The N Termini of Focal Adhesion Kinase Family Members Regulate
Substrate Phosphorylation, Localization, and Cell Morphology*
Jill M.
Dunty and
Michael D.
Schaller
From the Department of Cell and Developmental Biology, University
of North Carolina-Chapel Hill, Chapel Hill, North Carolina
27599
Received for publication, February 21, 2002, and in revised form, July 31, 2002
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ABSTRACT |
The focal adhesion kinase (FAK) and cell adhesion
kinase 
(CAK, PYK2, CADTK, RAFTK) are highly homologous FAK
family members, yet clearly have unique roles in the cell. Comparative
analyses of FAK and CAK have revealed intriguing differences in
their activities. These differences were investigated further through the characterization of a set of FAK/CAK chimeric kinases. CAK exhibited greater catalytic activity than FAK in vitro,
providing a molecular basis for differential substrate phosphorylation
by FAK and CAK in vivo. Furthermore, the N terminus may
regulate catalytic activity since chimeras containing the FAK N
terminus and CAK catalytic domain exhibited a striking high level of
catalytic activity and substrate phosphorylation. Unexpectedly, a
modulatory role for the N termini in subcellular localization was also
revealed. Chimeras containing the FAK N terminus and CAK C terminus
localized to focal adhesions, whereas chimeras containing the N and C
termini of CAK did not. Finally, prominent changes in cell
morphology were induced upon expression of chimeras containing the
CAK N terminus, which were not associated with apoptotic cell death, cell cycle progression delay, or changes in Rho activity. These results demonstrate novel regulatory roles for the N terminus of FAK
family kinases.
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INTRODUCTION |
Focal adhesion kinase
(FAK)1 and cell adhesion
kinase (CAK, also known as PYK2, CADTK, or RAFTK) constitute the
FAK family of cytoplasmic tyrosine kinases. The structural features of
this family include large N- and C-terminal domains that flank a
central tyrosine kinase domain (1). The sequences of FAK and CAK are 45% identical and 65% similar. The greatest homology exists between the catalytic domains (60% identity) and the extreme C termini, which
corresponds to the region of FAK that directs subcellular localization
(62% identity) (2). Regions within the N termini of both kinases have
homology with the band 4.1/ERM family of proteins within a region known
as the FERM domain (3). In addition, FAK and CAK share conserved
phosphorylated tyrosines and C-terminal proline-rich regions that
mediate interactions with SH2- and SH3-containing proteins (4).
FAK is expressed in nearly all tissues and cell types, and in a wide
variety of adherent cells FAK is discretely localized to focal
adhesions (5, 6). In contrast, CAK expression is restricted mainly
to the brain and hematopoetic cells, and its subcellular localization
is cell type-specific (4). In some cells, CAK is localized to focal
adhesions or focal adhesion-like structures (7, 8). CAK has also
been localized to specialized actin-containing structures such as the
podosomes of macrophages, the sealing zone of osteoclasts, and along
stress fibers in smooth muscle cells (9-11). It is also targeted to
membrane ruffles and lamellipodia in some spreading and motile cells
(12, 13). Alternatively, CAK staining has been described as diffuse,
perinuclear, or colocalized with the Golgi (7, 14-17). The
differential subcellular localization of FAK and CAK may underscore
important differences in biological function.
Focal adhesion targeting of FAK is mediated by conserved sequences
within the C terminus, designated the Focal Adhesion Targeting (FAT)
sequence (18), which shares extensive homology with the C terminus of
CAK. The C-terminal non-catalytic domain of CAK localizes
discretely to focal adhesions when autonomously expressed (7, 19).
Since full-length CAK exhibits focal adhesion localization in only a
subset of cells, a functional FAT sequence in the C terminus of CAK
appears to be masked in some cell types. This suggests that focal
adhesion localization may be regulated on multiple levels.
FAK is primarily activated through integrin-mediated cell adhesion to
an insoluble extracellular matrix. To a lesser extent, FAK is activated
by growth factors, neuropeptides, and bioactive lipids (20).
Conversely, activation of CAK occurs largely in response to soluble
extracellular factors, including signals that act through
G-protein-coupled receptors, cytokines, antigen receptors, and stress
signals (4). CAK is maximally activated in response to
integrin-mediated cell adhesion in only a subset of cells including hematopoetic cells (8, 21, 22). However, in many other cells, adhesion
induces a slight increase in tyrosine phosphorylation (7, 14, 23). Many
of the stimuli that activate CAK also elevate intracellular calcium
levels, and in fact, CAK activation is dependent upon the presence
of calcium (24-26). Interestingly, a chimeric protein consisting of
the N terminus and catalytic domain of CAK fused to the C terminus
of FAK was driven to focal adhesions and was strongly regulated by
adhesion to fibronectin (14). The positive correlation between focal
adhesion localization and activation by integrin-mediated adhesion has
been reported in the literature and highlights a potential mechanism
for differential regulation of FAK and CAK activity (27, 28).
Although the signals that lie upstream of FAK family kinases may
differ, many of the immediate consequences of activation are conserved.
These include recruitment of SH2 domain containing signaling molecules
such as Src and Grb2 into complex with the kinase, and tyrosine
phosphorylation of cytoskeleton-associated adaptor proteins, such as
paxillin and p130cas (1). Signaling via FAK and CAK is
also involved in the activation of MAP kinase family members (4, 20).
These data suggest that differential regulation of common signaling
events downstream of FAK and CAK are important for biological function.
Since FAK and CAK trigger tyrosine phosphorylation of some common
substrates, it may be predicted that the biological outcomes of
FAK/CAK signaling would be similar as well. In fact, both kinases
have been implicated in the processes of cell spreading, focal adhesion
turnover, and migration (1, 9, 13, 29, 30). Despite this commonality,
FAK and CAK clearly have divergent functions as well. The two
kinases have opposing effects on cell cycle progression, whereas FAK
accelerates progression into S phase, CAK delays this transition
(31, 32). In addition, FAK has been implicated in
adhesion-dependent cell survival (33, 34), while CAK has
been implicated in cell death pathways (35, 36). In neurons, the
integration of FAK and CAK-mediated signals may promote neurite
outgrowth and differentiation, and CAK may play a unique role in
maintenance of plasticity through modulation of ion channels (25, 37,
38). These results suggest that FAK and CAK may have both common and
distinct functions.
Although FAK and CAK are highly homologous, bind to a common subset
of proteins, and are capable of initiating a subset of common signaling
pathways, they clearly have unique and perhaps complementary roles in
the cell. Subtle differences in mode of activation, regulation,
subcellular localization, catalytic activity, substrate preference,
and/or scaffolding activity appear to be critical determinants of
differential signaling. In order to define the molecular basis of these
subtle yet critical differences, we have constructed, expressed, and
characterized a complete set of six chimeric FAK/CAK proteins in
chick embryo cells. CAK exhibited higher catalytic activity than FAK
in an in vitro kinase assay. Furthermore, chimeric kinases
revealed that coupling of the FAK N terminus and CAK catalytic
domain yielded a highly active kinase, suggesting that the N terminus
of FAK family kinases may be involved in regulation of catalytic
activity. Substrate phosphorylation by chimeric kinases in
vivo correlated perfectly with catalytic activity in
vitro, providing a molecular basis for differential substrate
phosphorylation by FAK and CAK in vivo. This analysis
revealed a potential role for the N terminus in modulating subcellular
localization, since chimeras with the FAK N terminus and CAK C
terminus targeted to focal adhesions, whereas chimeras with both the N-
and C termini of CAK did not. The CAK N terminus also mediated
striking changes in cell morphology that were independent of changes
RhoA activity. Although it was not strictly required, targeting to
focal adhesions enhanced the ability of this domain to alter morphology.
