1
Department of Pathology, University of Alabama at Birmingham, Birmingham, AL
35294, USA
2
Departments of Neurobiology, Pathology and Physical Medicine and
Rehabilitation, University of Alabama at Birmingham, Birmingham, AL 35294,
USA
*
Author for correspondence (e-mail:
wxiong{at}path.uab.edu
)
Accepted May 14, 2001
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SUMMARY |
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Key words: PYK2, FAK, Focal adhesions, Tyrosine phosphorylation, Actin cytoskeleton
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INTRODUCTION |
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Tyrosine phosphorylation is essential in integrin signaling and actin
cytoskeleton organization. Tyrosine phosphorylation of focal adhesion proteins
including FAK and paxillin is associated with focal adhesion assembly
(Burridge et al., 1988;
Kornberg et al., 1991
; Guan
and Shalloway, 1992
; Schaller
et al., 1992
). Inhibition of
protein tyrosine kinases disrupts focal adhesion assembly (Burridge and
Chrzanowska-Wodnicka, 1996
;
Chrzanowska-Wodnicka and Burridge,
1994
; Barry and Critchley,
1994
). By contrast, over
expression of protein tyrosine phosphatases inhibits focal adhesion assembly
(Persson et al., 1997
).
Interestingly, the reversal of the focal adhesion formation process
the disassembly of focal adhesions is also associated with tyrosine
phosphorylation. Focal adhesions are disrupted in cells transformed by the
Rous sarcoma virus or by the Fujinami avian sarcoma virus, whose oncogenes
encode tyrosine kinases (Tarone et al.,
1985
; Gavazzi et al.,
1989
). These cells form
podosomes, dot-shaped spots where cells adhere transiently to their matrix,
which contain actin and virtually all of the focal adhesion proteins (Tarone
et al., 1985
; Gavazzi et al.,
1989
). Concomitantly, tyrosine
phosphorylation of focal adhesion components including integrin, talin,
paxillin, Src and FAK is increased (Hirst et al.,
1986
; Smart et al.,
1981
; Schaller et al.,
1992
). These observations
indicate that regulated tyrosine phosphorylation of adhesion components seems
to be important in both assembly and disassembly of focal adhesions.
FAK is a major protein tyrosine kinase localized in focal adhesions
(Schaller et al., 1992;
Richardson and Parsons, 1996
;
Ilic et al., 1995
; Frisch et
al., 1996
). It contains a
central catalytic domain and large N- and C-terminal non-catalytic regions
that are devoid of SH2 or SH3 domains (Schaller et al.,
1992
; Hanks et al.,
1992
). Within the C-terminal
domain is the FAT domain that is both necessary and sufficient for the
targeting of FAK into focal adhesions (Hildebrand et al.,
1993
). Several lines of
evidence indicate that FAK plays an important role in regulating cell
migration and spreading. First, fibroblasts derived from FAK null mice
(fak-/-) exhibit reduced rates of cell migration and
altered cellular morphology (Ilic et al.,
1995
). Second, overexpression
of FAK in Chinese hamster ovary cells stimulates cell migration on fibronectin
(Carey, 1996; Carey, 1998). Third, overexpression of FAK-related non-kinase
(FRNK; a splice variant that contains only FAK's C-terminal domain and
functions as a dominant negative protein) inhibits cell spreading on
fibronectin (Richardson and Parsons,
1996
; Gilmore and Romer,
1996
).
PYK2 is a second member of the FAK family (Lev et al.,
1995; Sasaki et al.,
1995
; Avraham et al.,
1995
; Li et al.,
1996
); it is also known as
cellular adhesion kinase ß (CAKß), related adhesion-focal tyrosine
kinase (RAFTK) and calcium-dependent tyrosine kinase (CADTK). PYK2 catalytic
activity is regulated by tyrosine phosphorylation in a manner similar to FAK.
