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INTRODUCTION |
In rat liver epithelial cells, epidermal growth
factor-dependent tyrosine phosphorylation occurs in two
waves, the second wave is attributable, in part, to an epidermal growth
factor-stimulated rise in intracellular calcium (1, 2). G
protein-coupled receptor agonists initiate a greater intracellular
calcium signal and more robust tyrosine phosphorylation (1, 3, 4).
Purification and peptide sequencing of the responsible soluble tyrosine
kinase revealed a novel sequence highly related to the focal adhesion tyrosine kinase, FAK1 (5, 6).
We named this second member of the cytoskeleton-associated tyrosine
kinase family, the calcium-dependent
tyrosine kinase (CADTK) to denote a principle
mechanism of regulation. At the same time, four other groups identified
this kinase and named it Pyk2 (7), CAK
(8), RAFTK (9), and FAK2
(10). CADTK is activated by a wide variety of hormones and other G
protein-coupled receptor agonists as well as pharmacological agents
that raise intracellular calcium or activate protein kinase C (6, 7, 11-15). Additionally, growth factors (16-20), chemokines (21-24), cytokines (25, 26), cell stress signals (6, 27, 28), and in some
instances cell adherence (23, 29-31), have all been shown to activate
CADTK in some cell types. Once activated, CADTK has been implicated in
the regulation of ion channels (7), extracellular signal regulated
kinase (7, 12-14), c-Jun N-terminal kinase (JNK) (6, 18, 22, 25, 27,
28, 32), and p70 S6 kinase (p70S6K) (33). It has also been
reported that CADTK may play a role in Fyn-mediated T cell receptor
(34, 35), Syk-mediated Fc
RI receptor (36), and JAK3-mediated
interleukin-2 receptor signaling pathways (26). CADTK physically
associates with cytoskeletal proteins, such as paxillin and
p130Cas (37-40), as well as their homologues Hic5 (41),
leupaxin (42), and p105HEF1 (37), and may directly regulate
their tyrosine phosphorylation in response to agonists as diverse as
growth factors and cell adherence (16, 23).
CADTK and FAK are highly homologous, sharing an overall 45% amino acid
sequence identity with 60% identity in the catalytic domain. Several
tyrosine residues appear to be conserved between CADTK and FAK,
including a Src family tyrosine kinase SH2-binding site. Furthermore,
CADTK, like FAK, contains proline-rich motifs capable of SH3 domain
interaction and a putative focal adhesion targeting domain.
Immunostaining of CADTK and expression of GFP-tagged CADTK revealed
that CADTK localizes to the focal adhesion region. In cells expressing
both FAK and CADTK, both proteins appear to co-localize near the focal
adhesion membrane attachment sites, however, CADTK extends further into
the cell, e.g. CADTK extends onto actin stress fibers in
smooth muscle cells
(16).2
Given the high degree of structural and amino acid sequence similarity,
CADTK and FAK may well have some similar or even interchangeable biological functions. In fact, well studied mouse fibroblast cell lines
appear to express only FAK and not CADTK. In contrast, we and others
have recently shown that normal circulating monocytes, as well as T and
B cells, express only CADTK (23, 24). Thus certain cells can function
with only one of these two cytoskeleton-associated kinases. Conversely,
since both proteins are often co-expressed in mesenchymal cells,
multiple epithelial cells, neural cells and tissues, and endothelial
cells (6, 7, 14, 16, 39, 43), they presumably have distinct functions
as well. When expressed together the major difference between the two
enzymes was easily detected, FAK is constitutively active in resting,
adherent cells while CADTK is dephosphorylated and inactive until
stimulated by agonists, i.e. CADTK regulation is more
dynamic in the sense that it rapidly responds to extracellular signals.
In this report, we examined the tyrosine autophosphorylation and
tyrosine kinase activity of CADTK and variants constructed by
site-directed mutagenesis and compare them to previous
structure/function data obtained with FAK. CADTK and FAK were tyrosine
phosphorylated by Src family tyrosine kinases; the sites of CADTK are
not necessarily those previously thought to be Src targets on FAK.
Intriguingly, we showed that CADTK tyrosine phosphorylated FAK both
in vivo and in vitro while FAK did not
phosphorylate CADTK. Again, sites other than major autophosphorylation
or putative Src target site appeared to be involved. Last, by
transiently co-expressing the carboxyl terminus of CADTK (CRNK) and the
carboxyl terminus of FAK (FRNK) with wild type CADTK or FAK, we
demonstrated that FRNK inhibited both FAK and CADTK tyrosine
autophosphorylation, while CRNK inhibited only CADTK
autophosphorylation. Our results demonstrate that although CADTK and
FAK are regulated differentially in cells expressing both proteins,
they may, additionally, influence signal transduction from the other
family member.
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EXPERIMENTAL PROCEDURES |
Materials--
Yes and Y527F Src were kindly provided by Drs.