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EXPERIMENTAL PROCEDURES |
Cells, Viruses, and Plasmids--
Chick embryo (CE) cells were
isolated from 9-day-old embryos and maintained as described previously
(39). For expression of exogenous proteins in CE cells, constructs were
subcloned into a replication competent avian retroviral vector, RCAS A
(40). RCAS A constructs were transfected into CE cells using the
calcium phosphate method as described (39) or LipofectAMINE Plus
(Invitrogen) according to the manufacturer's recommended protocol.
Expression of RCAS A-encoded exogenous proteins was evaluated 7-9 days
post-transfection. For co-expression with c-Src, cells expressing wild
type or chimeric FAK family kinases were infected with RCAS B c-Src
virus on day 5 post-transfection and lysed on day 10 post-transfection
as described previously (41). pCMV-Myc RhoA Q63L and pCMV-Myc RhoA T19N
plasmids, which were the generous gifts of Dr. Krister Wennerberg, were transfected using LipofectAMINE Plus and analyzed 3 days
post-transfection. Cell cultures were viewed using a Nikon TMS inverted
microscope and imaged using a Nikon CoolPix 950 digital camera (×100 magnification).
PCR Mutagenesis and Cloning--
The DNA sequence corresponding
to the FAK N terminus (amino acids 1-332), catalytic domain
(332-690), and C terminus (690-1038), and the CAK N terminus
(1-336), catalytic domain (336-693), and C terminus (693-1009) were
amplified using PCR. At the junctions of these domains, restriction
sites were created. The N termini of FAK and CAK were amplified with
N- and C-terminal BamH1 sites. The catalytic domains were
flanked by an N-terminal BamH1 site and C-terminal
EcoR1 site. The C-terminal fragments were flanked by an
N-terminal EcoR1 site and C-terminal SalI site.
pBluescript KS+/FAK and pBluescript SK
/CAK (7, 42) were used as
templates, and VENT polymerase (New England Biolabs, Beverly, MA) was
used for PCR. Both template constructs had a C-terminal KT3 epitope tag, and therefore all resultant chimeric molecules were also C-terminal tagged. Fragments were ligated to generate sequences that
encoded eight chimeric proteins, each maintaining the three-domain structure. The clones were named using a three-letter system
corresponding to the three domains in the chimeric protein. The FFF and
CCC clones were reconstituted FAK and CAK molecules that contained the restriction sites that were engineered using PCR. Chimeric constructs were subcloned into RCAS A for expression in CE cells. All
amplified fragments were sequenced at the UNC-CH Automated DNA
Sequencing Facility on a model 377 DNA Sequencer (PerkinElmer, Applied
Biosystems Division) using the ABI PRISMTM Dye Terminator
Cycle Sequencing Ready Reaction Kit with AmpliTaq DNA Polymerase, FS
(PerkinElmer). Two unintended substitutions in FAK led to amino acid
changes: amino acid substitutions R724W and A872G. These mutations, as
well as the mutations engineered to create restriction sites, were
inconsequential in our analysis since the FFF and CCC chimeras behaved
like wild type FAK and CAK, respectively.
Antibodies--
The following antibodies were used for
immunoprecipitation, immunoblotting, and/or indirect
immunofluorescence. The KT3 monoclonal antibody (43), which recognizes
the C-terminal KT3 epitope tag on FAK, CAK, and chimeric proteins,
was the kind gift from Dr. J. T. Parsons (University of Virginia,
Charlottesville, VA). A commercially available KT3 antibody was used
for indirect immunofluorescence (Covance, Princeton, NJ). Monoclonal
antibody RC20 (Transduction Labortatories, San Diego, CA) and
polyclonal phosphospecific antibodies anti-PYK2 (pY402) and
anti-PYK2 (pY579/580) (BIOSOURCE
International,Camarillo, CA) were used to detect phosphotyrosine.
Monoclonal antibody anti-RhoA was purchased from Transduction
Laboratories. Anti-Src monoclonal antibody EC10 was purchased from
Upstate Biotechnology (Lake Placid, NY). Monoclonal anti-Myc antibody
(clone 9E10) was purchased from Sigma. Polyclonal antibody 8605, which
recognizes paxillin, was previously described (44).
Rhodamine-conjugated goat anti-mouse and fluorescein-conjugated donkey anti-rabbit antibodies were used for indirect immunofluorescence (Jackson ImmunoResearch Laboratories, West Grove, PA). The
AlexaFluor 488-conjugated anti-BrdUrd antibody (Molecular Probes,
Eugene, OR) was used for analysis of cell cycle progression.
Cell Lysis, Protein Analysis, and
Immunoprecipitation--
Confluent monolayers of cells were lysed in
ice-cold Triton X-100/radioimmune precipitation assay buffer as
described (44). Lysates were clarified, and protein concentrations were
determined using the bicinchoninic acid (BCA) assay (Pierce). For
immunoprecipitations, the paxillin antibody (2 ug), the KT3 antibody (6 ug), or the EC10 antibody (7 ug) were incubated with 0.2-0.8 mg of
cell lysate at 4 °C for 1 h. Immune complexes were precipitated
at 4 °C for 1 h with protein A-Sepharose beads (Sigma),
anti-mouse IgG-agarose beads (Sigma), or protein A-Sepharose beads
coated with AffiniPure rabbit anti-mouse IgG (Jackson
ImmunoResearch Labs). Immune complexes were washed twice with ice-cold
lysis buffer, and once with ice-cold PBS. Beads were resuspended in
Laemmli sample buffer and boiled to elute the proteins (45), and the
samples were analyzed by Western blotting.
In Vitro Kinase Assays--
Kinases were overexpressed in CE
cells and immunoprecipitated as described above with minor
modifications. To compensate for differences in expression level and to
promote recovery of comparable amounts of each kinase, some chimeric
proteins were immunoprecipitated from more lysate. Specifically, FAK,
CAK, FFF, FFC, FCF, FCC, and CCC were immunoprecipitated from 0.4 mg
of lysate, whereas mock, CCF, CFC, and CFF were immunoprecipitated from
0.8 mg of lysate. Immune complexes were washed two times with ice-cold
lysis buffer, once with ice-cold PBS, and once with kinase reaction buffer (20 mM PIPES, pH 7.2, 3 mM
MnCl2). Each reaction was resuspended in 20 µl of kinase
reaction buffer supplemented with 10 µCi of [
-32P]ATP (PerkinElmer Life Sciences). The reactions
were incubated at room temperature with periodic mixing for 10 min. The
reaction was stopped by adding 20 µl of 2× Laemmli sample buffer and
boiled to elute the protein. Samples were resolved on an
SDS-polyacrylamide gel. The gel was dehydrated and exposed to film to
reveal phosphorylated species.
