INTRODUCTION

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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) (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).

CAK 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 pp130^cas and related proteins (12, 13) and is functionally conserved in CAK residues 712-719, EPPPKPSR. A weak coimmunoprecipitation of pp130^cas 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).

CAK 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.

In a study to elucidate the upstream and downstream signaling pathways of CAK, 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

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Cloning of a cDNA Encoding a CAK-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).

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 pCAKTag as described previously (1). pCAK(S) and pCAKTag 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 pCAKTag 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 pCAKTag 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 pCAKTag. Mutation dl 741-903 was created by digesting pCAKTag 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.

	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 GenBank^TM 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 (GenBank^TM 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.

View larger version (68K): [in this window] [in a new window]   Fig. 1.   Comparison of amino acid sequences of Hic-5 and paxillin. The sequence of human Hic-5 was deduced from the nucleotide sequence of clone cbp-1. The numbers on the right indicate the amino acid residue numbers of human Hic-5 (Hic) and human paxillin (Pax) (40). The amino acid residues of human Hic-5 were tentatively numbered according to the numbering by Shibanuma et al. (23). The amino acid sequence encoded by the presumed (23) "5-untranslated region" of human Hic-5 cDNA are indicated by italic type. Amino acid residues of human paxillin identical with those of human Hic-5 are indicated by dashes. Dots represent gaps introduced to improve the alignment. The metal-liganding residues that define the LIM consensus sequence are boxed. The LD motifs (38) are underlined.

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).

View larger version (44K): [in this window] [in a new window]   Fig. 2.   Immunoprecipitation and immunoblotting of Hic-5 in WFB cells with anti-Hic-5. The WFB cell lysate was prepared in a lysis buffer containing 0.5% sodium deoxycholate and 0.1% SDS in addition to 1% Nonidet P-40. Hic-5 was immunoprecipitated from 1.2 mg of protein of the lysate with 1 µg of protein of affinity-purified anti-Hic-5 bound to protein A-Sepharose (10 µl of packed volume). Paxillin was immunoprecipitated from 0.4 mg of protein of the lysate with 1 µg of protein of anti-paxillin monoclonal antibody bound to anti-mouse IgG-agarose (7.5 µl of packed volume). The immunoprecipitates and 60 µg of protein of the WFB cell lysate (total lysate) were subjected to SDS-PAGE in a 10% gel. The separated proteins were blotted onto a PVDF membrane. Anti-Hic5 antibody and anti-paxillin monoclonal antibody were used for immunoblotting, which is indicated at the bottom of each lane. Positions of molecular mass markers (Sigma SDS-7B) are indicated on the right. i.p., immunoprecipitation.

View larger version (71K): [in this window] [in a new window]   Fig. 3.   Localization of Hic-5 at focal adhesions in WFB cells as shown by immunostaining with anti-Hic-5. a, colocalization of Hic-5 and FAK. WFB cells grown on glass coverslips coated with rat tail collagen were doubly stained with anti-Hic-5 and anti-FAK. Confocal laser-scanning micrographs of the cells are shown for staining of Hic-5 (green in A), FAK (red in B), and both Hic-5 and FAK (yellow in D). A transmitted light phase-contrast micrograph of the same cell (C) is also shown. Colocalization of Hic-5 and FAK was visualized as yellow signals in D. The bar represents 10 µm. b, double staining of Hic-5 and stress fibers. WFB cells grown on glass coverslips coated with rat tail collagen were doubly stained with anti-Hic-5 and rhodamine-conjugated phalloidin. Confocal laser-scanning micrographs of the cells are shown for staining of Hic-5 (green in A), stress fibers (red in B), and both Hic-5 and stress fibers (yellow in D). A transmitted light phase-contrast micrograph of the same cell (C) is also shown. Colocalization of Hic-5 and microfilaments was visualized as yellow signals in D. Both ends of stress fibers terminate at focal contacts where Hic-5 localizes. The bar represents 10 µm.

