Department of Microbiology, Health Science Center, University of Virginia, Charlottesville, Virginia 22908
Many cells (e.g., epithelial cells) require attachment to the extracellular matrix (ECM) to survive,
a phenomenon known as anchorage-dependent cell
survival. Disruption of the cell-ECM interactions mediated by the integrin receptors results in apoptosis. Focal
adhesion kinase (FAK), a 125-kD protein tyrosine kinase activated by integrin engagement, appears to be
involved in mediating cell attachment and survival. Proline-rich tyrosine kinase 2 (PYK2), also known as cellular adhesion kinase (CAK
) and related adhesion focal tyrosine kinase, is a second member of the FAK
subfamily and is activated by an increase in intracellular calcium levels, or treatment with TNF
and UV
light. However, the function of PYK2 remains largely
unknown. In this study, we show that over-expression of PYK2, but not FAK, in rat and mouse fibroblasts
leads to apoptotic cell death. Using a series of deletion
mutants and chimeric fusion proteins of PYK2/FAK,
we determined that the NH2-terminal domain and tyrosine kinase activity of PYK2 were required for the
efficient induction of apoptosis. Furthermore, the apoptosis mediated by PYK2 could be suppressed by
over-expressing catalytically active v-Src, c-Src, phosphatidylinositol-3-kinase, or Akt/protein kinase B. In
addition, it could also be suppressed by overexpressing an ICE or ICE-like proteinase inhibitor, crmA, but not
Bcl2. Collectively, our results suggest that PYK2 and
FAK, albeit highly homologous in primary structure,
appear to have different functions; FAK is required for
cell survival, whereas PYK2 induces apoptosis in fibroblasts.
HOMEOSTASIS of multicellular organisms is controlled not only by the proliferation and differentiation of cells but also by cell death (Raff, 1992 Apoptosis can be triggered by a variety of extrinsic and
intrinsic signals. Extrinsic inducers of apoptosis include
TNF family proteins (e.g., Fas ligand and TNF Integrin engagement and activation of FAK are implicated in a number of signaling pathways, including ones
that lead to anchorage-dependent cell survival (Burridge
and Chrzanowska-Wodnicka, 1996 Recently, a second member of the FAK subfamily, proline-rich tyrosine kinase 2 (PYK2), also known as cellular
adhesion kinase In this report we demonstrate that over-expression of
PYK2 in both fibroblastic and epithelial cell lines (e.g., rat-1,
mouse 10T1/2, swiss 3T3, quail QT6, and human embryonic kidney 293 cells [HEK 293]) leads to apoptosis. The
NH2-terminal domain and tyrosine kinase activity of PYK2
are required for its full apoptotic activity. Furthermore, we
show that the PYK2-mediated apoptosis can be suppressed by over-expression of catalytically active c-Src, v-Src, PI3 kinase, and Akt/PKB. It could also be suppressed by over-expressing crmA, but not Bcl2. These data demonstrate
that while PYK2 and FAK are structurally similar, each
has the capacity to mediate distinctly different signaling
responses.
Reagents and Cell Lines
Rabbit polyclonal antisera recognizing PYK2 were raised using a glutathione-S-transferase (GST) fusion protein containing the COOH-terminal 400 amino acids of rat PYK2 (amino acid 587 to 988). Monoclonal antibodies and goat polyclonal antibody recognizing NH2-terminal PYK2
domain were purchased from Santa Cruz Biotechnology (Santa Cruz, CA;
anti-myc and anti-PYK2) or Transduction Laboratories (Lexington, KY;
anti-phosphotyrosine). Propidium iodide was purchased from Sigma
Chemical Co. (St. Louis, MO). TUNEL kit (Apoptag kit) was purchased
from Boehringer Mannheim (Indianapolis, IN). GST-c-Jun was purchased
from Clonetech (Palo Alto, CA). LA29, a v-Src temperature-sensitive cell
line, was provided by M. Weber (University of Virginia, Charlottesville, VA). Mouse fibroblastic 10T1/2, 5HD47 (c-Src), pm430 (kinase inactive c-Src), and dl155 (SH2 defective c-Src) cell lines were provided by S. Parsons
(University of Virginia, Charlottesville, VA). HEK 293 cells were provided by Q.-H. Song (Columbia University, New York, NY).
Expression Vectors
The cDNAs of PYK2 (CAK Cell Culture and Transfections
Rat-1, mouse 10T1/2, and Cos-1 cells were maintained in DME containing
10% fetal calf serum, 100 µg/ml penicillin G, and 100 mg/ml Streptomycin
(GIBCO BRL, Gaithersburg, MD). LA29, 5HD47, pm430, and dl155 cell
lines were maintained in the above medium containing G418 (Sigma
Chemical Co.). HEK 293 cells were maintained in DME/F12 (1:1) medium with the same additives. Cells were plated 12 h before transfection at
a density of 104 cells/22 mm2 coverslip (VWR Scientific, West Chester, PA).
Cells were transfected with 30-40 µl of lipofectamine (GIBCO BRL) with
various constructs (3 µg DNA) with or without 1 µg of pCMV Apoptosis Assays
The morphology of transfected cells was examined by Immunoprecipitation
For immunoprecipitation, 500 µg of cell lysates was incubated with anti-PYK2 antibodies at 4°C for 1 h in a final volume of 1 ml RIPA buffer. After the addition of protein A-agarose beads, the reaction was incubated at
4°C for another hour. Immunocomplexes were purified and subjected to
immunoblotting using antiphosphotyrosine and anti-PYK2 antibodies.