Immunofluorescence--
Glass coverslips were coated with 50 µg/ml bovine plasma fibronectin (Sigma) in PBS for 1 h at
37 °C. Cells were plated onto the coated coverslips and maintained
at 37 °C for 16 h. Cells were fixed in 3.7% formaldehyde in
Universal Buffer (UB; 20 mM Tris, pH 7.6, 150 mM NaCl) for 8 min, washed twice in UB, permeabilized with
0.5% Triton X-100 in UB for 5 min, and washed twice more in UB.
Coverslips were incubated with primary antibody (KT3, 1:1000; 8605, 1:800) diluted in UB for 1 h at 37 °C in a humidified chamber, washed twice, and incubated with secondary antibody (anti-mouse rhodamine and anti-rabbit fluorescein, 1:1000) diluted in UB for 1 h at 37 °C in a humidified chamber. Coverslips were washed twice in
UB and once with distilled water, and mounted on slides for visualization using a Leitz Orthoplan fluorescence microscope. Images
were captured using a Hamamatsu digital camera and Metamorph imaging
software (Universal Imaging Corporation, West Chester, PA).
RhoA Activity Assay--
The level of RhoA activity in CE cells
was assessed as previously described (46). Briefly, lysates made from
confluent mock-transfected and CCF-expressing cells were incubated with
a GST-Rhotekin RBD fusion protein bound to glutathione-agarose beads
(Sigma). Complexes were washed and Western blotted with anti-RhoA and
anti-Myc antibodies to reveal the amount of precipitated RhoA. As
controls, CE cells were transfected with Myc-tagged RhoA Q63L
(constitutively active) or RhoA T19N (dominant negative) and
analyzed in parallel.
Apoptosis Assay--
In order to assess apoptosis, the Apoptag
Plus Fluorescein in situ apoptosis detection kit was used as
recommended by the manufacturer (Intergen Company, Purchase, NY).
Briefly, CE cells expressing wild type and chimeric kinases were plated
on fibronectin-coated coverslips as described above and grown to
confluence over 2 days. As a positive control for apoptosis,
mock-transfected cells were treated with 1 µM
staurosporine (Sigma) for at least 4 h. Cells were fixed in 3.7%
formaldehyde in PBS and permeabilized in ethanol/acetic acid (2:1) at
20 °C. Coverslips were then incubated with a
fluorescein-conjugated anti-digoxigenin antibody, and
4'-6-diamidino-2-phenylindone dihydrochloride (DAPI, 1 µg/ml,
Molecular Probes, Eugene, OR) to counterstain the nuclei. Coverslips
were mounted onto glass slides and visualized as described for
immunofluorescence experiments. In each experiment, 200 cells were
counted, and the percentage of apoptotic cells was determined.
Cell Cycle Progression Assay--
Differences in the rate at
which starved CE cells expressing wild type and chimeric constructs
entered S-phase following serum stimulation was assessed. Briefly,
cells were plated onto fibronectin-coated coverslips at low density
(~25% confluence) and incubated overnight in complete media. Cells
were then starved in Dulbecco's modified Eagle's medium (DMEM) +0.2%
chick serum (Invitrogen) for 48 h. After rinsing twice with DMEM,
complete CE media (DMEM+5% fetal bovine serum (Sigma), 1% chick
serum) supplemented with 100 µM BrdUrd
(5-bromo-2'-deoxyuridine, Sigma) was added back to the cells. As a
control, one coverslip was incubated in serum-free media supplemented
with BrdUrd to verify that serum starvation had arrested the cell
cycle. After 16 h, cells were fixed in 3.7% formaldehyde in PBS
and permeabilized in 0.5% Triton X-100 in PBS. Coverslips were blocked
in 2% bovine serum albumin in PBS for 1 h and subsequently
treated with 0.1 unit/µl DNase (Promega, Madison, WI) for 30 min at
37 °C. Slips were incubated serially with anti-BrdUrd antibody
(1:20), KT3 (1:500), and finally, a mixture of rhodamine-conjugated
anti-mouse antibody (1:500) and DAPI (1 µg/ml) for 1 h each at
37 °C, with washing following each antibody step. Coverslips were
then rinsed in water, mounted onto glass slides, and visualized as
described for indirect immunofluorescence. In each experiment, 200 cells were counted, and the percentage of cells that had entered S
phase was determined.
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RESULTS |
Construction of Chimeric Molecules--
In order to determine the
molecular basis of the functional differences between FAK and CAK, a
set of chimeric FAK/CAK molecules was constructed (Fig.
1). The three major domains of FAK and
CAK were individually amplified by PCR. Restriction sites were
created at the N and C termini of each domain to allow for assembly of the domains into chimeric molecules (Fig. 1, upper panel).
The central domains, which are referred to as the catalytic domains in
this manuscript, include both the catalytic domains and the binding
sites for the Src SH2 and SH3 domains (8, 47-49). A KT3 epitope tag
was engineered at the C terminus of each construct. Domains were
assembled to generate a full set of chimeric proteins that retain the
overall size and structure of the kinase. Chimeric proteins were named
using a three-letter system (Fig. 1, lower panel)
corresponding to the three domains, where an F represents a
FAK domain and a C represents a CAK domain. Creation of
restriction sites resulted in amino acid substitutions at the joints of
the chimeric proteins. To control for the presence of these
substitutions, two constructs, FFF and CCC, were built. These
correspond to wild type FAK and CAK except that they contain the
engineered restriction sites at the domain joints. The chimeric
constructs were subcloned into RCAS A, a replication-competent avian
retroviral vector, for expression in chick embryo (CE) cells. In order
to verify the domain structure of chimeric proteins, a panel of
antibodies that recognized specific domains of FAK and CAK was used
to probe Western blots of chimera-expressing CE cell
lysates.2 The results
confirmed the predicted domain structure of chimeric FAK/CAK
proteins.
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Fig. 1.
Schematic diagram of
FAK/CAK chimeric proteins. Chimeric
molecules were built as described under "Experimental Procedures."
Upper panel, N-terminal, catalytic, and C-terminal domains
of FAK (stippled boxes) and CAK (shaded boxes)
were individually amplified using PCR. The catalytic domains of the
chimera contained the catalytic domains and Src binding sites (FAK
pY397 and adjacent proline-rich region; CAK
pY402). Each construct had a C-terminal KT3 epitope tag.
Lower panel, chimeric molecules were named using a
three-letter system. The three letters correspond to the three domains
that constitute the chimeric protein. F represents a FAK
domain (stippled) and C represents a CAK
domain (shaded). FFF and CCC are reconstituted FAK and
CAK molecules that also contain the engineered restriction sites at
the domain joints. Gray boxes, phosphotyrosine;
striped boxes, proline-rich regions; FAT, focal adhesion
targeting domain.
