©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Cloning and Characterization of Cell Adhesion Kinase , a Novel Protein-tyrosine Kinase of the Focal Adhesion Kinase Subfamily (*)

(Received for publication, April 5, 1995)

Hiroko Sasaki (1) Kazuko Nagura (1) Masaho Ishino (1) Hirotoshi Tobioka (2) Kiyoshi Kotani (1) Terukatsu Sasaki (1)(§)

From the  (1)Department of Biochemistry, Cancer Research Institute, and the (2)Department of Pathology, Sapporo Medical University School of Medicine, South-1, West-17, Chuo-Ku, Sapporo 060, Japan

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

A second protein-tyrosine kinase (PTK) of the focal adhesion kinase (FAK) subfamily, cell adhesion kinase beta (CAKbeta), was identified by cDNA cloning. The rat CAKbeta is a 115.7-kDa PTK that contains N- and C-terminal domains of 418 and 330 amino acid residues besides the central kinase domain. The rat CAKbeta has a homology with mouse FAK over their entire lengths except for the extreme N-terminal 88 residues and shares 45% overall sequence identity (60% identical in the catalytic domain), which indicates that CAKbeta is a protein structurally related to but different from FAK. The CAKbeta gene is less evenly expressed in a variety of rat organs than the FAK gene. Anti-CAKbeta antibody immunoprecipitated a 113-kDa protein from rat brain, 3Y1 fibroblasts, and COS-7 cells transfected with CAKbeta cDNA. The tyrosine-phosphorylated state of CAKbeta was not reduced on trypsinization, nor enhanced in response to plating 3Y1 cells onto fibronectin. CAKbeta localized to sites of cell-to-cell contact in COS-7 transfected with CAKbeta cDNA, in which FAK was found at the bottom of the cells. Thus, CAKbeta is a PTK possibly participating in the signal transduction regulated by cell-to-cell contacts.


INTRODUCTION

Protein-tyrosine kinases (PTKs) (^1)that do not span the plasma membranes (so-called nonreceptor PTKs) have been classified into different subclasses (subfamilies) based on the sequence similarity and distinct structural characteristics(1) . Many nonreceptor PTKs participate in cellular signal transduction by associating with the intracellular portions of transmembrane receptors which do not themselves have PTK activity. Different nonreceptor PTKs play diverse and specific roles in mediating the signal transduction by different nonkinase receptors(2, 3, 4) .

Focal adhesion kinase (FAK) has been proposed as the prototype (and hitherto the sole member) of a new subfamily of nonreceptor PTK, represented by proteins with large N- and C-terminal domains flanking the catalytic domain but without Src homology 2 and 3 (SH-2 and SH-3) domains(5, 6, 7, 8, 9) . FAK is concentrated in focal adhesions(5, 6) , and its phosphorylation and activation are triggered by the ligand binding to integrins and by the stimulation of certain growth factor and neuropeptide receptors(6, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24) . The N- and C-terminal domains of FAK mediate its interactions with integrins, the Src-family kinases and paxillin, a focal adhesion associated protein(8, 9, 25, 26, 27, 28) . By these and other yet to be characterized interactions, FAK regulates signaling via different receptors. Because only one member of the FAK subfamily is known to date, we sought to identify a second PTK of the FAK subfamily by a homology-based cDNA cloning strategy. We describe here an isolation and characterization of a cDNA coding for a new member of the FAK family. The novel PTK described here is the second member, to our knowledge, of the FAK subfamily whose cDNA has been cloned and sequenced and is designated CAKbeta for cell adhesion kinase beta.


MATERIALS AND METHODS

Amplification of PTK Catalytic Domain cDNA Fragments by PCR

PTK cDNAs were amplified from adult rat brain RNA by reverse transcriptase-directed PCR. PCR primers were designed to recognize conserved regions in PTK catalytic domains: upstream ``EcoRI-FVHRDLA'' primers, 5`-G-GAATTC-TTT-GT(G/C)-CA(C/T)-(A/C)GN-GA(T/C)-CT (G/T)-GC-3`; downstream ``SDVWSFG- BamHI'' primers, 5`-TC-GGAT-CC-(G/A)(A/T)A-(G/A)CT-CCA-(G/C)AC-(G/A)TC-(G/A)CT-3`; where N = (A/C/G/T). RNA extracted from rat brain was reverse transcribed with the downstream primers and the Rous associated virus 2 (RAV-2) reverse transcriptase following the conditions of the manufacturer (Perkin-Elmer) in a 20-µl reaction. PCR was performed on the reverse transcriptase reaction product in a 50-µl reaction containing 50 mM KCl, 10 mM Tris-HCl (pH 8.3), 1.5 mM MgCl(2), 100 µg/ml gelatin, 0.2 mM dNTPs, 1.25 units of Taq polymerase (Perkin-Elmer), and 50 pmol each of the upstream and downstream primers. The thermocycling parameters used in PCR were as follows: annealing, 2 min at 55 °C; extension, 2 min at 72 °C; denaturation, 1 min at 94 °C. After 30 cycles, amplified cDNA products were digested with EcoRI and BamHI and electrophoretically separated on a 3% low melting agarose gel. An ethidium bromide-stained band at about 210 base pairs was cut out. The DNA was extracted from the gel and subcloned into pBluescriptII SK(+). Nucleotide sequences were determined for 100 inserts by the dideoxynucleotide chain termination method (29) using the BcaBEST dideoxy sequencing kit (Takara Shuzo, Otsu, Japan) and the Sequenase 2.0 kit (U. S. Biochemical Corp.), and compared with those in GenBank data base by the BLASTx program of NCBI (National Center for Biotechnology Information, Bethesda, MD).

Isolation of cDNA Clones Encoding CAKbeta

A clone, M9-3, isolated from the PCR library was labeled with [alpha-P]dCTP (Amersham Corp.) using a random primer labeling system (BcaBEST labeling kit, Takara Shuzo). The labeled probe was used to screen an oligo(dT)- and random-primed adult rat brain cDNA library constructed in the ZAP II vector (Stratagene, La Jolla, CA). Seven positive phage plaques were identified. To obtain clones covering the entire 4.0-kilobase transcript, it was necessary to rescreen the library with probes derived from the 5`- and 3`-ends of the initial cDNA isolates: 34 additional CAKbeta cDNA clones were obtained by screening about 8 10^5 independent clones. Nucleotide sequences were determined on both strands for selected overlapping clones and their derivatives prepared by exonuclease III/mung bean nuclease deletions to obtain the composite sequence (see ``Results''). Human CAKbeta cDNA was cloned by screening a human hippocampus cDNA library constructed in ZAP II vector (Stratagene, La Jolla, CA) with a probe derived from an ApaI/SacI fragment (nucleotides 60-544) of rat CAKbeta cDNA.