Expression of PYK2 Induced Morphological Changes
in rat-1 Cells
To study the function of PYK2, we attempted to generate
rat-1 fibroblastic cell lines stably expressing PYK2. After a
number of failures, we examined the phenotype of rat-1
cells after transient transfection using a vector carrying
cDNA encoding full length PYK2 with an NH2-terminal
c-Myc epitope tag under the control of the CMV promoter
(PYK2-WT). The expression of PYK2 as well as cell morphology were monitored by immunostaining of the cells using anti-PYK2 antibodies, anti-c-Myc (9E10) monoclonal antibodies, or by
Morphologically Altered rat-1 Cells Exhibit
Features of Apoptosis
The morphological changes in rat-1 fibroblasts induced by
PYK2 expression was indicative of apoptosis. We used
both propidium iodide staining and TdT-mediated dUTP-fluorescence nick end labeling (TUNEL) to determine
whether DNA condensation and fragmentation, a hallmark
of apoptosis, occurred in the cells expressing PYK2 (Fisher,
1994
The Full Apoptotic Activity of PYK2 Requires
NH2-terminal and Active Kinase Domains
PYK2 contains a central catalytic kinase domain (from
amino acid 419 to 679), flanked by noncatalytic NH2-terminal (amino acids 1 to 418) and COOH-terminal (amino acids
680 to 1009) domains (Fig. 3). Within the COOH-terminal
region, there are two proline-rich sequences and a putative
FAT domain (Fig. 3). To determine which domains of PYK2
were required for its apoptotic activity, we generated a series of PYK2 deletion mutants, whose structures are summarized in Fig. 3. Mutant proteins were tagged either with
GST or a c-Myc epitope at the NH2 terminus. Transfection
of individual mutant constructs into HEK 293 cells followed by Western blotting of cell extracts using anti-PYK2, antibodies to the c-Myc epitope or GST, confirmed
the expression of mutant proteins of the appropriate molecular sizes (Fig. 4 A). The apoptotic activity of individual mutant proteins was tested by transient transfection of rat-1 cells with each mutant construct in the presence or absence of pCMV
The PYK2-WT demonstrated the highest apoptotic activity or apoptotic index (0.97), while the basal apoptotic
index in rat-1 cells transfected with control vector alone
was 0.09-0.13. A significant reduction of the apoptotic index (0.3-0.5) was observed after transfection with the
NH2-terminal domain deletion mutants (e.g., PYK2
The putative FAT domain of PYK2 did not appear to be
critical for the apoptotic activity. Partial deletion of the
FAT domain, PYK2 To confirm that PYK2-KD was catalytically inactive, we
studied autophosphorylation of wild-type PYK2-WT and
mutants (PYK2-KD, PYK2 To further examine the mechanisms by which PYK2,
but not FAK, induces apoptosis, we constructed a chimeric protein (PYK2/FAK1) containing the NH2-terminal
domain of PYK2 (amino acids 2 to 385) and the catalytic
and COOH-terminal domains of FAK (amino acids 380 to
1,052) and a second chimeric protein (PYK2/FAK2) containing the NH2-terminal and kinase domains of PYK2
(amino acids 2 to 695) and the COOH-terminal domain of
FAK (amino acids 692 to 1,052; Fig. 6 A). The structure of
these chimeric constructs (PYK2/FAK1 and PYK2/FAK2)
was confirmed by Western blotting analysis of HEK 293 cell lysates overexpressing PYK2, PYK2/FAK1, PYK2/
FAK2, and FAK using antibodies against c-Myc epitope,
PYK2 NH2-terminal domain (anti-PYK2 from Santa Cruz
Biotechnology), and FAK COOH-terminal domain (mAb
2A7; Fig. 6 B). Apoptotic activities of these chimera were
examined in rat-1 cells. Both PYK2/FAK1 and PYK2/
FAK2 demonstrated similar apoptotic activities (apoptotic
index of 0.8 to 0.84) as wild-type PYK2, while the apoptotic activity of wild-type FAK was close to the basal level
(0.13; Fig. 6 C). These data indicated that the NH2-terminal domain of PYK2 was sufficient to render FAK indistinguishable from PYK2 in its ability to induce apoptosis.
PYK2-mediated Apoptosis Can
Be Suppressed by Over-Expression of Catalytically
Active Src, PI3 Kinase, and Akt
To examine the possible effects of Src on PYK2-mediated
apoptosis, we expressed PYK2 in LA29 cells, a temperature-sensitive v-Src rat-1 cell line. When PYK2-WT was
transiently expressed in LA29 cells grown at permissive
temperature (35°C), the apoptotic index was significantly
reduced from 0.97 to 0.25 (Fig. 7). No significant inhibition
of apoptosis was observed when LA29 cells expressing
PYK2 were grown at nonpermissive temperature (39°C) (data not shown), indicating that the tyrosine kinase activity of v-Src was required for this event. To examine whether
suppression of apoptosis could be mimicked by c-Src,
PYK2 was transiently expressed in mouse 10T1/2 cells
stably over-expressing c-Src (5H), as well as its parental
10T1/2 cells. 10T1/2 cells expressing full length PYK2 underwent apoptosis (apoptotic index of 0.95). The apoptotic
index was significantly reduced to 0.45 when PYK2 was
expressed in the c-Src (5H) cell line (Fig. 7). To determine if the reduced apoptotic index of c-Src was dependent on
kinase activity, PYK2 was transiently expressed in cells
stably over-expressing either catalytically inactive c-Src
(pm430 mutant) or an SH2-defective variant of c-Src
(dl155; Wilson et al., 1989
We next examined the effects of PI3 kinase on PYK2-mediated apoptosis. The plasmids encoding c-Myc-tagged
constitutively active or catalytically inactive P110/PI3 kinases, in which a truncated p85 subunit was linked to the
catalytic subunit (Hu et al., 1995
The serine/threonine protein kinase Akt (also called
protein kinase B, PKB) is believed to be one of the effectors in the PI3 kinase signaling pathway (Burgering and
Coffer, 1995 In this study, we have investigated the expression of the
FAK-related protein tyrosine kinase, PYK2, in several different fibroblastic and epithelial cell lines including rat-1,
mouse 10T1/2, and HEK 293 cells. In each cell line the
forced expression of PYK2, but not the related tyrosine kinase, FAK, rapidly (within 24 to 30 h) induced apoptosis.