Nancy Rabb-Traub, and Channing Der (University of North Carolina at
Chapel Hill), respectively. Fyn, Lck, Syk, and Zap70 were generously provided by Dr. Andre Veillette (McGrill University). Polyclonal anti-CADTK antibody was described previously (6), anti-HA monoclonal antibody and anti-FAK (C-20, A-17) were purchased from Boehringer Mannheim and Santa Cruz Biotechnology, respectively. Human 293(T) cells
were grown in Dulbecco's modified Eagle's medium with 10% fetal
bovine serum as described previously (39).
Site-directed Mutagenesis--
Rat wtCADTK cDNA was used as
a template to generate a series of CADTK mutants based on polymerase
chain reaction site-directed mutagenesis strategy (Stratagene). Mutated
CADTK cDNAs were amplified by Pfu DNA polymerase (Stratagene) with
complementary DNA mutagenic oligonucleotides for designed mutations
(K457A, D567N, Y402F, Y881F, K457A/Y402F, Y579F/Y580F CADTK). The human
FAK cDNA was used to make the mutants Y397F, Y576F/Y577F, and Y925F
FAK by the same method. All the mutants were confirmed by DNA sequence analysis (University of North Carolina, Sequence Facility).
Cell Lysate Preparation--
Cell lysates were prepared
essentially as described previously (5). Briefly, cells treated with
agonists were scraped into ice-cold cell lysis buffer (150 mM NaCl, 20 mM Tris (pH 7.5), 1% Triton X-100,
5 mM EDTA, 50 mM sodium fluoride, and 10%(v/v) glycerol with freshly added 1 mM
Na3VO4, 20 µg/ml phenymethylsulfonyl fluoride, 10 µg/ml leupeptin, and 100 kallikrein inhibitor units of
aprotinin/ml). Cell lysates were clarified by centrifugation and their
protein content determined by Coomassie protein assay reagent (Pierce).
cDNA Transient Expression in Human 293(T)
Cells--
pcDNA3 vector (Invitrogen), pcDNA3-CADTK,
pcDNA3-wtCADTK, pcDNA3-FAK, pCMV-FRNK, pcDNA3-Yes,
PXM139-Fyn, Lck, Syk, and ZAP70 were transfected or co-transfected into
human 293(T) cells with Fugene6 according to the manufacturer's
procedure (Boehringer Mannheim). After 48 h, transfected cells
were harvested and lysed, and the lysates were analyzed by
immunoprecipitation, followed by immunoblotting with anti-Tyr(P),
anti-CADTK, anti-FAK, or by a tyrosine kinase activity assay.
Immunoprecipitation and Immunoblotting--
In a typical
experiment, ~500 µg of cell lysate was immunoprecipitated by
incubation with the antibody for 2 h at 4 °C. 20 µl of
protein A-agarose beads were then added for 1 more hour. Immune
complexes were collected by centrifugation, washed three times with
lysis buffer, and resuspended in SDS-PAGE sample buffer. Samples were
subjected to SDS-PAGE, transferred to Immobilon (Millipore), and
incubated with the selected antibody. Immunoblots were developed with
ECL according to the manufacturer's procedure (Amersham). Immunoblots
were stripped in buffer (62.5 mM Tris (pH 6.8), 2% SDS,
100 mM
-mercaptoethanol) at 50 °C for 30 min and
reprobed with another antibody.
Tyrosine Kinase Activity Assay--
Immune complex tyrosine
kinase and autokinase assays were performed as previously reported (3).
Briefly, immune complex suspensions were preincubated for 5 min at
4 °C with 160 µg of the synthetic tyrosine kinase substrate
poly(Glu4:Tyr) (Sigma) or the control substrate poly(Glu).
Additional experiments were performed using a GST NH2
terminus of human FAK fusion protein encompassing amino acids 1 to 426 (5 µg per assay) as a substrate. Reactions (80 µl of total reaction
volume) were initiated by adding of 5 µCi of
[
-32P]ATP (5 µM) After 15 min at
25 °C, 50 µl of the reaction mixture was spotted on P81 Whatman
paper. The papers were washed once with 10 mM sodium
pyrophosphate in 10% trichloroacetic acid and twice with 5%
trichloroacetic acid, air-dried, and assayed by liquid scintillation
counting. For GST NH2-terminal FAK phosphorylation, the
assays were stopped with SDS stop solution, run on 8%
SDS-polyacrylamide gels, and subjected to autoradiography. Assessment
of the ability of CADTK and FAK to directly cross-phosphorylate was
performed using an in vitro tyrosine phosphorylation assay.