Northern Analysis of Expression

Total RNA was extracted from the tissues of adult rat (Sprague-Dawley strain) and the indicated cell lines using ISOGEN kit (Nippon Gene, Toyama, Japan) according to the manufacturer's protocol. RNA samples were electrophoresed through a 1.0% agarose, 2% formaldehyde gel and transferred to a nitrocellulose membrane. Hybridization to P-labeled fragments of CAKbeta cDNA, FAK cDNA, and actin cDNA was carried out in 50% formamide, 5 SSPE (1 SSPE = 0.18 M NaCl, 10 mM sodium phosphate, pH 7.7, 1 mM EDTA), 5 Denhard's solution, 5 mM EDTA, 0.1% SDS, and 100 µg/ml denatured salmon sperm DNA at 42 °C for 14-16 h. The filters were washed (final wash: 0.2 SSC and 0.1% SDS; 1 SSC = 0.15 M NaCl, 15 mM sodium citrate, pH 7.6) under conditions of either high stringency (final wash at 55 °C for 1 h) or low stringency (final wash at 43 °C for 1 h) as indicated in the figure legends. All DNA probes were radiolabeled by random priming. After hybridization, all blots were exposed to Kodak XAR film with an intensifying screen at -80 °C. cDNA probes were derived from StyI fragments of the CAKbeta cDNA (nucleotides 74-935 and 2990-3519, which are the 5`- and 3`-terminal regions). The expression of FAK was detected by hybridizing a probe derived from rat FAK cDNA, corresponding to the amino acid residues 342-600 of mouse and human FAKs(6, 7) . The rat FAK cDNA was cloned from rat brain PCR library prepared by the use of degenerated PCR primers designed from common amino acid sequences of FAK and CAKbeta. The actin probe was prepared from human beta-actin cDNA.

Production of Antiserum to CAKbeta and Affinity Purification of the Antibody

Digestion of rat CAKbeta cDNA (clone 24) with SphI and PstI restriction endonucleases generated a 688-base pair fragment encompassing nucleotides 2333 through 3020 of the CAKbeta cDNA. This fragment, encoding amino acid residues 779-1008 of CAKbeta, was inserted into pATH21 vector (ATCC 37701) (30) doubly digested with SphI and PstI at the polylinker site. Escherichia coli RR1 (ATCC 31343) transformed by this constract was grown and then induced to produce a TrpE-CAKbeta fusion protein(30) . The bacteria were lysed by sonication and the TrpE-CAKbeta fusion protein was purified by SDS-PAGE. The fusion protein was electroblotted onto a PVDF membrane (Immobilon, Millipore) and located by staining with Commassie Blue. The portion of the membrane where the fusion protein was located was broken to a powder in liquid nitrogen and used to prepare a water-in-oil emulsion in an adjuvant. Polyclonal antibodies directed against CAKbeta were prepared by immunization of New Zealand White male rabbits with the antigen. The antibody was affinity-purified by binding to a glutathione S-transferase fusion protein containing the CAKbeta C-terminal domain and by eluting with 0.5 M ammonium hydroxide, 3 M sodium thiocyanate (pH 11.0).

Cell Culture

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), BALB/3T3 clone A31 (ATCC CCL163), Swiss/3T3 (ATCC CCL92), NIH/3T3 (ATCC CRL 1658), Jurkat clone E6-1 (ATCC TIB 152), and PC-12 (ATCC CRL 1721) were obtained from American Type Culture Collection (Rockville, MD). A rat fibroblast line, WFB(33) , was obtained from the establisher of the line, Dr. N. Sato (Sapporo Medical University). Mouse neuroblastoma lines(34) , NIE-115 and NS-20Y, and a mouse neuroblastoma-rat glioma hybrid line, NG108-15(35) , were obtained from Mitsubishi Kasei Institute for Life Science (Machida, Japan). These cells were cultured in Iscove's modified Dulbecco'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. NG108-15 cells were grown in a medium containing hypoxanthine-aminopterin-thymidine medium supplement (Sigma).

Antibodies

Commercial sources of antibodies were as follows: anti-FAK monoclonal antibody 2A7(36) , anti-FAK rabbit polyclonal antibody, which was raised against a glutathione S-transferase fusion protein containing the residues 542-880 of human FAK, and anti-phosphotyrosine monoclonal antibody 4G10 (Upstate Biotechnology, Inc., Lake Placid, NY); goat anti-mouse IgG-agarose, monoclonal anti-rabbit immunoglobulins (clone RG-16) conjugated with alkaline phosphatase, and goat anti-mouse IgG (Fc-specific) conjugated with alkaline phosphatase (Sigma); anti-herpes simplex glycoprotein D-epitope tag monoclonal antibody (Novagen, Madison, WI).

Immunoprecipitation of CAKbeta and FAK

Confluent monolayer cultures of cells in 9-cm dishes were washed twice with phosphate-buffered saline (PBS) and then lysed on ice in 0.5 ml per dish of a lysis buffer (20 mM Tris-Cl (pH 7.4), 150 mM NaCl, 2.5 mM EDTA, 1% Nonidet P-40, 1% sodium deoxycholate, 0.1% SDS, 10% glycerol, 1% Trasylol, 20 µg/ml leupeptin, 1 mM phenylmethylsulfonyl fluoride, 50 mM NaF, 1 mM Na(3)VO(4), 20 mM Na(4)P(2)O(4)). A 2.5% rat brain lysate was prepared in the lysis buffer by the use of a Teflon pestle in a glass homogenizer. The lysates were subjected to centrifugation at 15,000 g for 20 min at 4 °C to obtain clarified lysates. CAKbeta was immunoprecipitated by mixing anti-CAKbeta bound to protein A-Sepharose with 1 mg of protein of clarified lysates and incubating for 2 h at 4 °C on a rotating platform. The anti-CAKbeta beads were prepared for each assay by mixing 2 µg of affinity-purified anti-CAKbeta protein with 10 µl (packed volume) of protein A-Sepharose and washing the Sepharose beads with the lysis buffer. As a control, preimmune serum beads were prepared for each assay by mixing 10 µl of preimmune serum with 10 µl of protein A-Sepharose. Four µg protein of anti-FAK monoclonal antibody, 2A7, bound to 10 µl (packed volume) of anti-mouse IgG-agarose were used to immunoprecipitate FAK from 1 mg of protein of clarified lysates. Two µg of protein of anti-epitope tag monoclonal antibody bound to 10 µl (packed volume) of anti-mouse IgG-agarose were used to immunoprecipitate epitope-tagged CAKbeta from 1 mg of clarified lysate protein. Immunoprecipitates were washed three times with the lysis buffer, and proteins were separated by SDS-PAGE according to the method of Laemmli and Favre(37) . The separated proteins were blotted onto PVDF membranes (Immobilon-P, Millipore, 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 30 min at 60 °C and then probed with the indicated primary antibody in TBST containing 1% bovine serum albumin for 1 h at room temperature. Affinity-purified anti-CAKbeta antibody and anti-FAK polyclonal antibody were used at 1 µg of protein per ml, and anti-epitope tag antibody was used at 0.4 µg of protein per ml. The membranes were washed with TBST three times and probed again with a second antibody conjugated with alkaline phosphatase in TBST for 1 h, followed by washing three times in TBST. Positive bands were detected by incubating in nitro blue tetrazolium (Sigma) and 5-bromo-4-chloro-3-indolyl phosphate (Sigma).