Apoptosis induced by expression of full length PYK2 was
very efficient; >90% of the cells expressing PYK2 undergo
programed cell death. In contrast, mutant forms of PYK2
deficient in kinase activity (PYK2-KD) or lacking the major site of autophosphorylation (PYK2-Y402F) were significantly less efficient in inducing apoptosis, reducing the
overall level of cell death from >90% to 40-58%. Chimeric proteins containing the NH2-terminal domain of
PYK2 and the kinase and COOH-terminal domains of
FAK were as efficient as wild-type PYK2 in the induction
of apoptosis. Co-expression of PYK2 with either active
Src, PI3 kinase, or the serine/threonine kinase, Akt, significantly reduced apoptosis. In addition, co-expression of
PYK2 with crmA, an inhibitor of the ICE or ICE-like proteinases, but not Bcl2, also significantly inhibited apoptosis. We conclude from these data that the efficient induction of cell death by PYK2 under our assay conditions,
requires specific interactions via sequences in the NH2-terminal domain of PYK2, PYK2 kinase activity, and the
ability to autophosphorylate at tyrosine residue 402. In addition, efficient induction of cell death requires that PYK2
functionally interacts with a downstream signaling pathway(s) (may be mediated in part by inhibition of the PI3
kinase pathway) to promote apoptosis.
The high apoptotic activity exhibited by the chimeric
constructs PYK2/FAK1 and PYK2/FAK2 point to the importance of the NH2-terminal domain of PYK2 in inducing
apoptosis. Furthermore, these observations argue that the
catalytic and COOH-terminal domains of PYK2 and FAK,
while necessary for the apoptotic process, are interchangable. Expression of the PYK2 NH2- and COOH-terminal
domains also induces apoptosis, albeit with an efficiency
significantly less than full length PYK2, but comparable to
catalytically inactive PYK2 (Fig. 3). While it is unclear
how these isolated domains induce apoptosis, studies of
FAK demonstrate that the NH2- and COOH-terminal domains interact with several well characterized binding
partners, linking FAK to both upstream and downstream
signaling pathways (Schaller and Parsons, 1994 Co-expression of full length PYK2 with either constitutively active PI3 kinase, Akt, or Src results in the partial
rescue of PYK2-induced apoptosis (Fig. 6). These data implicate the PI3 kinase pathway in the protection of apoptosis mediated by wild-type PYK2. Akt is a serine/threonine
protein kinase (also called protein kinase B or PKB), identified first as an oncogene; however, recent studies indicate that it is one of the major targets of PI3 kinase (Burgering and Coffer, 1995 The results presented herein are the first report of a role
for PYK2 in the induction of apoptosis. PYK2 is present at
relatively high levels in brain and is expressed in kidney,
liver, spleen, and a variety of cell lines (e.g., PC12 cells and
many hematopoietic cell lines; Avraham et al., 1995 In summary, PYK2 appears to mediate different functions from those of FAK, a related protein tyrosine kinase.
Ectopic expression of PYK2 led to apoptosis in several fibroblastic and epithelial cell lines, whereas over-expression
of FAK did not result in cell death in any of these cell lines
tested. The different effects of PYK2 and FAK on cell
growth suggest that these two protein tyrosine kinases, in
spite of similarity in overall structure, may mediate signaling via different pathways. Experiments to understand the
mechanisms for the opposite effects mediated by PYK2
and FAK are in progress.
).
Programmed cell death, or apoptosis, is characterized by
the presence of nuclear and cytoplasmic condensation and
segmentation and is an important regulatory event in embryogenesis, metamorphosis, endocrine-dependent tissue
atrophy, and normal tissue turnover (Raff, 1992
; Nagata
and Golstein, 1995
; Steller, 1995
; Muzio et al., 1996
). Disregulation of apoptosis contributes to the pathogenesis of
several diseases, including cancers, neurodegenerative disorders, immunodeficiency, and autoimmune diseases (Thompson, 1995
). Although the intracellular mediators that induce
apoptosis are beginning to be defined, relatively little is
known about the mechanisms by which cell death programs
are executed.
), calcium,
growth factor withdrawal, and loss of extracellular matrix
(ECM)1 attachment (Nagata and Golstein, 1995
; Thompson, 1995
). These extrinsic signals induce apoptosis in a
wide variety of cell types. Intrinsic inducers of apoptosis
comprise a number of genes conserved throughout evolution, including members of interleukin-1
converting enzyme (ICE) proteinase family, Bcl2 family (e.g., Bcl2s, Bad, and Bax), and p53 (Ellis et al., 1991
; Vaux et al., 1994
).
Apoptosis can be suppressed by a variety of extrinsic and
intrinsic signals, including growth factors (e.g., IGF1, NGF,
and CNTF), signaling molecules activated by these growth
factors (e.g., phosphatidylinositol-3-kinase [PI3 kinase]),
Bcl2 family proteins (e.g., Bcl2 and Bclxl), and proteinase
inhibitors (e.g., crmA; Kapeller and Cantly, 1994; Nagata
and Golstein, 1995
; Steller, 1995
; Thompson, 1995
; Yao and Cooper, 1995
).
; Frisch et al., 1996
;
Hungerford et al., 1996
; Parsons, 1996
; Xu et al., 1996
).