Briefly, CADTK-GFP, kdCADTK, kdFAK, and control vector were transiently
expressed in 293(T) cells, independently. Equal amounts of cell lysates were mixed before adding specific antibodies. Cell lysate mixtures were
incubated at 4 °C for 2 h followed by addition of protein A/G-agarose beads. Immunocomplexes were washed three times with lysis
buffer and once with kinase assay buffer before providing [
-32P]ATP. Reaction mixtures were incubated at
25 °C for 15 min. After removing the supernatant, SDS-PAGE sample
loading buffer was added to the immunocomplexes and they were subjected
to SDS-PAGE and autoradiography. These experiments were repeated using
wild type CADTK and separating phosphorylated CADTK (p115) and
FAK (p125) on 8% low bis-acrylamide gels and by immunoprecipitating
both CADTK-GFP and kdFAK, using buffers with 0.1% SDS in the washes.
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RESULTS |
Site-directed Mutagenesis: Analysis of Tyrosine Autophosphorylation
and Tyrosine Kinase Activity of CADTK Mutants--
Tyr397
is a major FAK tyrosine autophosphorylation site (44). Mutation of this
site dramatically inhibited FAK tyrosine autophosphorylation and
decreased tyrosine kinase activity by 50% (45). Autophosphorylated Tyr397 serves as an SH2 domain docking site for the
recruitment of Src family tyrosine kinases (44, 46-48), which in turn
may tyrosine phosphorylate FAK on Tyr925 (49) or other
tyrosine residues (45). Guided by amino acid sequence similarity to
FAK, we mutated homologous CADTK tyrosine residues to investigate their
effects on CADTK tyrosine phosphorylation and kinase activity. In
addition, to create "kinase deficient" CADTK, we separately mutated
two CADTK residues important in Mg2+-ATP binding,
Lys457 and Asp567, changing them to Ala and
Asn, respectively. For comparison, we also made similar mutations in
the human FAK cDNA.
Both wild type and mutant cDNAs were transiently expressed in
293(T) cells and their tyrosine autophosphorylation and kinase activity
was assessed. As shown in Fig.
1A, mutation of the CADTK Mg2+-ATP-binding site residues (K457A or D567N) abolished
tyrosine kinase activity as assessed by poly(Glu4:Tyr)
phosphorylation as well as CADTK tyrosine autophosphorylation (Fig.
1B), suggesting that these two residues are each required
for CADTK kinase activity. Mutation of Tyr402
(corresponding to the Src family binding site, Tyr397) also
abolished CADTK tyrosine autophosphorylation, severely depressing but
not completely inhibiting tyrosine kinase activity (~70% decrease).
These data suggest that Tyr402, like Tyr397 in
FAK, is the major tyrosine autophosphorylation site. Its
phosphorylation is important but not absolutely required for tyrosine
kinase activity. Mutation of adjacent tyrosine residues
Tyr579/Tyr580 in the catalytic domain of CADTK
(corresponding to FAK Tyr576/Tyr577)
significantly decreased CADTK tyrosine autophosphorylation and reduced
kinase activity by ~60%. These data, taken together with the Y402F
mutant data, suggest that Tyr579/Tyr580 are not
major tyrosine autophosphorylation sites but that transient tyrosine
phosphorylation of these sites may be required for full or continued
CADTK activation.

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Fig. 1.
Tyrosine kinase activity and
autophosphorylation of CADTK and its mutants. Wild type CADTK and
its mutants were independently transfected into 293(T) cells. After
48 h, each was immunoprecipitated with anti-CADTK antibody.
A, tyrosine kinase activity assays were performed using
poly(Glu4:Tyr) as a substrate and measuring 32P
incorporation as described under "Experimental Procedures."
B, immunoprecipitated samples were subjected to SDS-PAGE and
immunoblotted with anti-Tyr(P) antibody. The blot was then stripped and
reblotted with anti-CADTK antibody. These data are representative of
three independent experiments.
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Mutation of Tyr881 (corresponding to FAK
Tyr925, the site of Grb2 SH2 domain association) had little
or no effect on CADTK tyrosine autophosphorylation or tyrosine kinase
activity. Thus, autophosphorylation appears to involve
Tyr402 but not Tyr881. Finally, double mutants,
K457A/Y402F and Y402F/Y881F, produced the same effect as the single
mutants, K457A and Y402F, respectively, suggesting that the single
mutation of K457A and Y402F produced the dominant effect. CADTK
tyrosine kinase activity and tyrosine autophosphorylation data were
generally concordant, indicating that tyrosine phosphorylation
correlates with kinase activity. Comparison of the effects of
corresponding CADTK and FAK mutations (45), suggests that the
structure-function regulatory mechanisms of these two tyrosine kinases
are similar.
To further examine the regulation of these two kinases, the
kinase-deficient CADTK (kdCADTK), K457A CADTK, and kinase-deficient FAK
(kdFAK) K454A FAK were transiently co-expressed with the Src family
tyrosine kinase members, Src, Fyn, Yes, and Lck. As shown in Fig.