Transient Expression of CAKbeta in COS-7 Cells

The CAKbeta cDNA clone, 17N, was digested with EcoRI and subcloned into the simian virus 40-based expression vector pSRE(38) , which was derived by a modification of the original pcDL-SRalpha-296 vector(39) . The resulting construct, pCAKbeta(S), and a control construct, pCAKbeta(AS), in which the CAKbeta cDNA was subcloned in an inverted, antisense direction, were transfected into subconfluent COS-7 cells at 10 µg/9-cm dish using DEAE-dextran(40) . After 3 days, the cells were harvested and lysed on ice in the lysis buffer and the lysate was analyzed by immunoprecipitation.

The epitope-tagging vector, pHSV-Tag, was created by ligating a 50 mer oligonucleotide (Novagen, Madison, WI) encoding the 11 amino acid peptide (QPELAPEDPED) derived from herpes simplex virus glycoprotein D, followed by a termination codon, into pT7Blue-T (Novagen, Madison, WI) in a sense direction. The CAKbeta cDNA clone 17N was subcloned into pHSV-Tag by using a strategy that resulted in the epitope with additional N-terminal three amino acid residues, YGL, replacing the C-terminal residue, E, of CAKbeta. For expression in vivo, the derivative was subcloned into pSRE to obtain pCAKbetaTag. The 11-residue epitope tag is specifically recognized by the anti-epitope tag monoclonal antibody.

Immune Complex Kinase Assay

CAKbeta, epitope-tagged CAKbeta, and FAK were immunoprecipitated from the clarified lysates (0.4 mg of protein) of COS-7 cells, transfected with cDNA constructs, and 3Y1 cells as described above. The immune complexes were washed twice with 0.5 ml of the lysis buffer, once with 20 mM Tris-HCl (pH 7.4) containing 0.5 M LiCl, once with 20 mM Tris-HCl (pH 7.4) containing 1 mM EDTA, and once with a kinase assay buffer (20 mM Tris-HCl (pH 7.4), 10 mM MnCl(2), 1 mM dithiothreitol) and suspended in 20 µl of the kinase assay buffer containing 5 µCi of [-P]ATP (4500 Ci/mmol; ICN Pharmaceuticals, Inc., Irvine, CA). After incubation for 20 min at 20 °C, the incubation was terminated by the addition of 20 µl of 2 SDS-PAGE sample buffer. A 20-µl portion of the P-labeled immune complexes was subjected to SDS-PAGE in a 7.5% gel. The gel was dried, and the P-labeled proteins were made visible by autoradiography.

For assays of protein-tyrosine kinase activity using poly(Glu,Tyr) (4:1, 20-50 kDa; Sigma), the clarified lysates (0.4 mg of protein) of transfected COS-7 cells and 3Y1 cells were incubated in 20 µl of the kinase assay buffer with 5 µg/20 µl of the exogenous substrate along with 5 µCi of [-P]ATP, 5 µM unlabeled ATP, and 5 mM MgCl(2). The reactions were carried out for 10 min at 20 °C, and stopped by the addition of an equal volume of 2 SDS-PAGE sample buffer. The labeled substrate in a 20-µl portion of each assay was separated by SDS-PAGE in a 15% gel. P-Phosphorylated poly(Glu,Tyr) was then visualized and quantitated by bioimaging analysis of the dried gel with Bioimaging Analyzer (BAS 2000, Fuji Photo Film, Tokyo). The counts of photo-stimulated luminescence were corrected to reflect the incorporated radioactivity by subtracting the count of an appropriate control set in each series of kinase assays.

Analysis of CAKbeta Phosphotyrosine Content

To study changes in phosphotyrosine content of CAKbeta and FAK in response to trypsinization and plating cells on fibronectin and poly-L-lysine, confluent 3Y1 cells were cultured overnight in a medium containing 0.5% fetal calf serum and harvested by trypsinization, which was stopped by adding soybean trypsin inhibitor (Sigma). The cells were suspended in serum-free Iscove's modified Dulbecco's medium and plated on 9-cm tissue culture dishes coated with either bovine plasma fibronectin (Biomedical Technologies, Stoughton, MA) (0.1 mg/dish) or poly-L-lysine (0.2 mg/dish). The dishes were incubated at 37 °C for 50 min, and cells attached on dishes were analyzed. Cell lysates were prepared and clarified by centrifugation at 15,000 g for 20 min. The clarified lysates (0.6 mg protein per assay) were subjected to the immunoprecipitation with either anti-CAKbeta beads or anti-FAK beads. After SDS-PAGE and blotting to PVDF membranes, CAKbeta, FAK, and phosphotyrosine were detected by probing blots with anti-CAKbeta, anti-FAK polyclonal antibody, and anti-phosphotyrosine, 4G10.

Confocal Laser Scanning Microscopy of Immunostained COS-7 Cells

COS-7 cells, grown overnight on glass coverslips, were transfected with the indicated plasmid. After 3 days, the cells were rinsed in PBS and fixed for 15 min at 4 °C in 95% ethanol. Fixed cells were preincubated with fetal calf serum for 30 min at 20 °C and then incubated with primary antibodies for 1 h at 20 °C. The cells were then washed three times for 10 min each in PBS. The secondary antibodies were then applied for 1 h at 20 °C. After washing as above, the coverslips were mounted in PBS containing 50% glycerol and 0.02% 1,4-diazobicyclo-(2,2,2)-octane (Aldrich), which was added to delay fading of immunofluorescence during microscopy. The primary antibodies used were affinity-purified anti-CAKbeta (used at 20 µg of protein/ml), anti-epitope tag (used at 40 µg of protein/ml), and anti-FAK polyclonal antibody (used at 67 µg protein/ml). Secondary antibodies were fluorescein-conjugated goat anti-mouse and swine anti-rabbit immunoglobulins (DAKO-Japan, Tokyo) (used at a 100-fold dilution). Immunofluorescence was imaged with a Bio-Rad MRC-500 confocal laser scanning microscope. The microscope is fitted with 60 (numerical aperture, 1.4) objectives in connection with a Nikon Optiphot-2 upright fluorescence microscope. Digitalized fluorescence images obtained by illuminating with a 25-milliwatt multiline argon ion laser were filed in a 768 512 pixel frame memory. A series of optical sections through each cell was taken at vertical steps of 1 µm. Digital image files were stored on an optomagnetic disc and were subsequently recorded on 35-mm film.