FAK is a prototypic member of a family of nonreceptor
protein tyrosine kinases, containing a central catalytic domain and large NH2- and COOH-terminal noncatalytic regions that are devoid of SH2 and SH3 domains (Hanks et
al., 1992
; Schaller et al., 1992
). FAK is enriched in the
brain and expressed in most cell lines and tissues examined (Andre and Becker-Andre, 1993
; Grant et al., 1995
).
In some cell types, the COOH-terminal domain of pp125FAK
is expressed autonomously as a 41-kD protein termed focal adhesion kinase (FAK)-related nonkinase (FRNK)
(Schaller et al., 1993
). FAK is activated by many diverse
stimuli, including v-Src transformation (Schaller et al., 1992
),
attachment to the ECM (Guan and Shalloway, 1992
;
Schaller et al., 1992
), and exposure to growth factors (e.g., PDGF; Rankin and Rozengurt, 1994
), neuropeptides (e.g.,
bombesin; Rozengurt, 1991
; Zachary et al., 1992
), and
lysophosphatidic acid (Moolenaar, 1991
). Clustering of integrins through binding to the ECM leads to the tyrosine
phosphorylation of FAK (Guan and Shalloway, 1992
;
Schaller et al., 1992
). Phosphorylation of FAK on tyrosine
397 creates a high affinity binding site for the SH2 domains of Src and Fyn, both of which are associated with activated FAK (Cobb et al., 1994
; Schaller et al., 1994
).
Phosphorylation of FAK on tyrosine residues present in
the COOH-terminal domain leads to the association of
various other SH2 domain-containing signaling proteins,
including Grb2 (Schlaepfer et al., 1994
) and the p85 subunit of PI3 kinase (Chen and Guan, 1994b
; Guinebault et
al., 1995
). In addition, the proline-rich regions in the
COOH-terminal domain of FAK direct the binding to
p130cas (Crk-associated substrate) and Graf (GTPase regulator associated with FAK) in an SH3 domain-dependent
manner (Polte and Hanks, 1995
; Harte et al., 1996
; Hildebrand et al., 1996
). The COOH-terminal domain of FAK
can also associate with the cytoskeletal proteins paxillin
and talin (Bellis et al., 1995
; Chen et al., 1995
; Hildebrand et al., 1995
; Tachibana et al., 1995
). Inclusive within the
COOH-terminal domain is a 140-amino acid sequence
that is both necessary and sufficient for the targeting of
FAK to focal adhesions, called the focal adhesion targeting domain (FAT; Hildebrand et al., 1993
). FAK is believed to play an important role in regulating signaling events initiated by the activation of various membrane receptors that induce cytoskeletal rearrangement (Parsons,
1996
). Cells microinjected with reagents that attenuate the
activity of FAK (e.g., anti-sense oligonucleotides and anti-FAK antibodies) undergo apoptosis (Hungerford et al.,
1996
; Xu et al., 1996
). Cells expressing constitutively active
CD2-FAK are resistant to apoptosis when detached from
the ECM (Frisch et al., 1996
), suggesting that FAK may be
involved in mediating the anchorage-dependent cell survival.
(CAK
) and related adhesion focal tyrosine kinase (RAFTK), has been identified (Avraham et
al., 1995
; Lev et al., 1995
; Sasaki et al., 1995
). PYK2, a 116-kD
cytoplasmic protein tyrosine kinase, is rapidly phosphorylated on tyrosine residues in response to various stimuli,
including elevation of the intracellular calcium levels, activation of protein kinase C, and exposure to stress factors
(e.g., UV light, TNF
; Lev et al., 1995
; Tokiwa et al., 1996
). PYK2 is highly enriched in the brain and is expressed in fewer tissues and cell lines (e.g., PC12 cells, and
many hematopoietic cell lines; Avraham et al., 1995
; Lev
et al., 1995
; Sasaki et al., 1995
; Salgia et al., 1996
). The restricted expression of PYK2, as compared to FAK, suggests that they may mediate distinct functions. FAK and
PYK2 are highly homologous to each other, sharing 45%
overall sequence identity and 60% identity in the catalytic domain. Several tyrosine residues are conserved between
FAK and PYK2, including the binding site for the SH2 domains of Src and Fyn (Y397 in FAK, Y402 in PYK2) and the
putative binding site for the SH2 domain of Grb2 (Y925 in
FAK, Y881 in PYK2; Cobb et al., 1994
; Schaller et al., 1994
;
Schlaepfer et al., 1994
; Avraham et al., 1995
; Lev et al.,
1995
; Sasaki et al., 1995
; Dikic et al., 1996
). In addition,
PYK2 also contains the putative "FAT" domain and the
proline-rich sequences responsible for mediating the binding of p130cas and Graf. Given the high degree of sequence
similarity between PYK2 and FAK, it is possible that
PYK2 interacts with some or many of the FAK-binding
partners. Indeed, tyrosine-phosphorylated PYK2 is able to
bind to the SH2 domain of Grb2 in a similar manner to
FAK, which is thought to lead to the activation of MAPK
pathway in PC12 cells (Lev et al., 1995
). While PYK2 is
believed to play a role in regulating neurotransmission or
neuroplasticity by phosphorylating potassium channels
(Lev et al., 1995
), other functions of PYK2 are still unidentified.