2, Src, Fyn, and Yes dramatically
increased CADTK (Fig. 2A) and FAK (Fig. 2B)
tyrosine phosphorylation, while Lck had little effect (particularly on
CADTK). These data suggest that CADTK and FAK signaling may be
similarly affected by Src family tyrosine kinases, presumably by
phosphorylation of conserved tyrosine residues. However, Src and Fyn
were also capable of substantially phosphorylating the Y402F/Y881F
CADTK double mutant (Fig. 2C), suggesting that other
tyrosine residue(s) must be significant targets for Src family tyrosine
kinases. Moreover, at least when co-expressed, neither activated Src,
wild type Fyn, nor Yes required a Tyr402 docking site on
CADTK to tyrosine phosphorylate CADTK. Last, transient co-expression of
two important hematopoietic cytoplasmic tyrosine kinases, Syk or ZAP70,
with kdCADTK did not increase their tyrosine phosphorylation (data not
shown), suggesting that Syk and ZAP70 cannot directly phosphorylate
CADTK under these experimental conditions.

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Fig. 2.
Src family tyrosine kinases phosphorylate
both CADTK and FAK in vivo. Src family tyrosine
kinases (Src, Fyn, Yes, and Lck) were co-transfected with
kinase-deficient CADTK (kdCADTK) or kinase-deficient FAK
(kdFAK), respectively. kdCADTK (A) and kdFAK
(B) were immunoprecipitated, subjected to SDS-PAGE, and
immunoblotted with an anti-Tyr(P) antibody. Blots were stripped and
reblotted with anti-CADTK and anti-FAK antibodies, respectively.
C, kdCADTK or Y402F/Y881F CADTK were co-expressed with Src
or Fyn in 293(T) cells. CADTK was immunoprecipitated with anti-CADTK
antibody and subjected to anti-Tyr(P) followed by anti-CADTK
immunoblotting.
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CADTK Tyrosine Phosphorylates FAK in Vivo and in
Vitro--
Receptor tyrosine kinases from the epidermal growth factor
receptor family form heterodimers and cross-phosphorylate each other
(50). To test whether CADTK and FAK can also associate and
cross-phosphorylate, kdCADTK and kdFAK were transiently co-expressed in
human 293(T) cells with wild type FAK or CADTK, respectively. Immunoprecipitation with specific antibodies followed by
phosphotyrosine immunoblotting showed that CADTK significantly
increased kdFAK tyrosine phosphorylation (Fig.
3A). Similar results were
obtained with co-expression of either wtCADTK or CADTK-GFP (a fusion
protein with a green fluorescence protein fused to the CADTK carboxyl terminus) and kdFAK (Fig. 3A). In contrast, FAK failed to
phosphorylate kdCADTK (Fig. 3B). Interestingly, we could not
detect physical association between these two tyrosine kinases (data
not shown), i.e. immunoprecipitation (even using reduced
ionic strength buffer) of either enzyme from the dual transfected cells
did not reveal the other protein. Under the same conditions we have
easily detected complexes of CADTK and cytoskeletal proteins or other
SH3 containing proteins (39). Therefore, CADTK results in tyrosine
phosphorylation of kdFAK without forming lasting CADTK:FAK
heterodimers.

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Fig. 3.
CADTK phosphorylated FAK in vivo
and in vitro, but not vice
versa. A, CADTK or CADTK-GFP were
co-expressed with kdFAK in 293(T) cells. kdFAK was immunoprecipitated
with anti-FAK antibody. Immunocomplexes were subjected to SDS-PAGE and
immunoblotted with anti-Tyr(P) antibody. The blot was stripped and
reblotted with anti-FAK antibody. B, wtFAK were
co-transfected with kdCADTK in 293(T) cells. kdCADTK was
immunoprecipitated with anti-CADTK antibody and analyzed for
anti-Tyr(P) and anti-CADTK. The samples were also immunoprecipitated
with anti-FAK antibody and blotted with anti-Tyr(P) antibody, showing
that FAK was tyrosine phosphorylated by co-transfection. C,
CADTK-GFP, kdFAK, kdCADTK, and pcDNA3 vector were transfected into
293(T) cells, respectively. After lysis, equal amounts of cell lysates
were mixed as indicated, and immunoprecipitated with
NH2-terminal anti-CADTK and anti-FAK antibodies. After
washing, in vitro immunocomplex kinase assays were performed
labeling with [ -32P]ATP. Phosphotyrosine was analyzed
by SDS-PAGE, followed by autoradiography.
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Since the co-expression of CADTK could have activated an endogenous
third tyrosine kinase resulting in kdFAK phosphorylation, we
investigated CADTK:FAK cross-phosphorylation in vitro. To
separate CADTK and FAK on SDS-PAGE, we used CADTK-GFP, which has a
molecular mass of ~145 kDa, and similar tyrosine autophosphorylation
and kinase activity when compared with wtCADTK (data not shown).
Transient expression of CADTK-GFP, kdFAK, kdCADTK, and control vector
were performed individually in 293(T) cells. Equal amounts of cell lysate from the indicated pair of transfected cells were combined and
incubated with two antibodies, one directed against the NH2 terminus of CADTK and one against the NH2 terminus of FAK.