RESULTS AND DISCUSSION

Isolation of cDNA Clones Encoding a Second FAK

To identify novel members of the PTK gene family, a PCR-based approach was used. Reverse transcriptase-directed PCR was performed using RNA from adult rat brain and degenerated primers. A mixture of oligonucleotide primers coding for a PTK hallmark sequences allowed us to isolate eight different PTK catalytic domain cDNA fragments. Six of these PCR products coded for already known members of receptor PTKs. A computer-assisted sequence analysis of one other PCR fragment, M9-3, showed that it encoded a novel amino acid sequence with the conserved residues characteristic of PTKs. Among the known PTKs, the highest homology was found with the PTK catalytic domain of FAK (76% identity in the translated amino acid sequence). M9-3 is clearly related to but distinct from the cDNA of the mouse, human, and chicken FAKs(5, 6, 7) ; it was evident that M9-3 does not code for a rat homologue of FAK. M9-3, a 201-base cDNA fragment, was subsequently used as a probe for screening a commercially available rat brain cDNA library made by random and oligodeoxythymidylic acid (oligo(dT)) primers. The screening of about 6 10^5 independent clones allowed us to obtain seven overlapping cDNAs covering the major portion of the mRNA (Fig. 1). The cDNA clones covering 5`- and 3`-terminal regions of the mRNA were obtained by screening the library with the 5`- and 3`-portions of the initial overlapping cDNAs as probes. We present in Fig. 2a composite sequence of 4.05 kilobases deduced from these cDNA fragments, together with the sequence of the protein, which we call CAKbeta.


Figure 1: Organization of the cloned rat CAKbeta cDNA. The CAKbeta cDNA is illustrated as a line with the positions of restriction sites recognized by ApaI (A), HindIII (H), KpnI (K), SacI (Sa), ScaI (Sc), SphI (Sp), and XbaI (X). The extents of nine partial cDNAs that were isolated are also shown with their names indicated to the right. M9-3 is the original PCR product. Clones 24, 119, and 114 were isolated with the PCR product as probes. Clones 17N and 21N and clones 9C, 29C, 30C, and 6C were isolated with the 5`- and 3`-portions of clone 24 as probes. The heavy line below the cDNA clones represents the predicted translation product, the CAKbeta protein, with the catalytic domain of the protein kinase denoted by an open box.





Figure 2: Nucleotide sequence of rat CAKbeta cDNA and deduced amino acid sequence. Nucleotides and corresponding amino acids (single-letter code) shown above are numbered at the end of each lane. The catalytic domain of the PTK is indicated by a box. The translational stop codon is underlined. A variation of the polyadenylation signal, ATTAAA, near the 3`-end of the cDNA is also underlined. In the 5`-noncoding region, the in-frame stop codons are indicated in bold type.



CAKbeta Is a New Member of the FAK Subfamily of PTKs

The combined 4048-base pair cDNA contained a long open reading frame encoding a protein of 1009 amino acid residues with calculated molecular mass of 115,724 Da, which has all the characteristics of a nonreceptor PTK. The open reading frame is flanked by a 5`-untranslated sequence of 261 base pairs and a 3`-untranslated sequence of 757 base pairs. The 3`-extremity of the cDNA contains the polyadenylation signal 5`-ATTAAA-3`, followed closely by 12 consecutive terminal adenosine residues. The proposed initiation codon at nucleotides 1-3 is of the form 5`-GAGAGGATGTCC-3`, which represents a suboptimal primary sequence context for initiation of translation with a purine at position -3, but without a G at position +4(41) . The assignment of ATG at nucleotides 1-3 as the initiation codon is confirmed by the 5`-terminal sequence analysis of a human CAKbeta cDNA, which we cloned from human hippocampus cDNA library. The nucleotide sequences are well conserved in the putative coding regions of rat and human CAKbeta cDNAs, resulting in the almost identical N-terminal amino acid sequences of rat and human CAKbetas ( Fig. 3and 4). The 5`-noncoding regions of rat and human CAKbeta cDNAs contain an in-frame TAG stop codon at positions -18 to -16 and the rat and human cDNA sequences clearly diverge from the positions of about -60 to the 5`-extremity ( Fig. 3and 4).


Figure 3: Comparison of nucleotide sequences of the 5`-terminal portions of rat and human CAKbeta cDNAs and their deduced amino acid sequences. The 5`-terminal portion of human CAKbeta isolated from human hippocampus cDNA library was compared with the 5`-terminal portion of rat CAKbeta. Dots represent gaps introduced to improve the alignment. Nucleotides and corresponding amino acids (single-letter code) shown above are numbered at the end of each lane. The in-frame stop codons are indicated in bold type.



A protein kinase catalytic domain typical of the PTKs (1) and including the sequence identical to that encoded by the M9-3 PCR fragment encompasses amino acids 419-679 (Fig. 2). The deduced protein contains a 418 amino acid N-terminal and a 330 amino acid C-terminal noncatalytic domains. A comparison of the CAKbeta cDNA sequence by the BLASTx program of NCBI with those in GenBank data base revealed homology of CAKbeta and FAK over their entire lengths, and did not detect any sequence more closely related to CAKbeta. Comparison of the deduced amino acid sequence of the encoded protein with those of mouse, human, and chicken FAKs revealed that this cDNA encoded a FAK-related but distinct PTK (Fig. 5). The amino acid sequence and the structural organization of CAKbeta clearly indicate that CAKbeta is a PTK of the FAK subfamily. The unique overall architecture of FAK that the catalytic domain is flanked by large N-terminal and C-terminal domains (5, 9) is also found in CAKbeta. The amino acid sequence of the catalytic domain of CAKbeta is 60% identical with the catalytic domains of mouse and human FAKs (Fig. 6). The amino acid sequences of the N- and C-terminal domains of CAKbeta are 39 and 40% identical with those of mouse FAK (Fig. 5). As in FAK, CAKbeta contains neither SH-2 nor SH-3 domains. FAK are highly conserved evolutionary between species; human FAK shares 97% amino acid identity with mouse FAK and 95% identity with chicken FAK(7) . The result that rat CAKbeta shares only 45% amino acid identity with mouse FAK indicates that CAKbeta is the second PTK of the FAK subfamily. Indeed, we have amplified rat FAK in addition to CAKbeta from rat brain RNA by RT/PCR using degenerated oligonucleotide primers designed from the amino acid sequences common to both FAK and CAKbeta. (^2)


Figure 5: Comparison of amino acid sequences of rat CAKbeta and mouse FAK. The numbers on the right indicate the positions relative to the putative start methionine of CAKbeta and the amino acid residue number of mouse FAK(6) . The catalytic domains are boxed. Amino acid residues of FAK identical with those of CAKbeta are indicated by dashes. Dots represent gaps introduced to improve the alignment. The sequences of FAK at residues 861-882, 711-741, and 684-705 are duplicated at the sequences of CAKbeta where local homology are found with the duplicated FAK sequences. The tyrosine phosphorylation site discussed in the text is underlined.