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Catalytic Activity of FAK/CAK Chimeric Molecules in
Vitro--
CAK expression in CE cells results in greater substrate
phosphorylation than expression of FAK (7). To explore whether differences in intrinsic catalytic activity could explain this result,
the catalytic activity of FAK, CAK, and FAK/CAK chimeric kinases
was assessed in an in vitro immune complex kinase assay. From lysates made from confluent mock-transfected CE cells and cells
expressing FAK, CAK, and the chimeric proteins, kinases were
immunoprecipitated using the KT3 epitope tag antibody. One-half of each
immune complex was incubated with [-32P]ATP in kinase
reaction buffer for 10 min and separated by SDS-PAGE. Incorporation of
32P into the kinases was detected by autoradiography (Fig.
2, upper panel). The other
half of the immune complex was separated by SDS-PAGE and Western
blotted with the KT3 antibody to reveal the amount of kinase in each
reaction (lower panel). Under these assay conditions, CAK
exhibited higher catalytic activity than FAK. This result is
accentuated by the fact that less CAK was present in the reaction
than FAK. The catalytic activity of control chimeric proteins FFF and
CCC were similar to FAK and CAK, respectively, demonstrating that
amino acid substitutions in the chimeric kinases did not alter
catalytic activity. Overall, chimeric proteins that contained the
catalytic domain of CAK (including FCF, FCC, and CCF) exhibited
higher autophosphorylation activity than those that contained the FAK
catalytic domain (including FFC, CFC, and CFF). Unexpectedly,
juxtaposition of the FAK N terminus and catalytic domain of CAK
(i.e. FCF, FCC) resulted in an enhancement of catalytic activity above that of CAK itself. However, there was no apparent change in activity when the N terminus of CAK was combined with the
FAK catalytic domain (compare FAK, CFC, and CFF). These data implicate
the catalytic domain as the principal determinant of catalytic activity
in vitro and suggest that CAK may have higher intrinsic
catalytic activity relative to FAK. Furthermore, these results revealed
a potential modulatory role for the N terminus. This is the first
report of a direct comparison of FAK and CAK catalytic activity
in vitro, and identifies a biochemical basis for
differential substrate phosphorylation in vivo.
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Fig. 2.
Catalytic activity of
FAK/CAK chimeric kinases in
vitro. Cell lysates were made from mock-transfected CE
cells or cells expressing FAK, CAK, or FAK/CAK chimeric proteins.
Kinases were immunoprecipitated with the KT3 antibody. To compensate
for differences in levels of protein expression, FAK, CAK, FFF, FFC,
FCF, FCC, and CCC were immunoprecipitated from 400 µg of lysate and
mock, CCF, CFC, and CFF were immunoprecipitated from 800 µg of
lysate. Upper panel, one-half of each sample was subjected
to an in vitro kinase assay as described under
"Experimental Procedures." Kinase reactions were separated by
SDS-PAGE, and incorporation of 32P into the kinases was
visualized by autoradiography. Lower panel, the second half
of each immunoprecipitation was separated by SDS-PAGE and immunoblotted
with the KT3 antibody to show the amount of kinase present in each
reaction.
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Tyrosine Phosphorylation of Chimeric Kinases and Substrates in
Vivo--
It was previously shown that in adherent CE cells, CAK
expression elicited higher levels of whole cell phosphotyrosine than FAK expression. The major substrates that were differentially phosphorylated were paxillin, p130cas, and tensin.
Interestingly, the basal level of FAK phosphorylation was high, whereas
that of CAK was very low (7). These phosphorylation events were
investigated further using FAK/CAK chimeric kinases. Lysates were
made from confluent cultures of mock-transfected CE cells and cells
expressing FAK, CAK, and chimeric FAK/CAK proteins. To compare
cellular phosphotyrosine profiles, whole cell lysates (30 µg) were
Western blotted with an anti-phosphotyrosine antibody (Fig.
3A, upper panel). The membrane
was stripped and reprobed with the KT3 antibody to reveal the relative
amount of exogenous kinase in each lysate (lower panel).
Paxillin was also immunoprecipitated from these lysates. The immune
complexes were Western blotted for phosphotyrosine (Fig. 3B,
upper panel) and stripped and reprobed for paxillin to verify
equal protein loading (lower panel). As previously described
(7), FAK expression had little effect on cellular phosphotyrosine,
whereas CAK expression induced the phosphorylation of several
species (Fig. 3A). Likewise, paxillin phosphorylation was
only slightly elevated in cells expressing FAK but was dramatically
increased in CAK-expressing cells (Fig. 3B). Importantly,
FFF and CCC behaved as wild type FAK and CAK in these experiments,
suggesting that amino acid substitutions within the chimeric proteins
did not alter biochemical activity in vivo. Similar to FAK,
expression of chimeric proteins that contained the FAK catalytic domain
resulted in little change in cellular phosphotyrosine or paxillin
phosphorylation (Fig. 3, A and B; FFC,
CFC, and CFF). Chimeric kinases that contained the CAK catalytic domain were as efficient or more efficient than CAK
in elevating cellular phosphotyrosine and paxillin phosphorylation (Fig. 3, A and B; FCF, FCC, and
CCF). Interestingly, the N terminus modified substrate
phosphorylation in vivo, since chimeras containing the FAK N
terminus induced greater levels of cellular phosphotyrosine and
paxillin phosphorylation than their counterparts containing the CAK
N terminus (Fig. 3, A and B, compare
FFF and CFF, FFC and CFC, FCF and
CCF, FCC and CCC). Furthermore, the combination of the FAK N terminus and the CAK catalytic domain resulted in the
most potent induction of substrate phosphorylation (Fig. 3, A and B; FCF and FCC).
Tyrosine phosphorylation of p130cas and tensin was also
examined by immunoprecipitation and Western blot analysis, and the
results were similar to those seen with paxillin.2
Therefore, levels of whole cell phosphotyrosine and paxillin phosphorylation were determined primarily by the catalytic domain, whereby the CAK catalytic domain was more efficient than that of FAK
in elevating cellular phosphotyrosine. In addition, the N terminus of
the kinase may modify catalytic activity in vivo. Significantly, the level of substrate phosphorylation induced by the
kinases in vivo correlates well with catalytic activity in vitro (refer to Fig. 2).
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Fig. 3.
Tyrosine phosphorylation of substrates and
FAK/CAK chimeric kinases in
vivo. Cell lysates were made from mock-transfected CE
cells or cells expressing FAK, CAK, or FAK/CAK chimeric proteins.
A, whole cell lysates (30 ug) were separated by SDS-PAGE,
transferred to nitrocellulose, and Western blotted to reveal
tyrosine-phosphorylated species (upper panel). The membrane
was stripped and reprobed with the KT3 antibody to show the level of
kinase expression (lower panel). B, paxillin was
immunoprecipitated and Western blotted for phosphotyrosine (upper
panel). The membrane was stripped and reprobed to show the amount
of paxillin in each lane (lower panel). C, wild
type and chimeric kinases were immunoprecipitated from cell lysates
using the KT3 antibody. Immune complexes were Western blotted with an
anti-phosphotyrosine antibody (upper panel) and stripped and
reprobed with the KT3 antibody to reveal the amount of kinase in each
lane (lower panel).
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In CE cells growing in culture, FAK is heavily phosphorylated on
tyrosine while CAK exhibits very low phosphorylation (7). To define
the domains that mediate this dissimilarity, the phosphorylation state
of chimeric FAK/CAK proteins in vivo was determined.