Phosphorylation at tyrosine 402 (Tyr402) creates a binding site for the SH2
domains of Src family kinases, resulting in Src activation, which increases
the catalytic activity of PYK2 (Tokiwa et al.,
1996
). However, PYK2
activation seems to be different from that of FAK. PYK2 is activated by
various stimuli, including elevation of intracellular calcium levels,
activation of protein kinase C and exposure to stress factors (e.g. UV light,
tumor necrosis factor
) (Lev et al.,
1995
; Tokiwa et al.,
1996
; Li et al.,
1996
), whereas FAK is mainly
activated by integrin engagement (Guan and Shalloway,
1992
; Schaller et al.,
1992
). We have shown that PYK2
induces apoptosis when expressed in fibroblasts, whereas FAK seems to be
important for cell survival (Xiong and Parsons,
1997
; Frisch et al.,
1996
). The mechanisms
underlying the different effects of PYK2 and FAK, and the potential role of
PYK2 in regulating cytoskeleton organization remain unclear.
In this paper, we show that PYK2 was expressed in fibroblastic cell lines and co-localized with FAK at focal adhesions. Overexpression of PYK2, but not FAK, in fibroblastic cells led to reorganization of actin-associated cytoskeleton structures and cell rounding. PYK2-mediated actin cytoskeleton reorganization required the N terminus, kinase activity and focal adhesion targeting. Interestingly, PYK2's effects could be suppressed by overexpression of FAK, probably via inhibition of PYK2 kinase activity and focal adhesion targeting. Taking together, our results suggest that PYK2 and FAK coordinately regulate actin-cytoskeleton organization and cellular morphology, which are important for cell migration and survival.
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MATERIALS AND METHODS |
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Expression vectors
cDNAs of PYK2, FAK and FRNK were subcloned into expression vectors
downstream of a Myc epitope tag (MEQKLISEEDL) under the control of the
cytomegalovirus (CMV) promoter (pCMV-Myc) as described previously (Xiong and
Parsons, 1997). Chimeric
PYK2/FAK-1 that contained PYK2 N terminus (amino acids 2-385) and FAK's kinase
and C-terminal domains (amino acids 380-1052) has been described previously
(Xiong and Parsons, 1997
). All
mutants were generated using the polymerase chain reaction (PCR). The
authenticity of constructs was verified by DNA sequencing. Green fluorescent
protein (GFP) vector was purchased from Clontech.
Cell culture, microinjections and transfections
Swiss 3T3 and HEK 293 cells were maintained in Dulbecco's Modified Eagle's
Medium (DMEM) containing 10% fetal calf serum, 100 µg ml-1
penicillin G and 100 µg ml-1 streptomycin (GIBCO). FAK null
(fak-/-) cells, kindly provided by D. Ilic, were
maintained in DMEM containing 10% fetal calf serum, 100 µg ml-1
penicillin G, 100 µg ml-1 streptomycin (GIBCO), 4.5 mg
ml-1 D-glucose, 584 µg ml-1 glutamine and 49 µM
ß-mecaptoethonal (Ilic et al.,
1995). For microinjection,
Swiss 3T3 cells were plated at a density of 103 cells per 22 mm
cover slip 12 hours before injection. Plasmid DNAs (50 ng µl-1)
were microinjected into nuclei using the Eppendorf micromanipulation system.
Two hours after injection, cells were fixed and immunostained with specific
antibodies. For transfection, cells were plated at a density of 106
cells per 100 mm culture dish, allowed to grow for 12 hours and transfected
using the calcium phosphate precipitation method for HEK 293 cells and the
SuperFect method (Qiagen) for fak-/- cells. Cells were
lysed with modified RIPA buffer (50 mM Tris-HCL, pH 7.4, 150 mM sodium
chloride, 1% NP40, 0.25% sodium deoxycholate and proteinase inhibitors) 24
hours after transfection. Cell lysates were subjected to analyses by SDS-PAGE
and western blotting.
Analysis of focal adhesions and cellular morphology
Focal adhesions were characterized by immunostaining using antibodies
against focal adhesion proteins as previously described (Xiong and Parsons,
1997). Briefly, cells were
plated on fibronectin-coated coverslips (40 µg ml-1) for 2-4
hours, fixed with 4% paraformaldehyde, permeabilized with 1% Triton X-100 and
incubated with a primary antibody at room temperature for 1 hour and
subsequently incubated with a fluorescein-conjugated anti-rabbit or anti-mouse
secondary antibody (1:300 dilution) for 1 hour at room temperature.