Figure 6: Comparison of the PTK catalytic domain of CAKbeta with those of other PTKs. Identical residues are indicated by dashes. Dots represent gaps introduced to improve the alignment. Asterisks denote amino acid residues that are highly conserved among PTKs (1) and appear in CAKbeta. Residues which are highly conserved among PTKs, but different in CAKbeta, are indicated by the symbol, #. The number in parentheses is the percentage of residues that are identical with CAKbeta. PTK sequence data were taken from the GenBank data base and the accession numbers are given in parentheses; mouse FAK (M95408), human FAK (L13616), rat Flk (X13412), mouse ZAP-70 (U04379), mouse EGFR (X78987), human FER (J03358), human Arg (M35296), mouse Tec (X55663), rat Hck (M83666), chicken c-Src (V00402).



Homology of CAKbeta and FAK

The predicted amino acid sequence of CAKbeta contains isoleucine in one of the PTK-specific peptide sequences, Asp-Ile-Ala-Val-Arg-Asn (Figs. 5 and 6). The isoleucine at residue 550 is characteristic to FAK(5) ; leucine is found at the analogous position in other PTKs. The valine at residue 552 is unusual since alanine is found at the analogous position in most of the other PTKs including FAK (Fig. 6), with exceptions of several PTKs, which contain threonine or serine. CAKbeta contains other PTK-specific peptide sequences, Pro-Ile-Lys-Trp-Met and Ser-Asp-Val-Trp, and the structural motifs conserved in all protein kinases (1) including an ATP-binding site, three residues predicted to interact with the -phosphate group of the bound ATP, and the catalytic site Asp. In addition to the replacement of conserved leucine by isoleucine at the residue 550 of CAKbeta, three other residues highly conserved in most of the other PTK catalytic domains are not conserved in CAKbeta (Fig. 6). Two of these at the residues 536 and 626 of CAKbeta are not conserved in FAK as well. The other one at the residue 612 of CAKbeta is alanine. The corresponding residue in FAK is glycine, the conserved amino acid of this position in the PTK catalytic domain. Conversely, Met of CAKbeta is the residue highly conserved in the PTK catalytic domains but is replaced to leucine in FAK (Fig. 6).

Comparisons of the N- and C-terminal nonkinase domains between CAKbeta and FAK are shown in Fig. 5. Although the amino acid residues 89-418 of CAKbeta are highly homologous (47.6% identity) with the corresponding N-terminal domain of FAK, the sequence of the extreme N-terminal 88 residues of CAKbeta is entirely different from any portion of FAK (Fig. 5). This difference may imply specific binding of CAKbeta to the cytoplasmic domain of some receptors other than integrins. The binding site of FAK to integrins has been identified in the N-terminal domain(8) .

In FAK, the tyrosine 397 at the juncture of the N-terminal and catalytic domains is the site of autophosphorylation and is the major in vivo and in vitro site of tyrosine phosphorylation(28) . This phosphorylated tyrosine and the sequence around it are the binding site for a SH-2 domain of the Src family PTKs to FAK(27) . The sequence around the Tyr is Glu-Thr-Asp-Asp-Tyr-Ala-Glu-Ile in chicken, mouse, and human FAKs(5, 6, 7) . A homologous sequence, Glu-Ser-Asp-Ile-Tyr-Ala-Glu-Ile, is found in CAKbeta at the juncture of the N-terminal and catalytic domains ( Fig. 2and Fig. 5). The sequence, Tyr-Ala-Glu-Ile, common to both FAK (Tyr) and CAKbeta (Tyr) conforms to a consensus high affinity binding site for the SH-2 domains of the Src family of PTKs(42) .

The sequence of the C-terminal domain, a region following the kinase domain, is 46 amino acids shorter in CAKbeta as compared with FAK. In the sequence comparison presented in Fig. 5, three gaps were introduced in the C-terminal domain of CAKbeta to maximize the homology with FAK. As shown in Fig. 5, the C-terminal domain of CAKbeta immediately after the PTK catalytic domain (residues 699-720, 747-777, and 778-799) has local homologies with three C-terminal domain stretches of the FAK sequence (residues 861-882, 711-741, and 684-705) in a reverse order; more C-terminal sequences of FAK are homologous with more N-terminal sequences of CAKbeta. It should be noted that the residues 711-741 and 861-882 of FAK are the two most proline-rich stretches in the FAK sequence(5, 6, 7) . The C-terminal nonkinase region of CAKbeta contains two proline-rich stretches, residues 701-767 and 831-869, where the proline content exceeds 20%. The presence of proline-rich stretches has been recognized as a characteristic element of the FAK C-terminal domain(5) . The proline-rich stretches of CAKbeta may possibly function as ligands to the SH-3 domains of some proteins involved in the signal transduction. There is also a proline-rich stretch in the extreme N-terminal region of CAKbeta (residues 18-30).

The residues 869-999 of CAKbeta continuous with the C-terminal end of the proline-rich cluster are highly homologous (61.83% identity) with the residues 913-1043 of mouse FAK (Fig. 5). The region targeting FAK to focal adhesion was located to reside within the 159 residues of chicken FAK between amino acid positions 853 and 1012, which correspond residues 851-1011 of mouse FAK(26) . Thus the sequence of CAKbeta between positions 845 and 967 may possibly contain the targeting sequence of CAKbeta to a certain submembranous site. On the other hand, the CAKbeta sequence of the extreme C-terminal 10 amino acids, residues 1000-1009, is not homologous with the C terminus of FAK. It has been reported that a replacement of the extreme C-terminal 13 residues of FAK with an epitope tag blocks paxillin binding to FAK(8) . Therefore, CAKbeta may bind not to paxillin but to some other proteins associated with the cytoplasmic side of the surface membrane.