Materials and Methods
, kindly provided by T. Sasaki, Sapporo
Medical University, Sapporo, Japan), FAK, and FRNK were subcloned into expression vectors, either downstream of a c-myc epitope tag
(MEQKLISEEDL) under the control of the cytomegalovirus (CMV) promoter (pCMV-c-Myc; Evan et al., 1985
) or downstream of GST under the
control of the elongation factor 1
(EF1
) promoter (pEBG). The "FAT"
domain deletion mutant (PYK2
936-1009) was generated by inserting an
in-frame stop codon between XbaI site in PYK2 and the NheI site in
pCMV-c-Myc. The kinase and COOH-terminal domain deletion mutant
(PYK2
250-1009) was generated by inserting an in-frame stop codon between the Nsi site in PYK2 and the Nsi site in pCMV-c-Myc. NH2-terminal deletion mutants, the kinase inactive (lysine [K] 457 to alanine [A]),
and autophosphorylation site (tyrosine [Y] 402 to phenylalanine [F]) mutants were generated by PCR (Ho et al., 1989
). The chimeric constructs of
PYK2/FAK1 and PYK2/FAK2 were generated by in-frame ligation of individual PCR fragments. In construct PYK2/FAK1, the NH2-terminal domain of PYK2 (amino acids 2 to 385) was amplified by PCR and fused with the PCR fragments of the kinase and COOH-terminal domains of
FAK (amino acids 380 to 1052). In construct PYK2/FAK2, the NH2-terminal and kinase domain of PYK2 (amino acids 2 to 695) was ligated with
the COOH-terminal domain of FAK (amino acids 692 to 1052). The authenticity of all mutants was verified by DNA sequencing. The constructs
encoding constitutively active and inactive PI3 kinase and Akt were gifts
from Anke Klippel (Chiron Corporation, Emeryville, CA; Klippel et al.,
1996
).
-galactosidase in 1.6 ml of DME-serum free medium (GIBCO BRL). After incubation for 5 h, 1 ml of serum-containing DME media was added. 30 h
later, cells were fixed with 4% paraformaldehyde for immunostaining or fixed with 0.5% glutaraldehyde for
-galactosidase staining as described
below.
-galactosidase
staining or immunostaining of PYK2 using antibodies against PYK2 or the
c-Myc epitope tag. For
-galactosidase staining, fixed cells were washed
with PBS three times, each for 5 min, and incubated in PBS containing 20 mM K2Fe(CN)6, 20 mM K2Fe(CN)6.3H2O, 1 mM MgCl2, and 0.5 mg/ml
X-gal (5-bromo-4-chloro-3-indolyl-
-D-galactopyranoside) until a suitable
color developed (~1-2 h). For immunostaining, fixed cells were incubated
with the primary antibody against PYK2 (1:500 dilution) at 37°C for 1 h
and visualized with a fluorescence-conjugated anti-rabbit or anti-mouse
secondary antibody (1:300 dilution). Condensed or fragmented DNA was
detected in situ with propidium iodide or fluorescence-dUTP using terminal deoxynucleotidyl transferase (Apoptag) as described previously (Gabrieli et al., 1992
; Xiong and Montell, 1995
). These cells were double labeled
with anti-PYK2 antibodies to monitor the protein expression. Apoptotic
index was determined by counting the number of apoptotic cells, which expressed either
-galactosidase or PYK2, divided by the total number of
-galactosidase or PYK2-expressing cells. For each experiment, a minimum of 200 cells that expressed
-galactosidase or PYK2 was counted.
Each construct was examined at least three times.
Results
-galactosidase staining of cells
that were co-transfected with a reporter plasmid containing the cDNA encoding
-galactosidase (pCMV-
-gal).
As shown in Fig. 1, the transfection with a control pCMV
vector with or without pCMV-
-gal had no effect on cell
morphology (Fig. 1, A and B). However, rat-1 cells expressing the PYK2 protein, identified by both PYK2 or
Myc immunostaining or
-galactosidase staining, appeared
to be round with condensed cytoplasm, blebbed membrane, and poorly attached to the dish (Fig. 1, C and D).
These morphological changes were specific for PYK2-
expressing rat-1 cells, since the rat-1 cells overexpressing
FAK appeared similar to control cells (Fig. 1, E and F).
Fig. 1.
Morphological changes of rat-1 cells expressing PYK2.
Rat-1 cells were transiently transfected with pCMV vector (A
and B), pCMV-PYK2-WT (C and D), and pCMV-FAK (E and
F) plasmids without (A, C, and E) and with pCMV--galactosidase (B, D, and F). 30 h after transfection, the cells were fixed
with 4% paraformaldehyde and stained with antibodies against
c-Myc (9E10) epitope (A, C, and E), or fixed by 0.5% glutaraldehyde and stained for the
-galactosidase activity of the cells
cotransfected with pCMV-
-galactosidase (B, D, and F).
[View Larger Version of this Image (127K GIF file)]
; Muzio et al., 1996
). In parallel, the transfected cells
were also immunostained with either anti-PYK2 or anti-Myc (9E10) antibodies to monitor the protein expression.
Propidium iodide staining of cells expressing wild-type
PYK2 demonstrated condensed nuclei (Fig. 2, B and D).
This effect was not observed in cells transfected with vector alone (data not shown) or constructs encoding COOH-terminal PYK2 protein (PYK2
1-680; Fig. 3, A and C). In
addition, PYK2-expressing cells exhibited DNA fragmentation, detected using modified TUNEL (Fig. 2, F and H),
whereas no fragmentation was observed in cells transfected with the COOH-terminal PYK2, PYK2
1-680 (Fig.
2, E and G). The concomitant induction of cell death with
expression of wild-type PYK2 suggested that the expression of PYK2 may be directly responsible for the induction
of cell death. Similar morphological changes and apoptosis
were observed after transfection of PYK2 into mouse
10T1/2, swiss 3T3, quail QT6, and HEK 293 cells (data not
shown). In PC12 cells that express significant levels of endogenous PYK2, no detectable cell death was observed
when PYK2 was over-expressed (data not shown).
Fig. 2.