View larger version (75K): [in this window] [in a new window]   Fig. 4.   Localization at focal adhesions of Myc-tagged Hic-5 expressed in WFB cells. pHic5-Myc plasmid for expression of Myc-tagged Hic-5 was transfected into WFB cells at 2.2 µg/3.5-cm dish by the use of TfxTM-50 (Promega, Madison, WI). The transfected cells grown on glass coverslips coated with rat tail collagen were cultured for 2 days. Cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. The cells were doubly stained with anti-Myc (A) and with rhodamine-conjugated phalloidin (B) and were viewed under a fluorescence microscope. Pairs of photographs, A and B, were taken at the same height from the dish surface. Magnification was × 1361.

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.

View larger version (68K): [in this window] [in a new window]   Fig. 5.   Specific in vitro binding of the C-terminal regions of CAK and FAK to Hic-5 and the Hic-5 N-domain. Four µg each of purified GST-Hic5-Ndom and GST proteins and 5 and 20 µg of protein of total bacterial lysates expressing GST-Hic5(fl) and GST-Hic5-Cdom, respectively, were subjected in triplicate to SDS-PAGE in 7.5% gels and blotted onto PVDF membranes. Hic-5 immunoprecipitated from 1.5 mg of protein of the WFB cell lysate with anti-Hic-5 was also subjected to the procedures. After denaturation and renaturation of the GST fusion proteins, the proteins on the membrane were probed for binding by 32P-labeled GST-CAK-Ndom (data not shown), GST-CAK-Cdom (GSTCAKCdom) (B), GST-CAK-CdomA (GSTCAKCdomA) (E), GST-CAK-CdomB (GSTCAKCdomB) (C), GST-FAK-CdomA (data not shown), and GST-FAK-CdomB (GSTFAKCdomB) (F). These probes had 3 × 107 cpm/µg and were used at 3 × 106 cpm/ml. Autoradiograms are shown in B, C, E, and F. The stained protein patterns shown in A and D were obtained by staining of the gels with Coomassie Blue after electroblotting the separated proteins onto PVDF membranes. GST fusion proteins subjected to SDS-PAGE are indicated across the top. 32P-Labeled probes used in the blot overlay assay are indicated at the bottom of each panel.

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.

View larger version (82K): [in this window] [in a new window]   Fig. 6.   Specific in vitro binding of Hic-5 and paxillin from the WFB cell lysate to the GST fusion proteins of the full-length CAK and of the C-terminal regions of CAK and FAK. The binding of Hic-5 and paxillin (Pax) to the GST fusion proteins of CAK, its fragments, and a fragment of FAK is shown. Fifteen µg each of GST protein and the indicated GST fusion proteins (fusion proteins), GST-CAK(fl), GST-CAK-Ndom, GST-CAK-CdomA, GST-CAK-CdomB, and GST-FAK-CdomB was bound to glutathione-agarose (10 µl each of packed volume). The beads were mixed with 1.8 mg of protein of the WFB cell lysate (lysate) where indicated by plus signs, allowed to stand 4 h at 4 °C, and then washed three times with the lysis buffer. Bound proteins were separated by SDS-PAGE in a 10% gel, blotted onto a PVDF membrane, and probed with anti-Hic-5 (top), anti-paxillin (anti-Pax) (middle), and anti-GST (bottom) followed by visualization of positive bands by ECL. In the anti-Hic-5 blot of the proteins bound to GST-FAK-CdomB (top, lane 12), Hic-5 was separated into upper and lower bands due to the migration of GST-FAK-CdomB at the position between these two bands. In lane 13, Hic-5 immunoprecipitated from the WFB cell lysate with anti-Hic-5 was run as a reference. blot, immunoblot.

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.