Expression of the CAKbeta Gene Transcripts

We have searched for CAKbeta gene expression in rat tissues by hybridization of the cDNA fragments, 5`-coding region and 3`-coding/noncoding regions of the CAKbeta cDNA, to a Northern blot carrying RNA from the following adult rat tissues: whole brain without cerebellum, cerebellum, lung, liver, kidney, spleen, intestine, testis, epididymis, adrenal gland, pancreas, and skeletal muscle. The Northern blots were also probed with a rat FAK probe as a reference. Transcripts of about 4.4 kilobases, almost the same size as FAK mRNA, were detected in whole brain, intestine, kidney, spleen and epididymis ( Fig. 7and 8). The same results were obtained with the CAKbeta 5`- and 3`- cDNA probes. CAKbeta mRNA is particularly abundant in whole brain without cerebellum. The transcripts are scanty in cerebellum, testis, and adrenal gland; in these organs the FAK gene transcripts are abundant (Fig. 7). The 4.4-kilobase transcripts were also detected in rat fibroblast lines, WFB and 3Y1 (Fig. 8). These cell lines also express mRNA for FAK (Fig. 8). In a human T cell leukemia line, Jurkat, transcripts of 4.6 kilobases were detected with both CAKbeta and FAK probes (Fig. 8). No significant CAKbeta gene transcript was found in mouse fibroblast lines (Fig. 8), BALB/3T3, Swiss/3T3, or NIH/3T3, a monkey cell line, COS-7 (Fig. 8), or rat and mouse neural cell lines (data not shown), PC12, NIE115, and NG108-15. In 3T3 lines and COS-7, the expression of the FAK gene was confirmed (Fig. 8).


Figure 7: Northern hybridization analysis of CAKbeta transcripts in rat tissues. A 10-µg aliquot of total RNA from various rat tissues was fractionated by electrophoresis, transferred to nitrocellulose membranes, and hybridized with rat CAKbeta 5`-coding region cDNA probe (top), rat FAK cDNA probe (middle), and beta-actin cDNA probe (bottom) under conditions of high stringency. The positions of 28 S and 18 S ribosomal RNAs are indicated on the right.




Figure 8: Northern hybridization analysis of CAKbeta transcripts in cell lines. A 10-µg aliquot of total RNA from the indicated cells was fractionated by electrophoresis, transferred to nitrocellulose membranes, and hybridized with the same probes as in Fig. 7under conditions of low stringency. Total RNA from rat brain was also analyzed as a control (leftmost lane of each blot). The positions of RNA size markers along with those of 28 S and 18 S ribosomal RNAs are indicated on the right.



Detection of CAKbeta in Rat Brain and 3Y1 Cells

Anti-CAKbeta antiserum was raised by immunizing rabbits with a bacterially expressed TrpE fusion protein containing the extreme C-terminal 230 amino acids of CAKbeta (amino acid residues 779-1008). Anti-CAKbeta was affinity-purified on a column of a covalently bound glutathione S-transferase fusion protein of the CAKbeta C-domain to Sepharose. Anti-CAKbeta specifically immunoprecipitated and immunoblotted a protein of about 113-kDa (equivalent to the calculated mass of CAKbeta) from the lysates of rat brain, 3Y1 cells and SR-3Y1 cells, a src-transformed line of 3Y1 (Fig. 9, lanes 2, 4, and 5). In accordance with the calculated molecular masses, the immunochemically identified CAKbeta has a faster mobility in SDS-PAGE than FAK, which was immunoprecipitated from the 3Y1 cell lysate with anti-FAK monoclonal antibody, 2A7(36) , and immunoblotted with polyclonal anti-FAK antibody (Fig. 9, lane 9). Immunoblotting with anti-phosphotyrosine revealed a band at CAKbeta on the blotted membrane from a SDS-PAGE gel where the anti-CAKbeta immunoprecipitates from the lysates of rat brain, 3Y1 cells and SR-3Y1 cells were separated (Fig. 9, lanes 6-8). CAKbeta of SR-3Y1 cells was stained more strongly with anti-phosphotyrosine than CAKbeta of 3Y1 cells, indicating higher in vivo tyrosine-phosphorylation of CAKbeta in the src-transformed cells; compare the CAKbeta band density in lane 7 divided by that in lane 4 with that in lane 8 divided by that in lane 5.


Figure 9: Immunochemical identification of CAKbeta in lysates of rat brain, 3Y1 cells, and SR-3Y1 cells. The lysates of rat brain, 3Y1 cells and SR-3Y1 cells (1 mg of protein per lane) were mixed with the anti-CAKbeta (alphaCAKbeta) beads (lanes 2 and 4-8), the preimmune serum (pre) beads (lanes 1 and 3), or the anti-FAK, 2A7 (alphaFAK), beads (lane 9). Bound proteins were washed, and two-thirds of each immune complex was subjected to SDS-PAGE in a 7.5% gel. The resolved proteins were transferred to a PVDF membrane and probed with affinity-purified anti-CAKbeta (lanes 1-5), anti-phosphotyrosine antibody, 4G10 (lanes 6-8), and anti-FAK rabbit antibody (lane 9). Positions of molecular mass markers are indicated on the right. i.p., immunoprecipitation.



CAKbeta cDNA Encodes pp113

The pCAKbeta(S)-transfected COS-7 cells but not the pCAKbeta(AS), an antisense construct, transfected COS-7 cells or the mock-transfected COS-7 cells expressed CAKbeta of the same size as was detected in 3Y1 cells (Fig. 10A, lanes 1-5). Immunoprecipitation and immunoblotting with anti-CAKbeta was used to detect the expression. The CAKbeta expressed in COS-7 cells was also tyrosine-phosphorylated (Fig. 10A, lane 7). An epitope-tagged CAKbeta was expressed in the COS-7 cells transfected with pCAKbetaTag and contained an 11-amino acid epitope tag plus 3 amino acid residues in place of the C-terminal glutamic acid. The tagged CAKbeta was immunoprecipitated (Fig. 10B, lanes 4 and 8) and immunoblotted with anti-epitope tag monoclonal antibody (Fig. 10B, lanes 7 and 8). The tagged CAKbeta was also immunoprecipitated (Fig. 10B, lanes 3 and 7) and immunoblotted with anti-CAKbeta (Fig. 10B, lanes 3 and 4). The anti-epitope tag did not bind CAKbeta itself (Fig. 10B, lanes 2, 5, and 6).