Apoptosis of rat-1 cells expressing PYK2. Rat-1 cells were
transiently transfected with PYK2 COOH-terminal (PYK21-680;
A, C, E, and G) and wild-type PYK2 (PYK2-WT; B, D, F, and H)
plasmids. 24-30 h after transfection, the cells were fixed and immunostained with anti-PYK2 antibodies and propidium iodide (A-D).
In a separate experiment, the transfected cells were immunostained using antibodies against PYK2 and labeled with fluorescence-conjugated dUTP using terminal transferase (E-H). Nuclei
of rat-1 cells overexpressing PYK2
1-680 with normal morphology stained uniformly with propidium iodide, indicating intact
nuclei (C, arrows). Propidium iodide staining of rat-1 cells overexpressing PYK2-WT showed apoptotic nuclei (condensed, indicated by arrows) in a fraction of cells (D). While there was no
labeling of fluorescence-conjugated dUTP in cells expressing
PYK2
1-680 with normal morphology (G), there was labeling of
dUTP in cells expressing PYK2-WT (H).
[View Larger Version of this Image (66K GIF file)]
Fig. 3.
Schematic diagrams of PYK2, PYK2 mutants, FAK,
and FAK mutants. The numbers represent the amino acid residues deleted from wild-type PYK2. The shaded block represents
the kinase domain of PYK2. The putative FAT domain and proline-rich sequences are indicated. The constructs tagged with either c-Myc or GST are also indicated. Apoptotic index (mean ± SEM) was determined by counting the apoptotic PYK2 or FAK-expressing cells (or PYK2 positive) divided by total number of
PYK2 or FAK expression cells, and listed in the right-hand column.
[View Larger Version of this Image (37K GIF file)]
-galactosidase reporter. The "apoptotic
index" for each mutant was determined by counting the
apoptotic blue cells or apoptotic PYK2-expressing cells
and dividing by the total number of transfected cells (total
number of blue or PYK2-expressing cells).
Fig. 4.
Apoptotic activities and Western blotting of PYK2,
PYK2 mutants, FAK, and FAK mutants. (A) Western blotting of
HEK 293 cells expressing proteins of PYK2 and PYK2 mutants
using antibodies against PYK2 and c-Myc epitope. The position
of protein molecular weight markers is indicated at the right. (B)
Western blotting of HEK 293 cells expressing proteins of PYK2,
FAK, and FAK mutants using antibodies against c-Myc epitope.
The position of protein molecular weight markers is also indicated at the right. (C) Histograms of the apoptotic index of cells
transfected with constructs encoding PYK2, PYK2 mutants,
FAK, and FAK mutants. Apoptotic index (mean ± SEM) was
determined by counting the apoptotic PYK2 or FAK-expressing cells (or PYK2-positive) divided by total number of PYK2 or
FAK expression cells.
[View Larger Versions of these Images (65 + 36K GIF file)]
1-88
and PYK2
1-416; Figs. 3 and 4, A and C), suggesting that
the NH2-terminal domain of PYK2 was required for maximal apoptotic activity. This conclusion was further supported by the observation that transfection of cells with
the NH2-terminal domain alone (PYK2
250-1009) was
able to induce significant cell death (0.55; Figs. 3 and 4 C).
We next determined if kinase activity was required for
PYK2-induced apoptosis. The kinase-inactive PYK2 (K457A
[PYK2-KD]), containing a lysine (K) to alanine (A) mutation in the ATP-binding site (Fig. 5 A), was transfected
into rat-1 cells. The apoptotic index of rat-1 cells expressing the kinase-inactive mutant was 0.6, a significant decrease from that of the wild-type, suggesting that PYK2
catalytic activity was required for the maximal apoptotic
activity (Fig. 5 B). This conclusion was further supported by the observation that the apoptotic index of rat-1 cells
expressing the autophosphorylation mutant PYK2-Y402F,
containing a tyrosine (Y) 402 to (F) mutant (Fig. 5 A), was
also significantly reduced to 0.41 (Fig. 5 B). In contrast to
the cells expressing PYK2, cells expressing wild-type FAK,
an autophosphorylation mutant (Y397F), or the COOH-terminal domain of FAK (FRNK) exhibited a low level of
apoptosis (apoptotic index of 0.1-0.3; Figs. 3 and 4 C). The
expression levels of PYK2, FAK, FAK-Y397F, and FRNK were similar based on Western blot analysis of extracts
from the transfected cells (Fig. 4 B). The transfection efficiencies using these constructs were similar (data not
shown).
Fig. 5.
Catalytic and apoptotic activity of wild-type and
mutant PYK2. (A) Schematic
representation of PYK2 and
its mutants. The numbers represent the amino acid residues mutated from wild-type
PYK2. The shaded block represents the kinase domain of
PYK2. The putative FAT
domain and proline-rich sequences are indicated. The
constructs tagged with c-Myc
are also indicated. The tyrosine
phosphorylation and the apoptotic index of these mutants are listed in the right-hand column. (B) Tyrosine
phosphorylation of wild-type
PYK2 (PYK2-WT), kinase-inactive PYK2 (PYK2-KD),
the putative FAT domain deletion mutant (PYK2936-1009),
the NH2-terminal domain
deletion mutants (PYK2
1-88
and PYK2
1-416), and the
autophosphorylation site mutant (PYK2-Y402F). Cell
lysates from 293 cells
overexpressing PYK2-WT, PYK2-KD, PYK2
936-1009,
PYK2
1-88, PYK2
1-416,
and PYK2-Y402F proteins
were immunoprecipitated by
antibodies against PYK2. The immunoprecipitatied proteins
were then subjected to immunoblotting with the antibodies against phosphotyrosine
(anti-P-tyr) or PYK2 (anti-PYK2). (C) Histograms of
apoptotic index of PYK2
936-
1009, PYK2
1-88, PYK2-WT,
PYK2-KD, PYK2
1-416, and
PYK2-Y402F.