Subsequently, protein A/G-agarose beads were added and the indicated
pairs of transfected proteins or transfected protein and control were
precipitated, washed, and in vitro phosphorylation was
performed using [
-32P]ATP. Following SDS-PAGE,
autoradiography showed that CADTK-GFP facilitated the incorporation of
32P into kdFAK, suggesting direct in vitro
phosphorylation of FAK by CADTK-GFP (Fig. 3C).
Interestingly, CADTK-GFP only weakly phosphorylated kdCADTK in
vitro, suggesting that, at least for intermolecular phosphorylation, FAK may be a preferred substrate. Thus, CADTK can
tyrosine phosphorylate FAK both in vivo and in
vitro suggesting that agonist-dependent
(e.g. angiotensin II, lysophosphatidic acid, etc.) increases
in FAK phosphorylation in cells expressing and co-localizing both
enzymes may be attributed, in part, to CADTK-dependent FAK
tyrosine phosphorylation.
We next examined whether CADTK-dependent FAK tyrosine
phosphorylation resulted in increased FAK tyrosine kinase activity as measured by phosphorylation of poly(Glu4:Tyr). First,
co-expression of FAK and CADTK followed by immunoprecipitation of FAK
showed that CADTK can increase tyrosine phosphorylation of wtFAK (Fig. 4B) in addition to its
phosphorylation of kdFAK (Fig. 3). Second, assessment of in
vitro kinase activity showed an increase in FAK tyrosine kinase
activity (30-40%) (Fig. 4A). This was clearly less than
the ~5-10-fold CADTK-dependent increase in FAK tyrosine phosphorylation (Fig. 4B). This result (Fig. 4) obtained
with triplicate, independent transfections was typical of multiple experiments in which the increase in FAK tyrosine kinase activity was
much less than the increase in CADTK-dependent FAK tyrosine phosphorylation. Since poly(Glu4:Tyr) is not a a
physiologic substrate, the experiment, was repeated, using a GST FAK
NH2-terminal fusion protein that included
Tyr397 as a substrate for wild type FAK precipitated from
vector or CADTK transfected cells. The CADTK-dependent
tyrosine phosphorylation of FAK in co-transfected cells was again
increased dramatically (Fig.
5A), but autokinase activity
(32P incorporation into FAK in vitro, Fig.
5C) and substrate phosphorylation (32P
incorporation into GST NH2-terminal FAK, Fig.
5D) was only increased ~50-100%. Thus using 2 substrates, poly(Glu4:Tyr) and GST NH2-terminal FAK, CADTK-dependent FAK phosphorylation is greater than
the activation of FAK kinase. Because the kinase activity of FAK in
adherent cells has seldom been reported to change dramatically, it is
unclear what an ~50% change in kinase activity means. However, the
substantial increase in kdFAK or wtFAK tyrosine phosphorylation upon
co-transfection suggests that CADTK may indeed regulate FAK tyrosine
phosphorylation in vivo. Because FAK is in part a structural
protein whose function is to localize SH2-containing domain proteins,
increased FAK tyrosine phosphorylation may have physiologic
consequences.

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Fig. 4.
CADTK-dependent stimulation of
FAK tyrosine kinase activity and phosphorylation. In three
separate transfections, wtFAK was co-expressed with vector or CADTK in
293(T) cells. FAK was immunoprecipitated with anti-FAK antibody.
A, FAK kinase activity was measured by using
poly(Glu4:Tyr) as a substrate as described under
"Experimental Procedures." B, the immunocomplexes were
subjected to SDS-PAGE and blotted with anti-Tyr(P) antibody, then
stripped and reblotted with anti-FAK antibody.
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Fig. 5.
CADTK-dependent phosphorylation
and FAK autokinase activity. Following duplicate transfections
(vector and wtFAK (lanes 1 and 2) and CADTK and
wtFAK (lanes 3 and 4)), FAK was
immunoprecipitated and the immune complex analyzed. A,
phosphotyrosine immunoblotting demonstrated substantial
CADTK-dependent FAK tyrosine phosphorylation. B,
similar amounts of FAK were immunoprecipitated in each sample as
demonstrated by stripping and reprobing with anti-FAK (C20)
antibody. C, another portion of each immune complex was
incubated with [ -32P]ATP and GST
NH2-terminal FAK, (1-423) as described under
"Experimental Procedures. The autoradiograph shows FAK
autophosphorylation was increased slightly by a
CADTK-dependent action. D, FAK kinase activity
toward an exogenous substrate, GST NH2-terminal FAK was
also minimally increased. FAK parallel FAK-dependent
poly(Glu4:Tyr) phosphorylation by these immune complexes
showed an average of 14,366 cpm from samples 1 and 2 and 19,964 cpm
from samples 3 and 4.