View larger version (67K): [in this window] [in a new window]   Fig. 7.   Coimmunoprecipitation of CAK with Hic-5 from the WFB cell lysate and inhibition of this coimmunoprecipitation by GST fusion proteins of the C-domains of CAK and FAK. Hic-5, CAK, FAK, and paxillin were immunoprecipitated from 1.0 mg of protein of the WFB cell lysate with anti-Hic-5, anti-CAK(C-a), anti-CAK(N), and polyclonal anti-FAK bound to protein A-Sepharose and anti-paxillin, anti-CAK(N)-mAb, and anti-CAK(C)-mAb bound to anti-mouse IgG-agarose as indicated at i.p. As controls, normal rabbit Ig and normal mouse IgG1 bound to these beads were used for immunoprecipitation from the lysate (lanes 1 and 9). In the immunoprecipitation shown in lanes 5, 6, and 7, 20 µg of protein of GST-CAK-CdomB, GST-FAK-CdomB, or GST-CAK-Ndom was mixed with the cell lysate before immunoprecipitation as indicated (addition). The immunoprecipitates were subjected to SDS-PAGE in a 7.5% gel, and the separated proteins were blotted onto a PVDF membrane. The membrane was cut at the 89-kDa prestained marker protein into high and low molecular weight regions. The proteins in the high molecular weight region were first probed with anti-CAK(C-a) (anti-CAK) to obtain the data shown in the top, deprobed, and then reprobed with monoclonal anti-FAK to obtain the data shown at the bottom. The proteins in the low molecular weight region were first probed with anti-Hic-5 to obtain the data shown in the middle, deprobed, and then reprobed with anti-paxillin (data not shown). Binding of antibody probes was visualized either by alkaline phosphatase (bottom) or by peroxidase (ECL) (top and middle). The position of oligomeric GST-CAK-CdomB (CdomB) is indicated by an arrow above the position of CAK in the top. The positions of prestained molecular mass markers (Sigma SDS-7B) are indicated on the left. i.p., immunoprecipitation. blot, immunoblot. Ig, heavy chains of immunoglobulins clearly found when rabbit sera were used as antibody preparations (lanes 1, 3, and 8). mAb, monoclonal antibody.

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.

View larger version (61K): [in this window] [in a new window]   Fig. 8.   Expression of Myc-tagged Hic-5, CAK, and its deletion mutants in COS-7 cells from transfected cDNA constructs and coimmunoprecipitation of these proteins from the transfected cell lysates. COS-7 cells (one 10-cm dish each) were transfected with Myc-tagged Hic-5 cDNA in pcDNA3 (pHic5) (lanes 3 and 4) or HSV-tagged CAK cDNA in pSRE (pCAK) (lanes 5 and 6) or doubly transfected with Myc-tagged Hic-5 cDNA in pcDNA3 and either HSV-tagged CAK cDNA in pSRE (lanes 7 and 8) or its deletion mutants in pSRE (dl 741-903, dl 159-552, and dl 86-321) (lanes 9-14) as indicated. Three days after transfection, cells were harvested and lysed on ice in lysis buffer containing 1% Nonidet P-40. CAK and Hic-5 were immunoprecipitated from each lysate with anti-CAK(C-a) (anti-CAK) and anti-Hic-5. The immunoprecipitates were subjected to SDS-PAGE in a 10% gel, and the separated proteins were blotted onto a PVDF membrane. As a reference to show the position of the immunoglobulin heavy chain (Ig), anti-CAK(C-a) was run in lane 15. The proteins on the membrane were probed with anti-CAK(C-a) (top) and anti-Myc (bottom) antibodies. Binding of antibody probes was visualized by peroxidase (ECL). i.p., immunoprecipitation; t.f., transfection; blot, immunoblot.

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).

View larger version (49K): [in this window] [in a new window]   Fig. 9.   Tyrosine phosphorylation of Hic-5 in 3Y1 cells treated with pervanadate and in SR-3Y1 Src-transformed 3Y1 cells. Hic-5 was immunoprecipitated from 1.5 mg of protein (lanes 1-3) and 3.75 mg of protein (lane 4) of the lysates prepared from 3Y1 cells (lanes 1 and 2) and SR-3Y1 cells (lanes 3 and 4). These lysates were prepared in a lysis buffer containing 0.5% sodium deoxycholate and 0.1% SDS in addition to 1% Nonidet P-40. Cells used for lane 2 were treated with 1 mM pervanadate (P. Van.) for 10 min; pervanadate was prepared by mixing 200 µl of 100 mM sodium orthovanadate with 2 µl of 35% hydrogen peroxide, and, after being allowed to stand at room temperature for 20 min, the mixture was added to the medium at 1% volume. The immunoprecipitates were subjected to SDS-PAGE in a 9% gel. After transfer onto a PVDF membrane, the separated proteins were probed with anti-Hic-5 (A) and anti-phosphotyrosine 4G10 (B). The lower band seen at Hic-5 in lane 3 of A is the heavy chains of immunoglobulins used in the immunoprecipitation. blot, immunoblotting.