Immunoreactivity was visualized using a Nikon fluorescence microscope. Cell
morphology was determined by examining the cell length : cell width ratio of
fibroblasts. A ratio >2 was considered to be a normal elongated cell shape.
Abnormal or rounded cells usually had a ratio <2. More than 100
microinjected cells were examined for each protein. All the results are
presented as percentages of the mean + s.d. Statistical analyses were carried
out using Mann-Whitney u test.
Immunoprecipitation and western blotting
Immunoprecipitation of individual proteins was carried out as previously
described (Xiong and Parsons,
1997). Briefly, 1 mg of cell
lysates was incubated with anti-PYK2 antibodies (1-10 µg) in 1 ml of RIPA
buffer at 4°C for 1 hour with agitation. After the addition of
protein-A/agarose beads, the reaction was incubated at 4°C for another
hour. Immune complexes were collected by centrifugation and washed. Bound
proteins were resolved by SDS-PAGE and subjected to western blotting using the
indicated antibodies.
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RESULTS |
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Reorganization of actin cytoskeleton by PYK2 but not FAK
To study the role of PYK2 and FAK in regulating cytoskeleton organization,
we microinjected PYK2 cDNA into Swiss 3T3 cells, a typical mouse fibroblastic
cell line with well-characterized cytoskeleton structures and examined focal
adhesions and cell morphology in microinjected cells. Swiss 3T3 cells
expressing FAK showed normal focal adhesions
(Fig. 2D-F). By contrast, cells
expressing PYK2 exhibited altered cytoskeleton structures with either no focal
adhesions or abnormal `podosome-like' focal adhesions with peculiar ring- or
crescent-like adhesion sites at the periphery of cells
(Fig. 2A). The `podosome-like'
structures in PYK2 expressing cells could be labeled with antibodies against
various focal adhesion elements including paxillin or vinculin
(Fig. 2B). We next examined
whether the organization of stress fibers and microtubules were altered by
PYK2. In PYK2-expressing cells, stress fibers were disrupted in the central
region and enriched at the peripheral region of cells
(Fig. 2G-I). However, cells
expressing FAK exhibited normal stress fibers
(Fig. 2J-L). Microtubules
labeled by anti-ß-tubulin antibody seemed normal in
PYK2-expressing cells (data not shown).
|
These data demonstrate that actin-associated cytoskeleton structures, but
not microtubules, were reorganized by PYK2 in Swiss 3T3 cells. In addition to
altered focal adhesions and reorganized stress fibers, Swiss 3T3 cells
expressing PYK2 exhibited rounded morphology (ratio of length to width is
<2) with condensed cytoplasm (Fig.
2A-C,G-I), which was consistent with our previous report in Rat-1
cells (Xiong and Parsons,
1997), whereas cells
expressing FAK showed the normal elongated cell shape (ratio of
length over width is >2) (Figs
2D-F,J-K). Similar results were also observed in 3Y1 and 10T1/2
fibroblasts, and MDCK cells expressing PYK2
(Fig. 3). Moreover,
PYK2-induced cytoskeleton reorganization seemed to correlate with the level of
PYK2 expression. Cells overexpressing PYK2 at fivefold or
more that of endogenous PYK2, estimated by quantitative fluorescence
analysis, showed abnormal focal adhesions and morphology. These results
indicated that overexpression of PYK2, but not FAK, in Swiss
3T3 cells resulted in reorganization of actin cytoskeleton and changes of cell
morphology.