Kinases were immunoprecipitated using the KT3 antibody and Western
blotted for phosphotyrosine (Fig. 3C, upper
panel). The membrane was stripped and reprobed with the KT3
antibody to reveal the amount of protein in each lane (Fig.
3C, lower panel). As previously reported, FAK was
heavily phosphorylated in cells growing in culture, whereas the basal
level of tyrosine phosphorylation on CAK was low (7). Likewise, FFF
phosphorylation was high, and CCC phosphorylation was low.
Phosphotyrosine on FCF and CFF was equivalent to FAK. FCC
phosphorylation was similar to CAK. The remaining chimeric proteins,
including FFC, CCF, and CFC, exhibited an intermediate level of
phosphorylation. Tyrosine phosphorylation in vivo correlated best with the C terminus; chimeras containing the FAK C terminus (i.e. FFF, FCF, CCF, and CFF) were more highly
phosphorylated on tyrosine than their counterparts that contained the
CAK C terminus (i.e. FFC, FCC, CCC, and CFC,
respectively). In addition, chimeric proteins containing the FAK
catalytic domain were phosphorylated slightly better than those
containing the CAK catalytic domain. Thus, the C terminus and
catalytic domains are the primary and secondary determinants,
respectively, of tyrosine phosphorylation in vivo. It is
noteworthy that the level of tyrosine phosphorylation on chimeric
FAK/CAK kinases correlates with neither the relative catalytic
activity in vitro nor the ability to induce substrate phosphorylation in vivo.
Coupling of Chimeric Kinases with Src in Vivo--
FAK and Src
family kinases act coordinately to send downstream signals in the cell.
Therefore, the ability of FAK kinases to send strong signals downstream
in vivo may be attributable to both high intrinsic catalytic
activity and increased coupling to Src. In order to study this
interaction, the ability of wild type and chimeric kinases to
co-immunoprecipitate with exogenously expressed c-Src was determined.
Lysates were made from confluent cultures of CE cells expressing c-Src
alone (Mock) or co-expressing c-Src and FAK/CAK chimeric kinases.
From these lysates, c-Src was immunoprecipitated, and the immune
complexes were split in half. One-half was Western blotted with the KT3
antibody to reveal the amount of co-immunoprecipitated chimeric kinases
(Fig. 4A, upper
panel). The other half was blotted with the EC10 antibody to
verify equal recovery of Src in the immunoprecipitates (lower panel). A greater amount of FAK was found in complex with c-Src than CAK, and likewise more FFF was found in complex with c-Src than
CCC. Chimeras containing the FAK catalytic domain (FFF, FFC, CFC, CFF)
bound more efficiently to c-Src than those bearing the CAK catalytic
domain (FCF, FCC, CCC, CCF). Note that the chimeras were built such
that the central catalytic domains included all c-Src binding sites.
Thus, the catalytic domain was the primary determinant of c-Src
binding.
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Fig. 4.
Coupling of chimeric kinases with c-Src.
A, cell lysates were made from CE cells expressing c-Src
alone (mock) and cells coexpressing c-Src and FAK, CAK,
or FAK/CAK chimeric proteins. From these lysates, c-Src was
immunoprecipitated, and the immune complexes were split in half.
One-half was separated on an 8% gel and Western blotted with the KT3
antibody to reveal co-precipitated wild type and chimeric kinases
(upper panel). The second half was separated on a 12% gel
and Western blotted for c-Src (lower panel). As a control,
FFF lysate was incubated with secondary antibody conjugated to beads
alone (2° alone). To distinguish between c-Src and the antibody heavy
chain, antibodies used for the immunoprecipitation were incubated with
buffer and analyzed in parallel (1° + 2° Ab). B, cell
lysates were made from mock-transfected CE cells or cells expressing
FCF, FCC, CCC, or CCF. Kinases were immunoprecipitated with the KT3
antibody. To compensate for differences in levels of protein
expression, FCF and FCC were immunoprecipitated from 400 µg of
lysates, and mock, CCC and CCF were immunoprecipitated from 800 µg of
lysate. The immune complexes were split into thirds and Western blotted
for phosphorylated Tyr402 (pY402),
phosphorylated Tyr579/580 (pY579/580), and
phosphotyrosine (pY) as indicated. The pY blot was stripped and
reprobed with the KT3 antibody to reveal the amount of
immunoprecipitated kinase.
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Among those kinases containing the CAK catalytic domain, those that
also contained the FAK N terminus (FCF, FCC) bound better to c-Src than
those containing the CAK N terminus (CCC, CCF). This suggested that
the N terminus of FAK was permissive for c-Src binding in comparison to
the CAK N terminus. The major autophosphorylation site on FAK family
kinases also serves as the Src SH2-domain binding site. Therefore,
increased c-Src binding may be associated with elevated phosphorylation
at this site as well as sites that serve as Src targets, such as the
activation loop regulatory tyrosines. To further examine their tyrosine
phosphorylation, phosphospecific antibodies were used to determine the
level of phosphorylation on the major autophosphorylation site
(Tyr402) and regulatory tyrosines in the activation loop
(Tyr579/580) of chimeric kinases containing the CAK
catalytic domain (FCF, FCC, CCC, and CCF). Kinases were
immunoprecipitated using the KT3 antibody, and immune complexes were
split into thirds and Western blotted for phosphorylated
Tyr402 (pY402), phosphorylated
Tyr579/580 (pY579/580), and phosphotyrosine
(pY) as indicated in Fig. 4B. The phosphotyrosine blot was
stripped and reprobed with the KT3 antibody to reveal the amount of
recovered kinase (Fig. 4B, lower panel). FCF and FCC exhibited relatively higher levels of autophosphorylation than
their counterparts CCF and CCC, respectively (Fig. 4B,
pY402), providing a molecular basis for
increased Src binding to these chimeras. Furthermore, FCF and FCC
exhibited relatively higher levels of activation loop phosphorylation
than CCF and CCC (Fig. 4B, pY579/580).
Phosphorylation on activation loop tyrosines is associated with
increased catalytic activity (50, 51).
Induction of Morphological Changes by Selected
FAK/CAK Chimeric Proteins--
Expression of CAK in
CE cells results in sporadic cell rounding within the confluent
monolayer. Treatment of these cells with sodium orthovanadate, a
tyrosine phosphatase inhibitor, results in severe disruption of the
monolayer as evidenced by abundant cell rounding and loss of confluence
(7). Significantly, FAK expression has no effect on the integrity of
the monolayer, nor does treatment of FAK-expressing cells with sodium
orthovanadate. In order to identify the unique features of CAK that
mediate changes in cell morphology, the morphology of CE cells
expressing FAK, CAK, and FAK/CAK chimeric kinases was examined
(Fig. 5). As predicted, expression of FAK
or FFF had no effect on cell morphology, whereas expression of CAK
or CCC induced mild cell rounding, in agreement with the previous
report (7). FFC, FCF, or FCC expression had little or no effect on cell
morphology. Surprisingly, in the absence of vanadate treatment,
expression of several chimeric proteins had striking effects on cell
morphology that were reminiscent of vanadate-treated
CAK-expressing cells. Cells expressing CFC were extremely
elongated and spindly, and some cell rounding was also induced.
