Protein-tyrosine kinases (PTKs) (
)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 CAK
for cell adhesion
kinase
.
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
, 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 CAK
A
clone, M9-3, isolated from the PCR library was labeled with
[
-
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 CAK
cDNA clones were obtained by screening about 8
10
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
CAK
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 CAK
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 CAK
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 CAK
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 CAK
. The
actin probe was prepared from human
-actin cDNA.
Production of Antiserum to CAK
and Affinity
Purification of the Antibody
Digestion of rat CAK
cDNA
(clone 24) with SphI and PstI restriction
endonucleases generated a 688-base pair fragment encompassing
nucleotides 2333 through 3020 of the CAK
cDNA. This fragment,
encoding amino acid residues 779-1008 of CAK
, 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-CAK
fusion protein(30) .
The bacteria were lysed by sonication and the TrpE-CAK
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 CAK
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 CAK
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 CAK
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
VO
, 20 mM Na
P
O
). 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. CAK
was immunoprecipitated by mixing anti-CAK
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-CAK
beads were prepared for each assay by mixing 2 µg of
affinity-purified anti-CAK
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 CAK
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-CAK
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 CAK
in COS-7
Cells
The CAK
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-SR
-296 vector(39) . The resulting construct,
pCAK
(S), and a control construct, pCAK
(AS), in which the
CAK
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 CAK
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 CAK
. For
expression in vivo, the derivative was subcloned into pSRE to
obtain pCAK
Tag. The 11-residue epitope tag is specifically
recognized by the anti-epitope tag monoclonal antibody.
Immune Complex Kinase Assay
CAK
,
epitope-tagged CAK
, 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
, 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
. 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 CAK
Phosphotyrosine Content
To
study changes in phosphotyrosine content of CAK
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-CAK
beads or anti-FAK beads.
After SDS-PAGE and blotting to PVDF membranes, CAK
, FAK, and
phosphotyrosine were detected by probing blots with anti-CAK
,
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-CAK
(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
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 CAK
.
Figure 1:
Organization of the cloned rat CAK
cDNA. The CAK
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 CAK
protein,
with the catalytic domain of the protein kinase denoted by an open
box.
Figure 2:
Nucleotide sequence of rat CAK
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.
CAK
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 CAK
cDNA, which we cloned from human
hippocampus cDNA library. The nucleotide sequences are well conserved
in the putative coding regions of rat and human CAK
cDNAs,
resulting in the almost identical N-terminal amino acid sequences of
rat and human CAK
s ( Fig. 3and 4). The 5`-noncoding regions
of rat and human CAK
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 CAK
cDNAs and their deduced
amino acid sequences. The 5`-terminal portion of human CAK
isolated from human hippocampus cDNA library was compared with the
5`-terminal portion of rat CAK
. 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 CAK
cDNA sequence by the
BLASTx program of NCBI with those in GenBank
data base
revealed homology of CAK
and FAK over their entire lengths, and
did not detect any sequence more closely related to CAK
.
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 CAK
clearly indicate
that CAK
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 CAK
. The amino acid sequence of the catalytic domain of
CAK
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 CAK
are 39 and 40% identical with those of mouse FAK (Fig. 5). As in FAK, CAK
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 CAK
shares only 45%
amino acid identity with mouse FAK indicates that CAK
is the
second PTK of the FAK subfamily. Indeed, we have amplified rat FAK in
addition to CAK
from rat brain RNA by RT/PCR using degenerated
oligonucleotide primers designed from the amino acid sequences common
to both FAK and CAK
. (
)
Figure 5:
Comparison of amino acid sequences of rat
CAK
and mouse FAK. The numbers on the right indicate the
positions relative to the putative start methionine of CAK
and the
amino acid residue number of mouse FAK(6) . The catalytic
domains are boxed. Amino acid residues of FAK identical with
those of CAK
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 CAK
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
CAK
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 CAK
.
Residues which are highly conserved among PTKs, but different in
CAK
, are indicated by the symbol, #. The number in parentheses is the percentage of residues that are identical with CAK
.
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 CAK
and FAK
The
predicted amino acid sequence of CAK
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. CAK
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 CAK
, three other residues highly conserved in most
of the other PTK catalytic domains are not conserved in CAK
(Fig. 6). Two of these at the residues 536 and 626 of CAK
are not conserved in FAK as well. The other one at the residue 612 of
CAK
is alanine. The corresponding residue in FAK is glycine, the
conserved amino acid of this position in the PTK catalytic domain.