[View Larger Version of this Image (55K GIF file)]
936-1009, did not inhibit induction
of apoptosis (apoptotic index of 0.99; Figs. 3 and 4), yet
this mutant protein failed to bind to paxillin and localize to
focal contacts (data not shown). Paradoxically, transfection of mutant cDNAs encoding the entire COOH-terminal
domain (PYK2
1-680) or cDNAs encoding only the COOH-terminal FAT domain (PYK2
1-781 and PYK2
1-868)
still retained low but reproducible apoptotic activity (Figs.
3 and 4). These data suggested that while expression of either the NH2 or COOH terminus alone could induce limited apoptosis, the full apoptotic activity required both an
intact NH2 terminus and active kinase domain.
1-88, PYK2
1-416, PYK2
936-1009, and PYK2-Y402F) by immunoblotting using antiphosphotyrosine antibodies. While the PYK2-WT was
highly tyrosine phosphorylated, the PYK2-KD failed to
react with antiphosphotyrosine antibodies (Fig. 5 B), indicating that PYK2-KD was kinase inactive. The autophosphorylation site of PYK2 may be tyrosine 402 (Y402), a
residue also conserved in FAK (Y397) that became phosphorylated upon FAK activation. The Y402 mutant (PYK2-Y402F) and the NH2-terminal deletion mutant PYK2
1-416,
in which the autophosphorylation site (Y402) was deleted,
also did not react with antiphosphotyrosine antibodies
(Fig. 5 B). Interestingly, the partial NH2-terminal deletion
mutant (PYK2
1-88) with reduced apoptotic index also exhibited reduced tyrosine phosphorylation (Fig. 5, B and C).
Fig. 6.
Apoptotic activity
of PYK2/FAK chimeric fusion proteins. (A) Schematic
representation of PYK2,
PYK2/FAK1, PYK2/FAK2,
and FAK. The shaded block
represents the regions of
PYK2. The open block represents the regions of FAK.
The numbers represent the
number of amino acids in
FAK or PYK2. The kinase
domain sequence is indicated. The constructs tagged
with c-Myc are also indicated. NH2-terminal domain
of PYK2 (amino acid 2 to
385) was fused with the kinase and COOH-terminal
domains of FAK (amino acids 380 to 1,052) in construct
PYK2/FAK1. In construct
PYK2/FAK2, PYK2 (amino
acids 2 to 695) was fused with
FAK (amino acids 692 to
1,052). The apoptotic index
of these PYK2/FAK chimeric proteins was listed in
the right-hand column. (B)
Western blotting of HEK 293 cells expressing PYK2, PYK2/FAK1, PYK2/FAK2,
and FAK proteins using antibodies against cMyc epitope,
goat anti-PYK2 (from Santa
Cruz Biotechnology; recognizing epitope in PYK2 NH2-terminal domain), or FAK
(2A7, recognizing epitope in
FAK COOH-terminal domain). (C) Histograms of the
apoptotic index of rat-1 cells
transfected with FAK-WT,
PYK2/FAK1, PYK2/FAK2, and PYK2-WT.
[View Larger Version of this Image (36K GIF file)]
). The extent of cell death (apoptotic index of 0.98) mediated by PYK2 in cells expressing catalytically inactive Src, was not appreciably different
from the parental 10T1/2 cells (Fig. 5). However, in the
SH2-defective c-Src cell line (dl155), the apoptotic index
was significantly reduced to 0.55 (Fig. 7). These results
suggested that the catalytic activity, but not the SH2 domain of c-Src, appeared to be required for the suppression
of PYK2-mediated apoptosis in fibroblasts.
Fig. 7.
Suppression of the PYK2-induced apoptosis by overexpression of catalytically active Src. Wild-type PYK2 was transiently transfected into different cell lines stably expressing wild-type Src and various Src mutants. (A) Immunostaining of PYK2
with anti-c-Myc antibodies (9E10 mAB) in rat-1, temperature-sensitive v-Src (ts v-Src, LA29), 10T1/2, c-Src (5HD47), kinase-dead c-Src (430), and SH2-defective c-Src (c-Src dSH2, dl155) cell
lines expressing wild-type c-Myc-tagged PYK2. (B) Histograms
of apoptotic index mediated by wild-type PYK2 in different Src
cell lines.
[View Larger Versions of these Images (120 + 25K GIF file)]
), were cotransfected in
rat-1 cells with wild-type PYK2. Co-expression of the constitutively active PI3 kinase (PI3K) significantly reduced the apoptotic effects of PYK2 (apoptotic index of 0.59;
Fig. 8). The reduction in PYK2-induced apoptosis was not
observed when the catalytically inactive PI3 kinase (PI3K
)
was cotransfected into rat-1 cells (Fig. 8), indicating that
the catalytic activity of PI3 kinase was required for suppression of apoptosis. The expression of catalytically active (PI3K) and inactive (PI3K
) PI3 kinases was examined by immunostaining using antibodies against the c-Myc epitope and p85 subunit of PI3 kinase.
Fig. 8.
Suppression of PYK2-induced apoptosis by overexpression of active PI3 kinase and Akt. Full length PYK2 was
cotransfected into rat-1 cells with constitutively active and inactive PI3 kinase (PI3K, PI3K) and active and inactive Akt (Akt,
Akt
; at 1:1 molar ratio of DNA). (A) Cotransfected cells were
doubly immunostained using antibodies against PYK2 (a, c, e,
and g), p85 subunit for PI3 kinase (b and d), and HA-epitope for
Akt proteins (f and h). (B) Histograms of the apoptotic index mediated by cotransfection of PYK2-WT with vector alone (1), active PI3 kinase (2), inactive PI3 kinase (3), active Akt (4), inactive Akt (5), crmA (6), and Bcl2 (7).