Expression of CCF and CFF caused dramatic cell rounding, where CCF was
more potent than CFF. Furthermore, at the time when changes in
morphology were most striking, monolayers of CCF, CFC, and
CFF-expressing cells seemed to take longer to reach confluence than
other chimera-expressing cell cultures. These data implicate the N
terminus of CAK in the induction of changes in cell morphology. Furthermore, the C terminus of FAK may cooperate with the N terminus of CAK to promote the most dramatic alterations in morphology. Morphological changes did not correlate with high catalytic activity in vitro or an overall elevation of tyrosine
phosphorylation in vivo.
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Fig. 5.
Changes in cell morphology induced by kinase
expression. Mock-transfected CE cells and cells expressing FAK,
CAK, or FAK/CAK chimeric proteins were imaged using a Nikon TMS
inverted microscope and a Nikon CoolPix 950 digital camera (×100
magnification).
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Further Characterization of Cells that Exhibit Altered Cell
Morphology--
The morphological changes that were observed were
intriguing, and could be due to alteration of a number of cellular
processes. Cells with altered morphologies, i.e. cells
expressing CCF, CFC, and CFF, were further analyzed to determine the
basis of these changes. Since CAK expression promotes apoptosis in
some cell types, (35, 36) the viability of these cells was determined (Fig. 6A). At 7 days
post-transfection, cells were plated onto fibronectin coverslips and
allowed to reach confluence over 2 days. Similar to cells grown on
tissue culture-treated dishes, dramatic morphological changes were
evident. As a positive control for apoptosis, mock-transfected cells
were treated with staurosporine. The percentages of apoptotic and
non-apoptotic nuclei were calculated. The number of non-apoptotic
mock-transfected cells was quite high (98.5 ± 1.1%). FAK
(96.8 ± 2.9%), FFF (99.4 ± 0.7%), CAK (97.3 ± 2.1%), and CCC (98.7 ± 0.6%) exhibited similar high levels of non-apoptotic cells. Importantly, expression of chimeric kinases that
induced the greatest morphology changes did not increase the rate of
apoptotic cell death (CCF, 98.4 ± 0.7%; CFC, 97.1 ± 2.3%;
and CFF, 98.1 ± 1.2%). Therefore, cell rounding induced by
chimera expression was not associated with an elevation in the rate of
apoptosis.
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Fig. 6.
Analysis of apoptotic cell death and cell
cycle progression. A, mock-transfected CE cells and
cells expressing FAK, CAK, FFF, CCC, CCF, CFC, and CFF were plated
onto fibronectin-coated coverslips and grown to confluence at
approximately day 7 post-transfection. The percentage of non-apoptotic
cells was assessed using the Apoptag system as described under
"Experimental Procedures." The data are a summation of four
independent experiments. Error bars represent S.D.
B, mock-transfected CE cells and cells expressing FAK,
CAK, FFF, CCC, CCF, CFC, and CFF were adhered to fibronectin-coated
coverslips overnight and subsequently starved for 48 h in low
serum-containing media. Cells were then stimulated with complete media
containing 100 µM BrdUrd for 16 h. Nuclei were
scored for BrdUrd incorporation as described under "Experimental
Procedures." The data is expressed as percentage of BrdUrd-positive
cells and are a summation of three independent experiments. Error
bars represent S.D.
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While FAK expression has been reported to accelerate cell cycle
progression, CAK expression had an opposing effect (32). In order to
assess whether expression of morphology-altering chimeras was
associated with a decreased rate of cell cycle progression, a BrdU
incorporation assay was employed (Fig. 6B). Serum-starved cells were stimulated with complete media containing 100 µM BrdUrd for 16 h, and the percentage of
BrdUrd-positive nuclei was determined. A low level of BrdUrd
incorporation was seen in serum-starved cells (8.3 ± 2.8%).
Stimulation of mock-transfected cells with serum-containing media
prompted 38.5 ± 7.2% of cells to enter S phase. At the same time
point, more FAK-expressing cells (55.3 ± 3.1%) had incorporated
BrdUrd than mock cells, suggesting that, in accordance with
previous reports, FAK expression accelerated S phase entry (31).
However, in contrast to reports that it slows cell cycle progression
(32), CAK (48.7 ± 5.1%) accelerated S phase progression
almost as well as FAK in CE cells. FFF and CCC expression (59.3 ± 6.4% and 49.5 ± 2.6%, respectively) mimicked the effects of FAK
and CAK expression. Although each chimeric kinase had a slightly
different affect on cell cycle progression, all cells entered S phase
at a rate that was greater than mock-transfected cells (CCF, 43.3 ± 6.8%; CFC, 46.0 ± 7.8%; CFF, 48.3 ± 7.6%). Therefore,
altered cell morphology and the apparent disruption of the monolayers
were not due to a cell cycle delay.
Subcellular Localization of Chimeric Proteins--
In CE cells,
FAK is localized discretely to focal adhesions via its C-terminal focal
adhesion targeting (FAT) sequence. Although highly homologous to FAK
within its putative FAT sequence region, CAK localization is mainly
diffuse in CE cells (7). In order to elucidate the molecular mechanisms
of differential subcellular localization of FAK and CAK, indirect
immunofluorescence was used to study the localization of FAK/CAK
chimeric proteins in CE cells. Cells were plated onto
fibronectin-coated glass coverslips at low density and allowed to
adhere and spread for 16 h in complete media. Cells were then
processed and labeled with the KT3 antibody to reveal kinase
localization (Figs. 7 and
8). For the purposes of this analysis,
only well spread cells were studied.
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Fig. 7.
Subcellular localization of chimeras
containing the N terminus of FAK. Mock-transfected CE cells and CE
cells expressing FAK, CAK, FFF, FFC, FCF, and FCC were plated onto
fibronectin-coated coverslips and cultured in complete media for
16 h. Cells were then processed and labeled with the KT3 epitope
tag antibody to reveal the localization of the kinases as described
under "Experimental Procedures."
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Fig. 8.
Subcellular localization of chimeras
containing the N terminus of CAK. Mock-transfected
CE cells and CE cells expressing FAK, CAK, CCC, CCF, CFC, and CFF
were plated onto fibronectin-coated coverslips and cultured in complete
media for 16 h. Cells were co-stained with the KT3 epitope tag
antibody to reveal kinase localization and the anti-paxillin antibody
8605 to visualize focal adhesions.
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Mock-transfected cells exhibited a nearly undetectable level of
nonspecific staining (Figs. 7 and 8, Mock). FAK was clearly localized to focal adhesions, whereas CAK localization was largely cytoplasmic (Figs. 7 and 8, FAK and CAK). In a small percentage of
cells, CAK was present in focal adhesions and/or smaller focal adhesion-like structures. The control chimeras FFF (Fig. 7) and CCC
(Fig. 8) were localized as their wild type counterparts, demonstrating that the amino acid substitutions in the chimeric kinases did not alter
subcellular localization. As predicted, the FAK C terminus directed
focal adhesion localization of FCF, CCF, and CFF (Figs. 7 and 8). These
results demonstrate that the C terminus of FAK contains a potent focal
adhesion targeting sequence, since it can target the CAK N terminus
and catalytic domain (CCF) to focal adhesions. Like CAK, CFC
localization was diffuse (Fig. 8). However, quite interestingly, the C
terminus of CAK directed focal adhesion localization when the N
terminus of FAK was present (Fig. 7, FFC and FCC). This suggested that
the C terminus of CAK is also sufficient to direct focal adhesion
localization. Furthermore, substitution of the FAK N terminus may be
permissive for the focal adhesion targeting activity of the CAK C terminus.