Conversely, Met
of CAK
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 CAK
and FAK are shown in Fig. 5. Although
the amino acid residues 89-418 of CAK
are highly homologous
(47.6% identity) with the corresponding N-terminal domain of FAK, the
sequence of the extreme N-terminal 88 residues of CAK
is entirely
different from any portion of FAK (Fig. 5). This difference may
imply specific binding of CAK
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
CAK
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 CAK
(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 CAK
as compared with FAK. In the sequence
comparison presented in Fig. 5, three gaps were introduced in
the C-terminal domain of CAK
to maximize the homology with FAK. As
shown in Fig. 5, the C-terminal domain of CAK
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 CAK
. 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 CAK
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 CAK
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 CAK
(residues 18-30).
The residues 869-999 of
CAK
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 CAK
between
positions 845 and 967 may possibly contain the targeting sequence of
CAK
to a certain submembranous site. On the other hand, the
CAK
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, CAK
may bind not to paxillin but to some other proteins
associated with the cytoplasmic side of the surface membrane.
Expression of the CAK
Gene Transcripts
We
have searched for CAK
gene expression in rat tissues by
hybridization of the cDNA fragments, 5`-coding region and
3`-coding/noncoding regions of the CAK
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 CAK
5`- and 3`- cDNA probes. CAK
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 CAK
and FAK probes (Fig. 8). No significant CAK
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
CAK
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
CAK
5`-coding region cDNA probe (top), rat FAK cDNA probe (middle), and
-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
CAK
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 CAK
in Rat Brain and 3Y1
Cells
Anti-CAK
antiserum was raised by immunizing rabbits
with a bacterially expressed TrpE fusion protein containing the extreme
C-terminal 230 amino acids of CAK
(amino acid residues 779-1008).
Anti-CAK
was affinity-purified on a column of a covalently bound
glutathione S-transferase fusion protein of the CAK
C-domain to Sepharose. Anti-CAK
specifically immunoprecipitated
and immunoblotted a protein of about 113-kDa (equivalent to the
calculated mass of CAK
) 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 CAK
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 CAK
on
the blotted membrane from a SDS-PAGE gel where the anti-CAK
immunoprecipitates from the lysates of rat brain, 3Y1 cells and SR-3Y1
cells were separated (Fig. 9, lanes 6-8).
CAK
of SR-3Y1 cells was stained more strongly with
anti-phosphotyrosine than CAK
of 3Y1 cells, indicating higher in vivo tyrosine-phosphorylation of CAK
in the src-transformed cells; compare the CAK
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 CAK
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-CAK
(
CAK
) beads (lanes
2 and 4-8), the preimmune serum (pre)
beads (lanes 1 and 3), or the anti-FAK, 2A7 (
FAK), 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-CAK
(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.
CAK
cDNA Encodes pp113
The
pCAK
(S)-transfected COS-7 cells but not the pCAK
(AS), an
antisense construct, transfected COS-7 cells or the mock-transfected
COS-7 cells expressed CAK
of the same size as was detected in 3Y1
cells (Fig. 10A, lanes 1-5).
Immunoprecipitation and immunoblotting with anti-CAK
was used to
detect the expression. The CAK
expressed in COS-7 cells was also
tyrosine-phosphorylated (Fig. 10A, lane 7). An
epitope-tagged CAK
was expressed in the COS-7 cells transfected
with pCAK
Tag and contained an 11-amino acid epitope tag plus 3
amino acid residues in place of the C-terminal glutamic acid. The
tagged CAK
was immunoprecipitated (Fig. 10B, lanes 4 and 8) and immunoblotted with anti-epitope
tag monoclonal antibody (Fig. 10B, lanes 7 and 8). The tagged CAK
was also immunoprecipitated (Fig. 10B, lanes 3 and 7) and
immunoblotted with anti-CAK
(Fig. 10B, lanes 3 and 4). The anti-epitope tag did not bind CAK
itself (Fig. 10B, lanes 2, 5, and 6).
Figure 10:
Transient expression of CAK
in COS-7
cells. A, COS-7 cells (two 9-cm dishes) were transfected with
no DNA (mock) (lane 1), antisense CAK
cDNA in
pSRE (pCAK
(AS)) (lane 2), and sense CAK
cDNA in pSRE
(pCAK
(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-CAK
(
CAK
) 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-CAK
(lanes 1-5) or with
anti-phosphotyrosine, 4G10 (lanes 6 and 7). Positions
of molecular mass markers are indicated on the left. Arrow indicates CAK
. i.p., immunoprecipitation. B, COS-7 cells (two 9-cm dishes) were transfected with either
pCAK
(S) (lanes 1, 2, 5, and 6)
or epitope-tagged CAK
cDNA in pSRE (pCAK
Tag) (lanes
3, 4, 7, and 8). After 3 days, the
cells were lysed and CAK
was immunoprecipitated from 1 mg of
protein of the lysates with either
CAK
beads (lanes 1, 3, 5, and 7) or
anti-epitope tag (
Tag) 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-CAK
(lanes
1-4) or with anti-epitope tag (
Tag) (lanes
5-8). Arrow indicates CAK
. The lower band in lanes 6 and 8 represents the heavy chain of anti-tag
antibody.