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We investigated the potential CADTK-dependent tyrosine
phosphorylation sites on FAK by co-transfecting wtCADTK and FAK
mutants, kdFAK, Y397F, Y576F/Y577F, and Y925F FAK in 293(T) cells (Fig. 6). Transiently expressed CADTK
phosphorylated all FAK mutants in vivo suggesting
CADTK-dependent FAK tyrosine phosphorylation may occur on
other sites in addition to the well studied tyrosine residues. Taken
together, these data suggest that CADTK may potentially function as an
upstream regulator of FAK by directly phosphorylating FAK, slightly
increasing its tyrosine kinase activity, and generating more or even
new sites for the recruitment of SH2 containing proteins.

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Fig. 6.
CADTK phosphorylated FAK on tyrosine
phosphorylation sites other than Tyr397,
Tyr576/Tyr577, and Tyr925.
Several mutant FAK constructs were co-transfected with CADTK or
pcDNA3 vector in 293(T) cells as indicated. FAK was
immunoprecipitated and subjected to SDS-PAGE. After transfer and
immunoblotting with anti-Tyr(P), the blot was stripped and reprobed
with anti-FAK antibody.
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Overexpression of FRNK Inhibited FAK and CADTK Tyrosine
Autophosphorylation while Overexpression of CRNK Blocked Only CADTK
Tyrosine Phosphorylation--
Alternative RNA splicing of the FAK
primary transcript can yield a 43-kDa species encoding the carboxyl
terminus of FAK initiated just beyond the tyrosine kinase domain (51).
Overexpression of this protein, termed FRNK
(FAK-related
non-kinase), inhibits FAK activity, presumably
by displacing FAK (52) and in some manner blocking the formation of
focal adhesions on fibronectin (53, 54). FRNK probably acts by
competing for FAK binding partners through FRNK's focal adhesion
targeting domain, the sequence both necessary and sufficient for FAK
recruitment to the focal adhesion (55). CADTK shares 60% homology with
FAK in the carboxyl terminus. While not yet detected as an expressed
protein, CADTK has a methionine residue as a potential start site for
an alternatively spliced product analogous to FRNK. This has been
termed CRNK (CADTK or
CAK
-related
non-kinase, pronounced "crank") by our
colleague, Michael Schaller (56). Since CADTK activation, like FAK,
requires an intact engaged cytoskeleton (23, 39), we created a CRNK construct in pcDNA3 to investigate whether CRNK overexpression inhibited CADTK tyrosine autophosphorylation. When co-expressed CRNK
blocked CADTK tyrosine autophosphorylation (Fig.
7A); i.e. CRNK
functions similarly to FRNK and negatively regulates CADTK autophosphorylation. In contrast, co-expression of FAK and CRNK did not
alter FAK tyrosine autophosphorylation (Fig. 7B), suggesting that under these conditions of co-expression of CRNK, inhibition was
specific for CADTK, presumably because its sequence is not similar
enough to FRNK to displace FAK. As expected, FRNK inhibited FAK
activity when co-expressed (Fig. 7B) but, unexpectedly, FRNK co-expression dramatically inhibited CADTK tyrosine autophosphorylation (Fig. 7A). Thus, FRNK regulates both CADTK and FAK
activation under these conditions; however, the mechanisms by which
FRNK acts may not be the same for the two cytoskeleton-associated
kinases.

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Fig. 7.
Overexpression of CRNK only inhibited CADTK
autophosphorylation, while overexpression of FRNK inhibited both CADTK
and FAK tyrosine phosphorylation. wtCADTK or FAK were co-expressed
with CRNK, FRNK, or pcDNA3 vector in 293(T) cells, respectively.
A, CADTK was immunoprecipitated with anti-CADTK antibody and
analyzed with anti-Tyr(P) and anti-CADTK antibodies to determine CADTK
autophosphorylation, CADTK and CRNK expression. Samples were also
analyzed for FRNK expression. B, FAK was immunoprecipitated
with anti-FAK antibody, and its phosphorylation and expression level
was analyzed using anti-Tyr(P) and anti-FAK antibodies.
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The specific mechanisms of CADTK or FAK activation remain elusive. In
resting, adherent cells, FAK is constitutively activated by the process
of adherence, while CADTK is "off" in resting adherent cells, but
is rapidly activated by agonists. Transient overexpression of CADTK in
293(T) and other cells using a strong promoter results in CADTK
activation in resting cells without the addition of an agonist. This
presumably occurs by one of several mechanisms potentially including:
(i) overexpression-dependent movement of CADTK into an
activating binding site or (ii) by overcoming a saturable negative regulation system such as a constitutively activated tyrosine phosphatase that removes CADTK activating tyrosine phosphorylation. To
investigate this, we co-expressed CADTK and vector or CADTK and CRNK
and pretreated cells with pervanadate (5 min), a technique known to
inhibit most, if not all, tyrosine phosphatases. Treatment with
pervanadate resulted in a substantial increase (10-20-fold) in CADTK
tyrosine phosphorylation (Fig. 8),
indicating that there is a vast pool of underphosphorylated CADTK in
the transfected cells. CRNK, as previously shown, inhibited the
autophosphorylation of CADTK stimulated by overexpression but did not
significantly depress CADTK phosphorylation in cells pretreated with
pervanadate. That CRNK does not block activation by pervanadate
suggests that at least two mechanisms of CADTK activation, one
dependent on cellular binding and localization (this "site" can be
accessed by transient CADTK overexpression and blocked by CRNK
co-expression) and another mechanism dependent on suppression of
phosphatase activity. The latter is not detectably influenced by
CRNK.