View larger version (50K): [in this window] [in a new window]   Fig. 10.   Stimuli that activate CAK enhance tyrosine-phosphorylation of Hic-5 in WFB cells. Confluent WFB cells in 10-cm dishes were kept for 10 h in Iscove's medium without serum (lane 1, serum-starved). The cells were stimulated with 10% serum for 10 min (lane 2), 2 µM lysophosphatidic acid (LPA) for 3 min (lane 3), or 0.2 µM endothelin (endothelin I, Sigma) for 5 min (lane 5) or stimulated by osmotic stress (lane 4) via exposure of the cells to a medium containing 0.3 M sorbitol for 5 min. CAK and Hic-5 were immunoprecipitated from 1.5 mg of protein of each of the cell lysates prepared from these cells. The cell lysates were prepared in a lysis buffer containing 0.5% sodium deoxycholate and 0.1% SDS in addition to 1% Nonidet P-40. After blotting onto PVDF membranes, the separated proteins were probed with anti-Hic-5, anti-CAK(C-a) (anti-CAK) or anti-phosphotyrosine (anti-PTyr) as indicated. Anti-CAK(C-a) was used as an anti-CAK. Faintly stained heavy chains of rabbit immunoglobulins (Ig) overlap at the bottom of the Hic-5 band. i.p., immunoprecipitation; blot, immunoblotting.

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 pp130^cas.

View larger version (68K): [in this window] [in a new window]   Fig. 11.   CAK and Hic-5 were simultaneously tyrosine-phosphorylated in WFB cells either exposed to hypertonic osmotic stress or stimulated with lysophosphatidic acid. Sixteen 10-cm dishes of confluent and quiescent WFB cells of the same culture lot were prepared and divided into four groups. Cells on the dishes of the first group were stimulated in Iscove's medium with 2 µM lysophosphatidic acid (LPA) for 10 min at 37 °C (lanes 7 and 8). Cells on the dishes of the second group were exposed to hypertonic osmotic stress (Osm) for 20 min at 37 °C by replacing the culture medium with 2 ml of Iscove's medium containing 0.3 M sorbitol (lanes 9 and 10). Cells on the dishes of the third group were trypsinized (off) after washing with ATV solution (20); the cells were detached from dishes and allowed to stand at 37 °C for 20 min after the addition of trypsin inhibitor (lanes 3 and 4). Cells on the dishes of the fourth group were directly subjected to analysis without treatment (on) (lanes 5 and 6). Cell lysates were prepared from these four groups of cells. Hic-5, CAK, FAK, and paxillin (Pax) were immunoprecipitated from 1.2 mg of protein of these WFB cell lysates with anti-Hic-5 and anti-CAK(C-a) (anti-CAK) bound to protein A-Sepharose and anti-FAK and anti-paxillin monoclonal antibodies bound to anti-mouse IgG-agarose as indicated at i.p. As controls, normal rabbit Ig and normal mouse IgG1 bound to these beads were used for immunoprecipitation from the on cell lysate (lanes 1 and 12). In lane 2, anti-CAK(C-a) itself bound to protein A-Sepharose was run as a control. FAK and paxillin were immunoprecipitated from the lysate of on cells. The immunoprecipitates were subjected to SDS-PAGE in a 7.5% gel, and the separated proteins were blotted onto a PVDF membrane. The membrane was cut at the 89-kDa prestained marker protein into high and low molecular weight regions. The proteins in the high molecular weight region were first probed with anti-CAK(C-a) to obtain the data shown at the top. The proteins in the low molecular weight region were first probed with anti-Hic-5 to obtain the data shown at the top. The proteins in both regions were deprobed and then reprobed with anti-phosphotyrosine, 4G10, to obtain the data shown at the bottom; the heavy chains of the mouse immunoglobulins (Ig) used in the immunoprecipitation were stained at the position below Hic-5 in lanes 11, 12 and 13 of the bottom. Binding of antibody probes was visualized either by alkaline phosphatase (bottom) or by peroxidase (ECL) (top). Pax, paxillin; i.p., immunoprecipitation; blot, immunoblot.

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 am