[View Larger Versions of these Images (82 + 36K GIF file)]
; Franke et al., 1995
). Activation of Akt can
protect many cells against apoptosis, whereas inhibition of
Akt does not (Dudek et al., 1997
; Kauffmann-Zeh et al.,
1997
; Kulik et al., 1997
). To further confirm the suppressive effects of PI3 kinase in PYK2-induced apoptosis, we
determined if activated Akt was able to protect cells
against PYK2-induced apoptosis. The plasmids encoding
HA-tagged constitutively active Akt/PKB, in which Akt
was targeted to the membrane by myristoylation, or catalytically inactive Akt were cotransfected in rat-1 cells with
wild-type PYK2. Co-expression of the active Akt significantly reduced the apoptotic effects of PYK2 (apoptotic
index of 0.58; Fig. 8), whereas catalytically inactive Akt
did not (Fig. 8). These data demonstrated that active Akt
was able to at least partially block PYK2-induced apoptosis. In addition, we also examined the effects of other cell
death suppressors on PYK2-induced apoptosis. CrmA, an
ICE-like proteinase inhibitor, was able to efficiently suppress PYK2-induced cell death (apoptotic index of PYK2
was reduced to 0.4, when crmA was cotransfected with
wild-type PYK2; Fig. 8 B). In contrast, overexpression of
Bcl2, another cell death suppressor, did not block the
PYK2-induced cell death (Fig. 8 B).
Discussion
). Thus we
speculate that apoptosis induced by forced expression of
the chimeric proteins, the NH2-terminal, and COOH-terminal domains of PYK2 may result from the activation of
signaling pathways that lead to the apoptotic process, or/ and the inhibition of signaling pathways whose continuity
is required to maintain cell viability. What these pathways
might be remains unclear, particularly in light of the paucity of data regarding the function of PYK2 in different
cell types.
; Franke et al., 1995
). The fact that Akt can substitute for activated PI3 kinase in protection of
PYK2-induced apoptosis lends further support for the role
of PI3 kinase signaling in protection of PYK2-induced apoptosis. Finally, v- and c-Src also partially suppress PYK2-mediated apoptosis. The fact that PI3 kinase is activated
by Src (Fukai and Hanafusa, 1989
) and that catalytically
active Src is required for rescue of PYK2-induced apoptosis, leads us to speculate that activation of PI3 kinase by
Src may be a possible mechanism for Src-mediated rescue.
Thus one possible mechanism by which PYK2 might induce apoptosis is by inhibiting PI3 kinase either directly
(perhaps by a phosphorylation-dependent mechanism) or
indirectly by negatively regulating an upstream effector of
PI3 kinase activation. This suggestion is supported by recent observations that PI3 kinase activity is required for PC12 cell survival mediated by growth factors including
NGF and PDGF (Valius and Kazlauskas, 1993
; Yao and
Cooper, 1995
) and that active Akt is able to rescue the
apoptosis induced by c-Myc over-expression in fibroblasts
(Kauffmann-Zeh et al., 1997
), serum withdrawal in neuronal cells (Dudek et al., 1997
), and UV light in Cos-1 cells
(Kulik et al., 1997
).
; Salgia
et al., 1996
; Sasaki et al., 1995
). Elevated expression of
PYK2 has been reported in GN4, a transformed rat liver
epithelial cell line (Yu et al., 1996
). In PC12 and GN4 cells,
PYK2 activation appears to be linked to neurotransmitters, growth factors, and hormones that induce alterations
in calcium-dependent signaling pathways (Lev et al., 1995
;
Yu et al., 1996
). The cell lines used in this study (rat-1, mouse 10T1/2, and HEK 293 cells) exhibit low levels of endogenous PYK2 expression. Therefore it is unlikely that
PYK2 plays a role in regulating apoptotic pathways in
these cells. More likely is the possibility that PYK2 expression in these cells is mediating an inappropriate regulation
of an existing signaling pathway (such as PI3 kinase), leading to the perturbation of a signaling pathway required for
normal cell viability. Interestingly, no apoptosis was observed when PYK2 was overexpressed in PC12 cells where
there is significant level of endogenous PYK2. Whether
PYK2 can function to trigger apoptosis in cells in which it
is normally expressed remains to be examined.
Received for publication 20 March 1997 and in revised form 8 July 1997.
Address all correspondence to J. Thomas Parsons, Department of Microbiology, Box 441, Health Science Center, University of Virginia, Charlottesville, VA 22908. Tel.: (804) 924-5395. Fax: (804) 982-1071. E-mail: jtp{at}virginia.eduWe are grateful to Dr. T. Sasaki for providing cDNA of CAK, to Dr. A. Klippel for providing constructs of PI3 kinase and Akt, to Dr. S. Parsons
for providing cell lines transfected with c-Src and c-Src variants, to Dr. M. Weber for providing ts v-Src rat-1 cell line (LA29), and to Drs. Z.J. Luo
and J.Y. Wu for suggestions and constructs of Bcl2. We thank Z.-H.
Wang, M. Macklem, and C. Borgman for excellent technical assistance.
We also thank Drs. L. Mei, S. Weed, R. Malink, M. Hart, and G. Kulik for
helpful comments on the manuscript and discussions.
These studies were supported by Department of Health and Human Services grants CA 40042 and CA 29243 to J.T. Parsons; W.C. Xiong is supported by National Institutes of Health National Research Service Award fellowship NS 09918.
ECM, extracellular matrix; FAK, focal adhesion kinase; FRNK, FAK-related nonkinase; FAT, focal adhesion targeting domain; GST, glutathione-S-transferase; PI3 kinase, phosphatidylinositol-3-kinase.
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