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Fig. 8.
CADTK activation by pervanadate pretreatment
is not inhibited by CRNK. wtCADTK was co-expressed with vector or
CRNK in 293(T) cells. 48 h after transfection, cells were treated
with or without pervanadate (100 mM
H2O2/Na3VO4) for 5 min.
Cell lysates were prepared and immunoprecipitated with anti-CADTK
antibody. Immunocomplexes were analyzed with anti-Tyr(P) and anti-CADTK
antibodies.
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DISCUSSION |
The discovery and isolation of CADTK, the second member of the FAK
family of tyrosine kinases, raised questions about how this kinase is
regulated in comparison to FAK, and whether the two family members
interact as do some receptor tyrosine kinase family members. In this
report, we show that, 1) the structure-function relations governing
CADTK tyrosine autophosphorylation and kinase activity are similar to
those of FAK, at least superficially (Fig. 1); 2) CADTK and FAK are
both direct targets of Src family tyrosine kinases; and CADTK contains
Src-phosphorylated tyrosine residue(s) in addition to the
Tyr881, identified as a FAK Tyr925 equivalent
(Fig. 2); 3) CADTK directly cross-phosphorylates FAK slightly
increasing FAK tyrosine kinase activity and perhaps creating novel
phosphotyrosine sites as targets for SH2 group-containing proteins
(Figs. 3-6); 4) FAK does not cross-phosphorylate CADTK under the
conditions tested (Fig. 3B); 5) the carboxyl terminus of CADTK (CRNK)
can inhibit CADTK but not FAK tyrosine autophosphorylation, presumably
by displacing CADTK from its cytoskeleton-binding sites (Fig.
7A); and 6) overexpression of FRNK abolishes both FAK and CADTK tyrosine autophosphorylation (Fig. 7B).
The major CADTK tyrosine autophosphorylation site is
Tyr402; mutation of this site abolished CADTK tyrosine
autophosphorylation and substantially inhibited its kinase activity
(Fig. 1). The role of Tyr402 phosphorylation in full
activation of the kinase may result from two (or more) potential
mechanisms. First, tyrosine autophosphorylation of Tyr402
may modify CADTK structure by opening or keeping the catalytic cleft
open thereby promoting substrate access. Second,
phospho-Tyr402 may recruit Src family tyrosine kinases to
further increase CADTK kinase activity. We doubt that these results
(the difference between wtCADTK and Y402F CADTK) are confounded by
associated Src family tyrosine kinases (or other co-precipitated
endogenous kinases) because these experiments have been repeated using
RIPA buffer containing SDS, which reverses the association with other
molecules, e.g. Src family tyrosine kinases. We attempted to
test the first hypothesis by introducing a permanent negative charge at
this site by mutating Tyr402 to aspartic acid (Y402D). As
expected, the Y402D mutation was not autophosphorylated, but the
reduction of CADTK immunocomplex kinase activity was similar to that of
the Y402F mutant (data not shown). Whether this is an adequate test of
this hypothesis is unclear. The negatively charged aspartate which has
been shown to mimic activating phosphoserines in some kinases
(e.g. MEK) (57) may not be spatially equivalent to the
negative charge provided by phosphate on the tyrosyl ring.
In addition to Tyr402, full activation of CADTK requires
tyrosine phosphorylation of two other tyrosine residues
(Tyr579 and Tyr580) in the catalytic domain.
Mutation of these two residues decreased CADTK tyrosine
autophosphorylation and kinase activity by 60%. Unlike the Y402F
mutation, the Y579F/Y580F CADTK mutant exhibited partial tyrosine
autophosphorylation when overexpressed, suggesting that the residual
Y579F/Y580F CADTK kinase activity autophosphorylates Tyr402. Surprisingly, there is no
Tyr579/Tyr589 tyrosine phosphorylation in the
Y402F mutant despite detectable tyrosine kinase activity (Fig.
1A). Tyrosines 579 and 580 lie within the catalytic domain
at a position that is conserved among many but not all protein tyrosine
kinase families (58). In some instances, phosphorylation of these
residues is obligatory for tyrosine kinase activation (e.g.
insulin receptor) (59, 60). As noted above, it has been proposed that
tyrosine phosphorylation of FAK Tyr397 provides an
SH2-binding site for Src family tyrosine kinases (44, 46-48) which in
turn phosphorylate Tyr576 and Tyr577 and
further increase tyrosine kinase activity (45). While CADTK Y579F/Y580F
does have slightly higher autophosphorylation and kinase activity than
Y402F, it seems unlikely that the difference is due to endogenous
Src/Fyn activation of overexpressed CADTK. It seems more likely that
both Tyr402 and Tyr579/Tyr580
tyrosine phosphorylation are independently important for opening and/or
maintaining the active conformation of the kinase. Construction and
assessment of a
Tyr402/Tyr579/Tyr580 triple mutant
will help to resolve this issue.