The morphology changes observed in cells expressing certain chimeric
kinases raised questions regarding the integrity of their focal
adhesions. To visualize focal adhesions, cells were co-stained for
paxillin (Fig. 8). Chimeric proteins that contained the N terminus of
FAK were well spread and exhibited characteristic staining of paxillin
in focal adhesions.2 Surprisingly, even in apparently well
spread cells expressing CAK, CCC, CCF, CFC, and CFF, paxillin
localization was altered to varying degrees, suggesting that expression
of these kinases altered focal adhesion structure. The Rho family of
GTPases plays a central role in regulating the actin cytoskeleton (52).
In particular, Rho activity is important for generating tension which is associated with the formation of actin stress fibers and focal adhesions (53). Therefore, the changes in cell shape and focal adhesion
structure observed in certain chimera-expressing cells might be
mediated by down-regulation of Rho activity. Since CCF cells exhibited
the most dramatic morphological changes, the level of activated,
endogenous RhoA in mock and CCF cells was assessed using the GST-RBD
pull-down assay (Fig. 9). The amount of
active RhoA recovered from mock and CCF cells was equal, suggesting
that there was no difference in the activation state of RhoA between these cells (Fig. 9, Rho blot). As controls for this assay,
RhoA Q63L (constitutively active) and RhoA T19N (dominant negative) were analyzed. The GST-RBD fusion protein bound to RhoA Q63L, but not
RhoA T19N (Fig. 9, Myc blot). Therefore, it appears that alteration of cell morphology is not mediated by changes in RhoA activity.
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Fig. 9.
RhoA activity is similar in mock and CCF
cells. The activity of RhoA in confluent mock-transfected CE cells
and cells expressing CCF, as well as cells expressing Myc-RhoA Q63L
(constitutively active, positive control) and Myc-RhoA T19N (dominant
negative, negative control) was assessed as described under
"Experimental Procedures." Membranes were Western blotted for Rho
(upper panel) and stripped and reprobed with anti-Myc
antibody to reveal exogenous Rho proteins (lower
panel).
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DISCUSSION |
A comparative analysis of FAK and CAK revealed both
similarities and intriguing differences in their function (7). The goal
of this study was to define the sequences within FAK and CAK that
mediate their unique properties. Our approach was to construct and
comprehensively characterize a full set of chimeric FAK/CAK
proteins. Chimeric proteins have been used previously to study the
activities of FAK and CAK (14, 32, 35, 54). These studies have
provided insight into selected activities of FAK and CAK. However,
additional valuable information was obtained through the biochemical
and biological analysis of novel chimeric molecules. The results,
which are summarized in Table
I, revealed an unexpected role for the
N-terminal domain in regulating substrate phosphorylation, focal
adhesion localization and cell morphology, and provided an explanation
for enhanced phosphorylation of substrates by CAK relative to
FAK.
Tyrosine phosphorylation of substrates in vivo correlated
perfectly with the origin of the catalytic domain, whereby the
catalytic domain of CAK induced greater levels of phosphotyrosine
than the catalytic domain of FAK. Unexpectedly, the N terminus had a
striking impact on catalytic activity. Chimeras with the N-terminal domain of FAK and catalytic domain of CAK (FCF and FCC) elicited even higher levels of substrate phosphorylation than CAK itself. The
degree to which chimeric kinases elevated substrate phosphorylation in vivo was proportional to their level of catalytic
activity in an in vitro kinase assay.
The mechanism by which the FAK and CAK catalytic domains support
different levels of catalytic activity is unclear. One obvious possibility was that elevated catalytic activity was the result of
enhanced coupling to Src family kinases. Two lines of evidence suggest
that this was not the case. First, chimeras with the FAK catalytic
domain bound more c-Src, yet exhibited lower activity in
vitro. Second, the inclusion of PP2, a Src-specific inhibitor (55)
in the in vitro kinase assays had no effect upon the
autophosphorylation activity of the chimeras.2 Thus, the
presence of Src kinases in these immune complexes had a negligible
contribution to the observed differences in catalytic activity in
vitro. Rather, the intrinsic activity of the kinase itself
determines the baseline level of catalytic activity.
Interestingly, exchanging the CAK N terminus for the FAK N terminus
in the context of the CAK catalytic domain (i.e. CCC, CCF
to FCC, FCF) resulted in elevated c-Src binding. Increased c-Src
binding correlated with higher levels of catalytic activity of these
chimeras in vitro and in vivo. Further, FCF and
FCC exhibited higher levels of autophosphorylation on
Tyr402 and increased phosphorylation on activation loop
regulatory tyrosines Tyr579/580, which when phosphorylated,
promote maximal catalytic activity (50, 51). Therefore, modulation of
Src binding by the N terminus in the context of the CAK catalytic
domain was associated with elevated phosphorylation of key regulatory
tyrosines, providing a potential mechanism by which the N terminus may
regulate catalytic activity.
It was previously suggested that the N-terminal domain of FAK kinases
might impinge upon and negatively regulate catalytic activity, since
deletion of the N terminus of FAK led to elevated catalytic activity in
some cases (56, 57). However, deletion of the N terminus in other cases
has little effect upon catalytic activity (18). A recent study of JAK3
also suggests a mechanism by which the N-terminal FERM domain of FAK
kinases could regulate catalytic activity through an intramolecular
interaction. The N-terminal FERM domain of JAK3 physically associates
with the C-terminal kinase domain, and this interaction is important
for catalytic activity (58). Similar intramolecular interactions within
CAK or the chimera could have inhibitory or stimulatory effects on
catalytic activity. Alternatively, the N terminus of CAK could
recruit negative regulators, e.g. phosphatases, or the N
terminus of FAK may recruit positive regulators into complex with the
chimeras. While the molecular details remain to be established, it is
clear that the presence of the N terminus of FAK with the catalytic
domain of CAK resulted in increased Src binding, which was
associated with pronounced effects on the tyrosine phosphorylation of
these kinases, as well as their catalytic activity.
Tyrosine phosphorylation on the chimeric kinases correlated, albeit not
perfectly, with two domains: the C termini and catalytic domains.
Chimeric kinases containing the C-terminal domain of FAK tended to be
more highly phosphorylated. All chimeras containing this domain
efficiently targeted to focal adhesions and thus enhanced tyrosine
phosphorylation may be due to subcellular localization. These results
are consistent with the conclusion drawn previously from a study using
FAK/CAK chimeric proteins (14). It has been suggested that
localization and complex formation in focal adhesions may be sufficient
for the activation and phosphorylation of FAK. This may explain why
CAK phosphorylation is low in cells in which it is diffusely
localized (14, 59). Interestingly, further analysis revealed that
localization to focal adhesions cannot be the sole determinant of a
high level of tyrosine phosphorylation, since the FCC chimera targeted
to focal adhesions, yet is poorly tyrosine phosphorylated. Thus the
C-terminal domain of FAK may play a role in addition to targeting to
promote tyrosine phosphorylation.