In Vitro Phosphorylation of CAK
in Immune Complex
Kinase Assays and Demonstration of PTK Activity
Immune complexes
formed by incubating cell lysates with anti-CAK
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 CAK
, 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-CAK
(Fig. 11, lane 8), of that with
anti-CAK
from the pCAK
(S)-transfected COS-7 cell lysate (Fig. 11, lane 3), of that with anti-CAK
from the
pCAK
(S)Tag-transfected COS-7 cell lysate (Fig. 11, lane
5), and of that with anti-epitope tag from the
pCAK
(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-CAK
from the pCAK
(AS)-transfected COS-7 cell lysate (Fig. 11, lane 1), of that with anti-epitope tag from
the pCAK
(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 vitro
P-phosphorylation is the CAK
autophosphorylated in vitro.
Figure 11:
P-Phosphorylation of
CAK
in an immune complex kinase assay. CAK
was
immunoprecipitated with the anti-CAK
(
CAK
)
beads from the lysates of COS-7 cells transfected with pCAK
(AS),
pCAK
(S), and pCAK
Tag (lanes 1, 3, and 5), and from the 3Y1 cell lysate (lane 8). CAK
was also immunoprecipitated with the anti-epitope tag (
Tag) beads from the lysates of COS-7 cells transfected
with pCAK
Tag and pCAK
(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 (
FAK) 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 CAK
, the
immune complexes formed with anti-CAK
and anti-epitope tag were
assayed for the kinase activity with poly(Glu,Tyr) as an exogenous
substrate. CAK
and the tagged CAK
immunoprecipitated from the
transfected-COS-7 cells efficiently catalyzed the phosphorylation of
the substrate (Table 1). CAK
and FAK immunoprecipitated from
3Y1 cells also catalyzed the phosphorylation (Table 1).
Tyrosine Phosphorylation of CAK
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
CAK
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 CAK
by immunoblotting with
anti-phosphotyrosine. The tyrosine-phosphorylated state of CAK
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-CAK
verified that equal amounts of CAK
were present in the lysates (Fig. 12A, lanes 1-4). The results
indicate that CAK
is not activated in response to cell
interactions with fibronectin, suggesting the association of CAK
with a cell surface molecule other than integrin.
Figure 12:
Analysis of phosphorylation of CAK
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-CAK
. Immunoblotting was either with
anti-CAK
(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).
CAK
Localizes to Sites of Cell-to-Cell Contact but
Not to Sites of Focal Adhesion
Anti-epitope tag antibody and
affinity-purified anti-CAK
antibodies were used to localize
CAK
in the pCAK
Tag-transfected COS-7 cells by immunostaining.
Confocal laser scanning microscopy made it possible to locate FAK at
the base of the cells and CAK
at the cell-to-cell contact (Figs.
13-15). As shown in Fig. 8, COS-7 cells express FAK mRNA
but not CAK
mRNA. Therefore, the endogenous FAK and CAK
expressed from the transfected pCAK
Tag were examined by the
immunostaining. In the confluent pCAK
Tag-transfected cells, both
anti-epitope tag and anti-CAK
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
pCAK
-transfected cells, a control, on the anti-tag staining (Fig. 13D), or in the mock-transfected cells on the
anti-CAK
staining (Fig. 14D). The nuclear
immunofluorescence was not specific to the pCAK
Tag-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-CAK
( 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 CAK
in the
pCAK
Tag-transfected COS-7 cells with anti-epitope tag viewed with
optical sections obtained by confocal microscopy. The
pCAK
Tag-transfected COS-7 cells (A and B) and
the pCAK
-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 CAK
in the
pCAK
Tag-transfected COS-7 cells with anti-CAK
viewed with
optical sections obtained by confocal microscopy. The
pCAK
Tag-transfected COS-7 cells (A and B) and
the mock-transfected COS-7 cells, a control (C and D), were immunostained with anti-CAK
. 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
pCAK
Tag-transfected COS-7 cells with anti-FAK viewed with optical
sections obtained by confocal microscopy. The pCAK
Tag-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 CAK
to be
answered is the identification of the cell adhesion molecule, which
associates with CAK
, and activates and tyrosine-phosphorylates
CAK
. Localization of CAK
to the sites of cell-to-cell contact
and not to focal adhesions suggests an association of CAK
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 CAK
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 CAK
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 CAK
. 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 CAK
. Therefore, fakB is not likely
to be CAK
.