and we suggest that phosphorylation of ICAP-1 could play a role in these events.

    ACKNOWLEDGEMENTS

We thank Dr. R. Brent (Massachusetts General Hospital, Boston) for providing the yeast strains and plasmids for the yeast two-hybrid genetic screening. We also thank Dr. D. Chang (UCLA, Los Angeles) for sharing information about ICAP-1 before publication and for antibody to ICAP-1.

    FOOTNOTES

* This work was supported by National Institutes of Health Grant GM46526.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.

Dagger To whom correspondence should be addressed: Dana-Farber Cancer Institute, Rm. D-1410, 44 Binney St., Boston, MA 02115. Tel.: 617-632-3410; Fax: 617-632-2662; Email: Martin_Hemler{at}dfci.harvard.edu.

    ABBREVIATIONS

The abbreviations used are: RACK, receptor for activated protein kinase C; CHO, Chinese hamster ovary cell line; FCS, fetal calf serum; FN, fibronectin; GST, glutathione S-transferase; dhfr, dihydrofolate reductase; HEK293, human embryonic kidney 293 cell line; HA, hemagglutinin; ICAP-1, integrin cytoplasmic domain-associated protein-1; mAb, monoclonal antibody; PCR, polymerase chain reaction; PLL, poly L-lysine; X-gal, 5-bromo-4-chloro-3-indolyl beta -D-galactopyranoside; MEM, minimum Eagle's medium; DMEM, Dulbecco's modified Eagle's medium; PBS, phosphate-buffered saline..

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