The identity of the catalytic domain of the chimera also contributed to
levels of phosphotyrosine on chimeric kinases in vivo. Chimeras with the FAK catalytic domain tended to be more highly phosphorylated than chimeras with the CAK catalytic domain, despite the fact that CAK catalytic activity in vitro and
in vivo is greater than that of FAK. Therefore, relative
tyrosine phosphorylation in vivo is not an indication of
relative catalytic activity. In support of this observation, a previous
report demonstrated that FAK catalytic activity was low when NIH3T3
cells in culture were serum-starved, and maximal after replating on
fibronectin, although FAK was highly phosphorylated in both cases (60).
These data suggest that the FAK catalytic domain could promote kinase
phosphorylation via another mechanism. Since more c-Src was found in
complex with FAK than CAK, it is possible that FAK binds Src kinases
with a higher affinity than CAK, resulting in elevated
phosphorylation of Src-dependent sites on FAK/CAK. FAK
contains a consensus Src SH3 domain binding site
(368RALPSIPKL376)
in the proximity of the Src SH2 domain binding site which is not highly
conserved in CAK
(374NSLPQIPTL382).
Alternatively, enhanced Src binding might impair dephosphorylation of
the major autophosphorylation site in vivo.
FAK and FRNK localize prominently to focal adhesions in adherent cells
(1). Conversely, there are conflicting reports regarding the
subcellular localization of CAK. The autonomously expressed C-terminal domain of CAK (CRNK/PRNK) localizes to focal adhesions (7, 19). However, in CE cells, only a subset (~10%) of CAK expressing cells exhibit focal adhesion staining. These data suggest that the FAT sequence within the CAK C terminus is functional, but
may be masked in the context of the full length protein (7). Several
groups have employed FAK/CAK chimeric proteins to explore differences in FAK and CAK localization. The FAK C terminus was universally capable of directing chimeric kinases to focal adhesions. Like CFF and CCF, chimeric proteins PFhy1 and Pyk2/FAK-CT (which resemble CCF) were targeted to focal adhesions (14, 32, 54). These
results demonstrate that the FAK FAT sequence was dominant over a
potential opposing localization signal in the CAK N terminus or
catalytic domain.
While there is consensus that the FAT sequence of FAK can target CAK
to focal adhesions, there is conflicting data regarding the ability of
the C terminus of CAK to target chimeric proteins to focal
adhesions. Clearly, FFC and FCC localized to focal adhesions in CE
cells. The CAK FAT sequence might be slightly less efficient than
that of FAK since FFF and FCF exhibited slightly more discrete focal
adhesion localization than FFC and FCC. That the C terminus of CAK
can direct focal adhesion targeting contrasts with previous reports of
chimeric proteins FPhy2 (similar to FFC) and FAK/Pyk2-CT (similar to
FCC), which were diffusely localized in NIH3T3 cells and FAK null
cells, respectively (32, 54). Discrepant results regarding the
localization of similar chimeric proteins may reflect slight
differences in domain structure or the use of different cell systems.
Since CE cells are primary cells that form well-defined focal
adhesions, we believe that it is a system well suited for these analyses.
The C terminus of CAK was capable of directing chimeric kinases
containing the N terminus of FAK to focal adhesions (FFC and FCC), but
not those that contained the N terminus of CAK (CCC and CFC). A
potential explanation for this phenomenon is that the N terminus of FAK
houses a second focal adhesion targeting activity that compensates for
the absence of a strong C-terminal CAK FAT sequence. In support of
this notion, the N terminus of FAK contains a 1 integrin
binding site (61). Additionally, the N termini of both FAK and CAK
contain a putative FERM domain, which has been implicated in linkage to
transmembrane proteins (3, 62, 63). However, an ancillary targeting
sequence in the N terminus of FAK must be weak, since it cannot
independently target FAK to focal adhesions (18). Alternatively, the N
terminus of CAK may oppose the focal adhesion targeting activity of
the C terminus of CAK. The CAK N terminus could fold back onto
the molecule and block the FAT sequence. It may also direct the protein to or anchor the protein in another location in the cell.
It was shown previously that treatment of cells expressing CAK, but
not FAK, with the tyrosine phosphatase inhibitor vanadate induced
dramatic changes in cell morphology (7). In an effort to determine
which domain of CAK mediated changes in cell morphology, the
morphology of cells expressing chimeras was observed. Expression of
kinases containing the N terminus of CAK resulted in a profound loss
of the typical fibroblastic morphology. Strikingly, these morphological
changes occurred in the absence of vanadate. One explanation for the
morphology change is that expression of certain chimeras induced
apoptosis. CAK is activated by a variety of stress signals and has
been directly implicated in signaling pathways downstream of known
apoptotic agents (25, 26, 36, 64). Further, Xiong and Parsons (1997)
showed that expression of CAK/Pyk2 and chimeric proteins Pyk2/FAK1
and Pyk2/FAK2 (similar to CFF and CCF, respectively) in Rat-1
fibroblasts resulted in apoptosis in a high percentage of cells. These
data suggested that the N terminus of CAK and catalytic activity
were required for maximum induction of cell death. Although
morphological changes that occur in CE cells are also dependent upon
the N terminus of CAK, cell rounding was not associated with an
increase in apoptotic cell death. Therefore, it seems more likely that
these kinases induce cell rounding by eliciting changes in cytoskeletal structure.
The notion that FAK family kinases impinge upon the actin cytoskeleton
is not a new one. Both associate physically and biochemically with
actin-based structures and actin cytoskeleton regulatory proteins (1,
65). The importance of FAK family kinases in regulation of the
cytoskeleton is underscored by the fact that FAK-null cells have
increased stress fiber formation and a transient migration defect. This
affect has been attributed to a deregulation of Rho signaling (29, 66).
Attenuation of CAK expression in osteoclast-like cells causes cell
retraction and a dramatic decrease in the area of the cell (67).
Similar to CE cells, it was recently reported that CAK expression
induced cell rounding in Swiss 3T3 cells, and that this effect was
dependent upon the N terminus of the kinase. This morphology was
attributed to changes in actin cytoskeletal structure (68).
The mechanism by which chimeras containing the N terminus of CAK
mediate dramatic cell shape changes in CE cells is unclear. The primary
effect is not decreased adhesion or an inability to spread, since there
were no detectable difference in the ability of these cells to
initially attach or spread onto fibronectin, and the kinetics of
spreading were not altered.2 However, these cells may have
a defect in maintenance of focal adhesions or cell shape. This may be
due to direct affects on the structure of focal adhesions and/or the
actin cytoskeleton. In support of this hypothesis, paxillin staining
was altered even in well spread cells, suggesting that changes in focal
adhesion structure may precede gross morphological changes. It is
interesting to note that RhoA activity was unaltered in CCF cells,
suggesting that an alternate signaling pathway may be involved in
regulating cell morphology. The results in CE cells are clearly
phenomenological since CAK is not expressed in this cell type.
However, these finding may provide insight into the physiological
function of
TOP
ABSTRACT
INTRODUCTION