|
Need for intact N-terminal and FAT domains for PYK2-induced
cytoskeleton reorganization
To understand mechanisms of PYK2-mediated cytoskeleton reorganization, we
attempted to map the domains required for this event. PYK2 contains a central
catalytic domain (amino acids 419-679), flanked by non-catalytic N-terminal
(amino acids 1-418) and C-terminal (amino acids 680-1009) domains
(Fig. 4b). The C-terminal
region has two proline-rich sequences and the FAT domain
(Fig. 4b). We generated a
series of PYK2 deletion mutants and studied their cytoskeleton-reorganizing
activity and focal adhesion targeting in Swiss 3T3 cells
(Fig. 4). Whereas Swiss 3T3
cells expressing PYK2 demonstrated abnormal focal adhesions, Swiss
3T3 cells expressing the FAT-domain deletion mutant (PYK2936-1009),
which failed to target to focal adhesions, exhibited normal focal adhesion
structures (Fig. 4a). This
result suggests that the FAT domain of PYK2, or PYK2 focal adhesion targeting,
was required for PYK2-mediated reorganization of focal adhesions. However,
PYK2 focal adhesion targeting was not sufficient to induce reorganization of
focal adhesions. Swiss 3T3 cells producing PYK2 FAT domain or C-terminal
regions, which localized to focal adhesions, failed to induce reorganization
of focal adhesions (Fig. 4a).
This result suggests that, in addition to the FAT domain, PYK2 N terminus and
kinase domain were also required for focal adhesion reorganization. The
requirement for PYK2 N terminus by this event was further demonstrated by the
finding that cells expressing PYK2 N-terminal deletion mutants
(PYK2
1-88 or PYK2
1-416) showed normal focal adhesions
(Fig. 4a). Interestingly,
although the C-terminal domain of PYK2 can localize to focal adhesions, the
partial N-terminal deletion mutant failed to localize to the focal adhesions
(Fig. 4a), even though they
contain the intact FAT domain, implying that the PYK2 FAT-domain-mediated
focal adhesion targeting might be regulated by PYK2 kinase domain and /or N
terminus. Taken together, these results indicated that both PYK2 N terminus
and FAT domain were required for PYK2-mediated reorganization of focal
adhesions.
|
Effects of PYK2 catalytic activity on cytoskeleton
reorganization
To examine whether the catalytic activity of PYK2 was required for
PYK2-induced cytoskeleton reorganization, a catalytically inactive mutant
(PYK2-KD) was generated that contained a lysine (K) to alanine (A) mutation in
the ATP binding site (Fig. 5).
Being unable to bind to ATP, this mutant showed little if any catalytic
activity when expressed in mammalian cells by in vitro kinase assays or
anti-phosphotyrosine blotting (Fig.
5B). In addition, tyrosine phosphorylation of several proteins was
increased in cells expressing wild-type PYK2 but not PYK2-KD
(Fig. 5C). One of these
proteins was identified as p130Cas, a constituent in the focal
adhesions that is known to be a potential substrate of PYK2
(Fig. 5C). Swiss 3T3 cells
microinjected with the plasmid DNA encoding PYK2-KD were analyzed in parallel
with the cells expressing wild-type PYK2. As shown in
Fig. 5A, unlike those
expressing PYK2-WT, which had abnormal or no focal adhesions, a
fraction of PYK2-KD-expressing cells (33%) exhibited normal
focal adhesions. In addition, a fraction of PYK2-KD-expressing cells
(
45%) seemed elongated in shape, with a ratio of length to width of
>2. Fig. 5D summarizes
results from three independent experiments. Significantly more cells showed
normal focal adhesions or cell shape (a ratio of length to width of >2) in
PYK2-KD-expressing cells than in PYK2-WT-expressing cells.
These results indicated that the catalytic activity of PYK2 contributed to,
but was not absolutely required for, the induction of cytoskeleton
reorganization. In addition, the PYK2-Y402F mutant, which contains a tyrosine
(Y402F) mutation, also showed decreased cytoskeleton reorganization activity
(Fig. 5D), suggesting that
autophosphorylation of PYK2 might also contribute to this event.