Figure 10: Transient expression of CAKbeta in COS-7 cells. A, COS-7 cells (two 9-cm dishes) were transfected with no DNA (mock) (lane 1), antisense CAKbeta cDNA in pSRE (pCAKbeta(AS)) (lane 2), and sense CAKbeta cDNA in pSRE (pCAKbeta(S)) (lanes 3, 4, and 7). After 3 days, the cells were washed and then lysed in the lysis buffer. The 3Y1 cell lysate was prepared from a confluent culture (lanes 5 and 6). Proteins were immunoprecipitated from 1 mg protein of the lysates with either preimmune (pre) serum beads (lane 3) or with anti-CAKbeta (alphaCAKbeta) beads (lanes 1, 2, and 4-7). Two-third of each immune complex was subjected to SDS-PAGE in a 7.5% gel, and the resolved proteins were transferred to a PVDF membrane and immunoblotted with affinity-purified anti-CAKbeta (lanes 1-5) or with anti-phosphotyrosine, 4G10 (lanes 6 and 7). Positions of molecular mass markers are indicated on the left. Arrow indicates CAKbeta. i.p., immunoprecipitation. B, COS-7 cells (two 9-cm dishes) were transfected with either pCAKbeta(S) (lanes 1, 2, 5, and 6) or epitope-tagged CAKbeta cDNA in pSRE (pCAKbetaTag) (lanes 3, 4, 7, and 8). After 3 days, the cells were lysed and CAKbeta was immunoprecipitated from 1 mg of protein of the lysates with either alphaCAKbeta beads (lanes 1, 3, 5, and 7) or anti-epitope tag (alphaTag) beads (lanes 2, 4, 6, and 8). SDS-PAGE and transfer to a PVDF membrane were done as described above. The membrane were immunoblotted with affinity-purified anti-CAKbeta (lanes 1-4) or with anti-epitope tag (alphaTag) (lanes 5-8). Arrow indicates CAKbeta. The lower band in lanes 6 and 8 represents the heavy chain of anti-tag antibody.



In Vitro Phosphorylation of CAKbeta in Immune Complex Kinase Assays and Demonstration of PTK Activity

Immune complexes formed by incubating cell lysates with anti-CAKbeta and anti-epitope tag were assayed for protein kinase activity with [-P]ATP as the phosphate doner without adding exogenous acceptor, and the P-labeled immune complexes were analyzed by SDS-PAGE. A protein of about 113-kDa, the size of CAKbeta, was found to become P-phosphorylated (Fig. 11). The P labeling of the protein was found in the kinase assays of the immunoprecipitate from the 3Y1 cell lysate with anti-CAKbeta (Fig. 11, lane 8), of that with anti-CAKbeta from the pCAKbeta(S)-transfected COS-7 cell lysate (Fig. 11, lane 3), of that with anti-CAKbeta from the pCAKbeta(S)Tag-transfected COS-7 cell lysate (Fig. 11, lane 5), and of that with anti-epitope tag from the pCAKbeta(S)Tag-transfected COS-7 cell lysate (Fig. 11, lane 6). The P labeling of the 113-kDa protein was not found in the kinase assays of the control immunoprecipitate with anti-CAKbeta from the pCAKbeta(AS)-transfected COS-7 cell lysate (Fig. 11, lane 1), of that with anti-epitope tag from the pCAKbeta(S)-transfected COS-7 cell lysate (Fig. 11, lane 4), or of the immunoprecipitates prepared by preimmune serum (Fig. 11, lanes 2 and 7). When an immunoprecipitate with anti-FAK from the 3Y1 cell lysate was subjected to the in vitro kinase assay, a 125-kDa protein was P-phosphorylated and tentatively identified as the autophosphorylated FAK (Fig. 11, lane 9). These results indicate that the 113-kDa protein revealed by the in vitroP-phosphorylation is the CAKbeta autophosphorylated in vitro.


Figure 11: P-Phosphorylation of CAKbeta in an immune complex kinase assay. CAKbeta was immunoprecipitated with the anti-CAKbeta (alphaCAKbeta) beads from the lysates of COS-7 cells transfected with pCAKbeta(AS), pCAKbeta(S), and pCAKbetaTag (lanes 1, 3, and 5), and from the 3Y1 cell lysate (lane 8). CAKbeta was also immunoprecipitated with the anti-epitope tag (alphaTag) beads from the lysates of COS-7 cells transfected with pCAKbetaTag and pCAKbeta(S) (lanes 6 and 4). Immunoprecipitates with preimmune (pre) serum bound to protein A-Sepharose were used as controls (lanes 2 and 7). FAK was immunoprecipitated from the 3Y1 cell lysate with the anti-FAK (alphaFAK) beads (lane 9). 0.4 mg of protein of the COS-7 or 3Y1 cell lysate was used for each assay. The immune complexes were subjected to the kinase assay with [-P]ATP as the substrate. The labeled proteins in the immune complexes were separated by SDS-PAGE and made visible by exposing the gel to a XAR film as described under ``Materials and Methods.'' Positions of molecular mass markers are indicated on the right. i.p., immunoprecipitation; S, sense; AS, antisense.



To confirm the PTK activity of CAKbeta, the immune complexes formed with anti-CAKbeta and anti-epitope tag were assayed for the kinase activity with poly(Glu,Tyr) as an exogenous substrate. CAKbeta and the tagged CAKbeta immunoprecipitated from the transfected-COS-7 cells efficiently catalyzed the phosphorylation of the substrate (Table 1). CAKbeta and FAK immunoprecipitated from 3Y1 cells also catalyzed the phosphorylation (Table 1).



Tyrosine Phosphorylation of CAKbeta Is Not Enhanced in Response to Plating 3Y1 Cells onto Fibronectin

As has been shown in 3T3 cells (6, 43) , the phosphotyrosine content of FAK decreased on detachment of 3Y1 cells by trypsinization (Fig. 12B, lanes 5 and 6) and regained on plating the cells onto fibronectin but not on plating onto poly-L-lysine (Fig. 12B, lanes 5, 7, and 8). To test the possible interaction of CAKbeta with integrin and fibronectin, we investigated the effects of trypsinization and plating of 3Y1 cells on fibronectin-coated dishes on the tyrosine-phosphorylated state of CAKbeta by immunoblotting with anti-phosphotyrosine. The tyrosine-phosphorylated state of CAKbeta was not affected by trypsinization (Fig. 12A, lanes 5 and 6) or by plating the cells onto fibronectin or poly-L-lysine (Fig. 12A, lanes 5, 7, and 8). A control blot probed with anti-CAKbeta verified that equal amounts of CAKbeta were present in the lysates (Fig. 12A, lanes 1-4). The results indicate that CAKbeta is not activated in response to cell interactions with fibronectin, suggesting the association of CAKbeta with a cell surface molecule other than integrin.