CADTK phosphorylates FAK but does not form heterodimers in
vivo. These data suggest that CADTK may be an
agonist-dependent regulator of FAK. Agonist stimulation in
most cells slightly increases FAK tyrosine phosphorylation (by ~50%
in rat liver epithelial and smooth muscle cells) (5, 16, 39). In
addition, agonist-dependent tyrosine phosphorylation of
paxillin, tensin, and p130Cas) is better correlated with CADTK
than FAK activation (39). The increase in FAK phosphorylation
complicates the analysis of which enzyme is responsible for
agonist-dependent paxillin tyrosine phosphorylation. It may
even be that increased CADTK (Tyr402) and FAK
(Tyr397) phosphorylation induces Src activation which is
primarily responsible for agonist-dependent cytoskeletal
protein phosphorylation. CADTK can phosphorylate FAK on sites in
addition to Tyr397 and Tyr925 (Fig. 6) raising
the possibility that new docking sites for SH2 containing proteins are
generated on FAK. The increase in CADTK-dependent FAK
phosphorylation is clearly greater than its effect on FAK kinase
activity. In summary, agonist-dependent CADTK activation may alter FAK activity and function, adding more complexity to cytoskeleton regulation in cells expressing both enzymes. While, we
cannot rule out that CADTK-dependent FAK tyrosine
phosphorylation in vivo may be due to another tyrosine
kinase, it is a direct effect in vitro in an immune complex.
In addition, the fact that autophosphorylated wtFAK, which associates
with Src family tyrosine kinases, fails to induce CADTK tyrosine
phosphorylation in vivo, suggests, that the role of Src
family tyrosine kinases in CADTK and FAK cross-phosphorylation is limited.
Like FAK, CADTK tyrosine phosphorylation can be regulated by its CRNK
(Fig. 7A). Neither finding (FRNK inhibiting FAK or CRNK inhibiting CADTK) can explain the physiological mechanism of activation by adhesion or agonists, but these results suggest that access to a
specific binding site is crucial for activation. While CRNK only
inhibited CADTK autophosphorylation, FRNK inhibits both FAK and CADTK
autophosphorylation. This initially suggested that the FRNK sequence is
better able to displace both FAK and CADTK from crucial sites. However,
we previously showed that cytoskeleton engagement is necessary for
agonist-dependent CADTK activation; i.e. CADTK
is not activated in suspended monocytes, rat liver epithelial, or
smooth muscle cells (23). Thus, FRNK could influence CADTK through its
established action, displacing FAK from focal contacts. This might
change focal contact function or structure, preventing CADTK activation
by inhibiting a crucial "permissive step." CADTK and CRNK have a
higher affinity for paxillin than does FAK (39); talin, which
physically associates with FAK (61), does not interact with CADTK (62).
This reinforces a model in which CRNK and FRNK, which have different
target affinities, may act differently to regulate CADTK. Said simply,
CRNK may displace CADTK from its site of activation, while FRNK may
displace FAK, making the cells behave as if they were in suspension.
It is unclear why overexpression of CADTK results in its activation,
but it does so in 293(T) (Fig. 8), as well as in HeLa, MCF10, and
NIH3T3 cells (data not shown). Our best explanation is that
overexpressed CADTK molecules access activation sites or effectors that
overcome the physiologic proclivity to be "off" in resting adherent
cells. Substantial additional CADTK activation by phosphatase
inhibition (pervanadate) demonstrates that only a small proportion of
CADTK is activated by overexpression. A large inactive pool of
overexpressed CADTK, perhaps located in the cytoplasm, can be activated
by pervanadate (Fig. 8). The small pool of activated CADTK can be
completely inhibited by CRNK, presumably by displacement from
activating protein-protein interactions. Although we cannot rule out a
mechanism by which CRNK activates a CADTK-specific phosphatase, we
favor the above explanation.
In summary, our results suggest that CADTK and FAK, at some level,
participate in "cross-talk." CADTK directly phosphorylates FAK,
possibly generating more SH2 docking sites. This may enhance physiologic control mechanisms in response to agonists in some cells.
In turn, FAK may be necessary for CADTK activation in some adherent
cells. This requirement cannot be absolute since FAK is not present
when adherence activates CADTK in monocytes and the T cell receptor
activates CADTK in T cells. Nevertheless, epithelial, neural, smooth
muscle, and endothelial cells, may use these two mechanisms of
CADTK-FAK interaction to orchestrate cytoskeleton regulation of
morphology and signaling in response to agonist stimulation and
extracellular matrix.