|
Suppression of PYK2-induced cytoskeleton reorganization by FAK
Considering that FAK is important for regulating focal adhesion turnover
(Ilic et al., 1995),
cytoskeleton reorganization by PYK2 but not FAK led us to determine whether
FAK could regulate PYK2's effect. Swiss 3T3 cells microinjected with cDNAs
encoding PYK2 and FAK or GFP were examined for focal adhesions and cell
morphology. Unlike cells expressing PYK2 alone, cells expressing both
FAK and PYK2 exhibited normal focal adhesions and cell
morphology, suggesting that FAK could inhibit PYK2-induced cytoskeleton
reorganization and cell rounding. This inhibitory effect was specific, in that
co-injection of GFP failed to rescue the phenotype
(Fig. 6A). Moreover, the rescue
of the PYK2 phenotypes by FAK seemed to be dose dependent. When the ratio of
injected cDNAs of FAK to PYK2 was increased, FAK inhibitory activity was
enhanced from
20% (for 1:0.5 ratio) to 70% (1:2 ratio)
(Fig. 6B). Finally, FAK's
inhibitory effect on PYK2 did not require FAK autophosphorylation, because the
mutant of the autophosphorylation site (Tyr397) (FAK-Y397F) could suppress
PYK2-induced cytoskeleton reorganization
(Fig. 6B). Interestingly,
co-expression of FRNK seemed to be able to rescue PYK2-induced alterations
only in focal adhesions, not in cell morphology
(Fig. 6), suggesting that
different mechanisms might be involved in PYK2-induced alternations in focal
adhesions and cell morphology. These results suggest that PYK2 and FAK,
although highly homologous in structure, might mediate different (and possibly
opposing) roles in cytoskeleton organization.
|
Inhibition of PYK2 autophosphorylation by FAK
PYK2's catalytic activity was required for cytoskeletal reorganization and
FAK could inhibit PYK2's effect. We thus hypothesized that FAK might regulate
PYK2's catalytic activity. Phosphorylation of PYK2 at Tyr402 is crucial for
PYK2 kinase activity in vivo (Lev et al.,
1995; Dikic et al.,
1996
). We tested whether FAK
affected phosphorylation of PYK2 Tyr402. As shown in
Fig. 7A, Tyr-402
phosphorylation detected by the anti-PY402 antibody was significantly reduced
when PYK2 was co-expressed with FAK in HEK 293 cells. Interestingly, FRNK, the
splice variant containing the C-terminal domain of FAK, did not seem to have
an effect on PYK2 autophosphorylation (Fig.
7A), suggesting that the FAK N-terminal region and/or kinase
domains are required for the inhibitory effect. We next examined whether FAK
N-terminal region was required for the inhibition of PYK2 autophosphorylation.
Whereas PYK2 autophosphorylation is significantly decreased in cells
co-expressing FAK and FAK
C20 (a C-terminal FAT domain deletion mutant),
FAK
N-term (a mutant with a deletion of FAK's N-terminal 1-412 amino
acids) was unable to mediate the inhibitory effect, suggesting again that the
N-terminal domain (amino acids 1-412) of FAK was required
(Fig. 7A). To further confirm
this finding, we generated a chimeric construct, PYK2/FAK-1, in which the
N-terminal domain of FAK (amino acids 2-380) was replaced with the N-terminal
domain of PYK2 (amino acids 2-385). Expression of this chimeric protein did
not seem to affect PYK2 autophosphorylation
(Fig. 7A). These data suggested
to us that the FAK N-terminal domain (2-380) was essential for the inhibition
of PYK2 autophosphorylation in HEK 293 cells.
|
PYK2 activity can be regulated by tyrosine phosphorylation. Phosphorylation
at Tyr402 of PYK2, like Tyr397 in FAK, creates a binding site for the SH2
domain of Src. Src binding to PY402 leads to activation of Src, which in turn
phosphorylates and activates PYK2 (Dikic et al.,
1996). Overexpression of FAK,
which has similar binding property, might absorb Src or a Src-like kinase that
is essential for PYK2 activation and thus decreases PYK2 autophosphorylation.
To test this hypothesis, we determined whether FAK tyrosine phosphorylation,
including PY397, was required for the inhibition of PYK2 autophosphorylation.