Figure 12: Analysis of phosphorylation of CAKbeta and FAK in 3Y1 cells before and after trypsinization and 50 min after plating the cells onto fibronectin or poly-L-lysine. The 3Y1 cell lysates were prepared by adding the lysis buffer either directly to the washed cells on dishes (lanes 1, 3-5, 7, and 8) or to the trypsinized cells (lanes 2 and 6) (Off Dish). The lysates of cells on dishes were prepared either from cells before trypsinization (On Dish), from cells 50 min after plating on fibronectin, or from cells 50 min after plating on poly-L-lysine. A, immunoprecipitation with anti-CAKbeta. Immunoblotting was either with anti-CAKbeta (lanes 1-4) or with anti-phosphotyrosine (lanes 5-8). B, immunoprecipitation with anti-FAK. Immunoblotting was either with anti-FAK (lanes 1-4) or with anti-phosphotyrosine (lanes 5-8).



CAKbeta Localizes to Sites of Cell-to-Cell Contact but Not to Sites of Focal Adhesion

Anti-epitope tag antibody and affinity-purified anti-CAKbeta antibodies were used to localize CAKbeta in the pCAKbetaTag-transfected COS-7 cells by immunostaining. Confocal laser scanning microscopy made it possible to locate FAK at the base of the cells and CAKbeta at the cell-to-cell contact (Figs. 13-15). As shown in Fig. 8, COS-7 cells express FAK mRNA but not CAKbeta mRNA. Therefore, the endogenous FAK and CAKbeta expressed from the transfected pCAKbetaTag were examined by the immunostaining. In the confluent pCAKbetaTag-transfected cells, both anti-epitope tag and anti-CAKbeta stained the sites of cell-to-cell contact at the middle to upper portions of the cells (Figs. 13B and 14B). The confluent cells were about 7 µm tall, and the images of optical sections 4 µm above the base of the cells were presented in Fig. 13B and 14B to show the pericellular stainings. Such pericellular staining was not found in the pCAKbeta-transfected cells, a control, on the anti-tag staining (Fig. 13D), or in the mock-transfected cells on the anti-CAKbeta staining (Fig. 14D). The nuclear immunofluorescence was not specific to the pCAKbetaTag-transfected cells but was also found in the control cells (Fig. 13, B and D, and 14, B and D), and thus considered to be mostly nonspecific. FAK was immunolocalized at the bottom of COS-7 cells in a patchy distribution around the base of nucleus (Fig. 15A). The sites of immunostained FAK probably represent focal adhesions of the cells. The presence of focal adhesions at the bottom of COS-7 cells was confirmed by confocal interference reflection microscopy (44) (data not shown). Neither significant staining at the cell bottom with anti-epitope tag (Fig. 13, A and C) and with anti-CAKbeta ( Fig. 14A and C) nor significant staining with anti-FAK at the middle of the cells, optical sections at 4 µm, (Fig. 15B) was found in images of these optical sections.


Figure 13: Immunostaining of CAKbeta in the pCAKbetaTag-transfected COS-7 cells with anti-epitope tag viewed with optical sections obtained by confocal microscopy. The pCAKbetaTag-transfected COS-7 cells (A and B) and the pCAKbeta-transfected COS-7 cells, a control (C and D), were immunostained with anti-epitope tag. Images of optical sections at 0 µm (A and C) and 4 µm (B and D) above the surface of coverslips were obtained with a confocal laser scanning microscope. The calibration bar in C represents 10 µm.




Figure 14: Immunostaining of CAKbeta in the pCAKbetaTag-transfected COS-7 cells with anti-CAKbeta viewed with optical sections obtained by confocal microscopy. The pCAKbetaTag-transfected COS-7 cells (A and B) and the mock-transfected COS-7 cells, a control (C and D), were immunostained with anti-CAKbeta. Images of optical sections at 0 µm (A and C) and 4 µm (B and D) above the surface of coverslips were obtained with a confocal laser scanning microscope. The calibration bar in C represents 10 µm.




Figure 15: Immunostaining of FAK in the pCAKbetaTag-transfected COS-7 cells with anti-FAK viewed with optical sections obtained by confocal microscopy. The pCAKbetaTag-transfected COS-7 cells (A and B) were immunostained with anti-FAK. Images of optical sections at 0 µm (A) and 4 µm (B) above the surface of coverslips were obtained with a confocal laser scanning microscope. Omission of the primary antibody yielded no significant staining of the cells (data not shown). The calibration bar in A represents 10 µm.



One of the most important questions on CAKbeta to be answered is the identification of the cell adhesion molecule, which associates with CAKbeta, and activates and tyrosine-phosphorylates CAKbeta. Localization of CAKbeta to the sites of cell-to-cell contact and not to focal adhesions suggests an association of CAKbeta with some cell-to-cell adhesion molecule. Thus, FAK is the first cell adhesion kinase participating in the signal transduction triggered by cell interactions with the extracellular matrix, while CAKbeta is the second cell adhesion kinase with a possibility to participate in the signal transduction pathway regulated by cell-to-cell adhesions.

The finding that WFB and Jurkat express CAKbeta as well as FAK raises an interesting question: whether any receptor known to be present in these cells is coupled to the activation and phosphorylation of CAKbeta. WFB responds to vasopressin, endothelin, bombesin, prostaglandin F, and PDGF with the activation of phospholipase C and an elevation of intracellular free Ca concentration(45) . In Jurkat cells, tyrosine kinase pathways are activated by the ligation of the T cell receptor complex(46) . Recently, Kanner et al.(47) reported that ligation of the antigen receptors on T and B lymphocytes rapidly augments the tyrosine phosphorylation of a FAK-related protein, which they denote fakB. The human FAK peptide sequence, against which they raised fakB-reactive antiserum, 714, is only 3 out of 20 residues identical with the corresponding sequence, residues 683-701, of rat CAKbeta. Therefore, fakB is not likely to be CAKbeta.


FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequences reported in this paper have been submitted to the GSDB, DDBJ, EMBL, and NCBI nucleotide sequence data bases with accession numbers D45854 [GenBank]and D45853[GenBank].

§
To whom correspondence should be addressed: Dept. of Biochemistry, Cancer Research Institute, Sapporo Medical University School of Medicine, South-1, West-17, Chuo-Ku, Sapporo 060, Japan. Tel.: 81-11-611-2111 (ext. 2380); Fax: 81-11-612-5861.

(^1)
The abbreviations used are: PTKs, protein-tyrosine kinases; FAK, focal adhesion kinase; CAKbeta, cell adhesion kinase beta; PCR, polymerase chain reaction; SH-2, Src homology 2; SH-3, Src-homology 3; PAGE, polyacrylamide gel electrophoresis; PVDF, polyvinylidene difluoride; PBS, phosphate-buffered saline.

(^2)
T. Sasaki, K. Nagura, and H. Sasaki, manuscript in preparation.


ACKNOWLEDGEMENTS

We thank Dr. Michio Mori (Department of Pathology, Sapporo Medical University School of Medicine) for helpful discussions and for valuable advice in using the confocal laser scanning microscope of his laboratory.


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