As shown in Fig. 7B, mutations
of key tyrosine residues in FAK did not seem to alter its inhibitory effect on
PYK2. These results demonstrated that tyrosine phosphorylation of FAK is not
required for FAK-mediated inhibition of PYK2 autophosphorylation and ruled out
the possibility that inhibition of PYK2 by FAK resulted from a decrease in Src
that effectively interacts with PYK2.
Reduction of PYK2 focal adhesion targeting by FAK
PYK2 was localized with FAK to focal adhesions in fibroblasts
(Fig. 1) and PYK2 focal
adhesion targeting was required for PYK2-mediated cytoskeletal reorganization
(Fig. 4). We thus hypothesized
that FAK might rescue PYK2's phenotypes by regulating PYK2 focal adhesion
targeting. To test this hypothesis, we examined the PYK2 focal adhesion
localization in fak-/- fibroblasts transiently transfected
with FAK or FRNK. Whereas PYK2 localizes with paxillin to focal adhesions in
fak-/- fibroblasts
(Fig. 8aA-aC,b), PYK2 focal
adhesion targeting and the number of focal adhesions seemed to be decreased in
cells expressing FAK compared with cells that are not expressing FAK
(Fig. 8aD-aF,b). Similar
results were also observed in fak-/- fibroblasts
expressing FRNK (Fig.
8aG-aI,b) or FAK-Y397F (data not shown), suggesting that
FAK C-terminal domain is sufficient for the reduction of PYK2 focal adhesion
targeting. These results suggested that overexpressed FAK might displace
endogenous PYK2 from focal adhesions, probably via the C-terminal FAT
domain.
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DISCUSSION |
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Although PYK2 has been found to be located at cytoplasm or perinuclear
regions (Schaller and Sasaki,
1997; Zheng et al., 1997; Sieg
et al., 1998
), its presence at
focal adhesions has not been documented. Here, we report that, in addition to
cytoplasmic and perinuclear regions, PYK2 localizes with FAK to focal
adhesions in fibroblastic cells. The anti-PYK2 antibody used in our studies
does not cross-react with FAK. Western blot analysis using this antibody
recognizes a single band that is different from that of FAK and does not
cross-react with overexpressed FAK in HEK293 cells. In
immunohistochemical studies, this antibody could label focal adhesion
structures in fak-/- cells, in which FAK was not
expressed. Moreover, in fak-/- cells overexpressing
FAK, it did not stain overexpressed FAK. These results convincingly
demonstrated that the anti-PYK2 antibody is specific and does not cross-react
with FAK. The reasons why PYK2 has not been reported in focal adhesions are
complex. The antibodies available for previous studies might not be as
sensitive for immunostaining of endogenous PYK2. This might explain the
failure to observe focal adhesion localization of endogenous PYK2 using the
polyclonal anti-PYK2 antibody (amino acids 587-988) (Xiong and Parsons,
1997
). In studies in which
PYK2 was overexpressed (Sasaki et al.,
1995
; Schaller and Sasaki,
1997
; Zheng et al.,
1998
), it could be difficult
to visualize its subcellular localization. We found that only 10-20% of 10T1/2
cells overexpressing PYK2 exhibited focal adhesion targeting, in agreement
with a previous report (Schaller and Sasaki,
1997
). Furthermore, we found
that PYK2 focal adhesion targeting could be regulated by fibronectin
stimulation or expression of FAK or FRNK. PYK2 focal adhesion localization
became obvious when cells were plated on fibronectin-coated coverslips for 2-4
hours. In cells with high level of FAK or FRNK, PYK2 might not be localized at
focal adhesions.
Our results suggest that PYK2 focal adhesion targeting has the following properties. First, PYK2 FAT domain was required and sufficient for its focal adhesion targeting. Second, PYK2 focal adhesion targeting seemed to be negatively regulated by its N terminus and catalytic activity. Third, PYK2 focal adhesion targeting was inhibited by FAK. The mechanism by which FAK or FRNK regulated PYK2 subcellular localization remains unclear. It is intriguing to speculate that the subcellular location of PYK2 might be regulated by FAK or FRNK, perhaps via competition for a common binding protein(s) whose association with PYK2 is necessary for PYK2 focal adhesion targeting, or by its catalytic activity, perhaps by modification of targeting sequences like the C-terminal FAT domain.
The finding of PYK2 at focal adhesions suggests that PYK2 might play an
important role in focal adhesion signaling and/or regulate the formation and
turnover of this subcellular structure. Indeed, we found that endogenous PYK2
autophosphorylation (assayed by PY402 antibody) is increased when 10T1/2 cells
are plated on fibronectin-coated plates. The increase in PY402 is time
dependent, and peaked around 1 hour grown on fibronectin-coated dishes (data
not shown). Moreover, overexpression of PYK2 in fibroblasts changed cell shape
and focal adhesion structures. PYK2-mediated cytoskeletal reorganization
required its catalytic activity and focal adhesion targeting. These results
support a role for PYK2 as a regulator of focal adhesion assembly. Altered
focal adhesions mediated by PYK2 are characterized by podosome-like
morphological structures. Podosome-like structures have been identified in
v-Src-transformed cells (Tarone et al.,
1985; Gavazzi et al.,
1989
) and non-transformed
cells including peripheral mononuclear lymphocytes, macrophages and
osteoclasts, in which PYK2 is highly expressed (Avraham et al.,
1995
; Duong et al.,
1998
). The abundant expression
of PYK2 in these cells, the localization of PYK2 to podosomes (Duong
et al., 1998
) and our
observation that expression of PYK2 results in cytoskeleton reorganization in
Swiss 3T3 fibroblasts suggest that PYK2 might play an important regulatory
role in the formation of podosome-like structures in vivo (Gavazzi et al.,
1989
; Boyles and Bainton,
1979
; Boyles and Bainton,
1981
).
Interestingly, FAK, a tyrosine kinase related to PYK2, could rescue
PYK2-induced actin cytoskeleton reorganization, suggesting that PYK2 and FAK
do not only mediate different and possibly opposing roles in regulating focal
adhesion organization and cell morphology. What are the mechanisms for
FAK-mediated suppression of PYK2's effects? Because FAK inhibits PYK2
autophosphorylation and focal adhesion targeting, which are required for
PYK2-mediated cytoskeleton reorganization, we propose that FAK-mediated
suppression of PYK2-induced cytoskeleton reorganization might be due to its
inhibition of PYK2 autophosphorylation and/or focal adhesion localization. FAK
inhibition of PYK2 autophosphorylation is consistent with previous reports
that FAK and the FAK autophosphorylation mutant FAK-Y397F inhibit
integrin-mediated PYK2 activation in fak-/- cells (Owen et
al., 1999). The mechanism of
FAK-mediated inhibition of PYK2 activity remained unclear. FAK might inhibit
PYK2 tyrosine phosphorylation by displacing PYK2 focal adhesion targeting.
This might be a mechanism underlying FAK inhibition of integrin-mediated PYK2
tyrosine phosphorylation (Owen et al.,
1999
). However, FAK inhibition
of PYK2 autophosphorylation in HEK 293 cells expressing PYK2 seemed
to be caused by a different mechanism. The FAT domain deletion mutant,
FAK
C20 (which failed to localize to focal adhesions), but not FRNK
(which decreases PYK2 focal adhesion targeting) could inhibit PYK2
autophosphorylation in HEK 293 cells. These results indicate that FAK
inhibition of PYK2 autophosphorylation does not correlate with FAK focal
adhesion targeting. Furthermore, our results suggest that FAK N-terminal
region, which contains a Band 4.1 domain (Giranlt, J. A. et al.,
1998
), is essential for the
inhibition of overexpression-induced PYK2 autophosphorylation.
PYK2 and FAK play important roles in regulating actin-associated
cytoskeletal organization, and a balance between PYK2 and FAK tyrosine kinases
is important for the determination of cytoskeletal organization and cell
morphology. These data, together with our previous results (Xiong and Parsons,
1997), suggest that FAK and
PYK2 could play an opposing roles in the overall regulation of the actin
cytoskeleton organization and cell morphology, death and survival, which is
reminiscent of the Yin-Yang principle.
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ACKNOWLEDGMENTS |
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