From the Yale University School of Medicine, New
Haven, Connecticut 06510, the ¶ Fox Chase Cancer Center,
Philadelphia, Pennsylvania 19111, and the
Department of
Medicine, Pulmonary, Allergy, and Critical Care Division, University of
Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15213
Received for publication, June 13, 2002, and in revised form, December 12, 2002
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ABSTRACT |
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Keratinocyte growth factor (KGF), a member of the
fibroblast growth factor (FGF) family (also known as FGF-7), is an
important protective factor for epithelial cells. The receptor for KGF
(also called FGFR2-IIIb), which has intrinsic tyrosine kinase activity, is expressed specifically on epithelial cells and in the lung epithelium. Administration of KGF has been shown to protect the lung
from various insults, but the mechanism of protection is not well
understood. To understand the mechanism by which KGF exerts protective
functions on epithelial cells, we used the yeast two-hybrid assay to
identify proteins that interact with the KGF receptor (KGFR). Here we
show that the cytoplasmic domain of KGFR interacts with p21-activated
protein kinase (PAK) 4, which is a new member of the PAK family. The
PAKs are regulated by the Rho-family GTPases Rac and Cdc42. PAK4 is
the most divergent member of the PAK family of proteins and may have
distinct functions. However, stimuli that regulate PAK4 activity are
not known. Our data show that PAK4 can associate with the KGFR, which
is dependent on KGFR tyrosine kinase activity. We show that a dominant
negative mutant of PAK4 blocks KGF-mediated inhibition of caspase-3
activation in epithelial cells subjected to oxidant stress. Our data
demonstrate that PAK4 is an important mediator of the anti-apoptotic
effects of KGF on epithelial cells.
Fibroblast growth factors
(FGFs)1 are a large family of
growth factors that control cell proliferation, differentiation, and organ development (1-5). The activities of FGFs are mediated by
binding to receptors that have tyrosine kinase activity. To date, four
FGF receptors have been described, namely FGFR1, FGFR 2, FGFR 3, FGFR
4, and their splice variants (3-5). Each FGF receptor contains an
extracellular domain composed of 2-3 immunoglobulin (Ig)-like loops, a
transmembrane segment, and an intracellular domain with tyrosine kinase
activity. In the case of FGFR1-3, the third Ig loop is encoded by two
exons, an invariant exon called IIIa that is spliced to either exon
IIIb or IIIc, yielding receptors with distinct ligand binding
abilities. FGFR2-IIIb binds FGF1, FGF3, FGF7 (also called keratinocyte
growth factor or KGF), and FGF10 (6, 7). It is expressed specifically
on epithelial cells and is diffusely expressed in the day 11 lung (8).
Gene-targeting studies have revealed an indispensable role for this
receptor in mouse lung development. Transgenic mice expressing a
dominant negative mutant of FGFR2-IIIb under the control of a
lung-specific promoter exhibit embryonic lethality with grossly
abnormal lung development with only two primordial epithelial tubes and
no branching morphogenesis (9). Similarly, mice with homozygous
mutations in FGFR2-IIIb are viable until birth but show severe defects
with no evidence of lung formation (10). A key observation in these mice is that although the growth of organs such as lungs and limbs is
initiated, further development is not sustained in the face of
extensive apoptosis in the tissues (10).
Interactions between FGFR2-IIIb and its ligands are just as important
in adults. For example, FGF7/KGF has been shown to be essential in
wound reepithelialization after skin injury (11). The administration of
KGF has been shown to protect the lungs of animals from a variety of
insults, including oxygen (12), radiation, chemotherapy (bleomycin)
(13, 14), and acid (15). However, the detailed downstream signaling
pathways that contribute to KGF-FGFR2-IIIb-mediated cell protection are
not well understood.
Here we show that FGFR2-IIIb interacts with a protein that shares 86%
identity with a member of the PAK family of kinases, namely PAK4,
previously identified in human cells (16). PAK proteins were originally
described as downstream targets for the Rho GTPases Rac and Cdc42 (17,
18). The PAK proteins can be essentially divided into two categories,
Groups I and II, based on their structures (19). In Group I are the
proteins PAK1, PAK2, and PAK3. Each of these proteins has an
amino-terminal regulatory domain and a carboxyl-terminal kinase domain.
The regulatory domain also includes the GTPase-binding
domain, which mediates binding to Rac and Cdc42. These PAK
members also bind to SH3 domain-containing adapter proteins such as Nck
through the proline residues in the regulatory domain (17, 18). The
carboxyl terminus interacts with yeast G protein
Interaction Cloning of Murine PAK4--
Duplex-ATM
yeast two-hybrid system (OriGene Technologies, Inc., Rockville, MD) was
used to identify KGFR-interacting proteins. The KGFR cytoplasmic domain
containing the tyrosine kinase domain was cloned into the yeast
expression plasmid pEG202-NLS, and the resulting bait construct was
named pNEGKGFRc. Expression of the cytoplasmic domain of the KGFR in
the yeast strain EGY194 led to autophosphorylation of the receptor as
judged by immunoprecipitation with an anti-phosphotyrosine antibody and
Western blotting with an anti-KGFR antibody. We first ensured that the
bait construct, pNEGKGFRc containing the LexA-KGFRc fusion, did not
activate reporter genes due to autoactivation. Autoactivation of the
reporter gene lacZ was checked by co-transforming EGY194
with pNEGKGFRc and the reporter plasmid pSH18-34. No activation of
lacZ was observed with the LexA-KGFRc fusion protein. To
identify KGFR-interacting proteins, EGY194 was first transformed with
pNEGKGFRc and pSH18-34, and the pretransformed EGY194 was next
transformed with a cDNA expression library constructed from
cDNA derived from a 19-day-old post-coital mouse embryo fused to
the B42 activation domain HA-tagged expression vector pJG4-5. After
selecting clones potentially expressing interacting proteins, the
specificity of the interaction was confirmed by a yeast-mating assay.
The expression plasmid isolated from the putative positive clone was
introduced into EGY194 (a strain), and a combination of the bait
plasmid (pNEGKGFRc) and reporter plasmid (pSH18-34) was introduced
into EGY40 ( Plasmids--
The chimeric trk/KGFR plasmid was kindly provided
by Martin Sachs (Akt-r-29; MDC Max-Delbruck Center for Molecular
Medicine, Berlin, Germany). The mutagenesis of the tyrosine residues of trk-KGFR was performed using the QuikChangeTM site-directed
mutagenesis method (Stratagene). The sequences of the primers used for
mutagenesis were as follows: (i) trk/KGFR(Y542F,Y543F), 5'-GGGATATCAACAACATAGA CttctttAAAAA GACCA CAA A TGGGCG-3' and 5'-CGCCCATTTGTGGTCTTTTTaaagaaGTCTATGTTGTTGATATCCC-3'; (ii)
trk/KGFR(Y352F), 5'-CCACTTTGGATCCTCTGGCAACTCaaaCTCGGAGACCCCTGC-3' and
5'-GCAGGGGTCTCCGAGtttGAGTTGCCAGAGGATCCAAAGTGG-3'; and (iii)
trk/KGFR(Y655F), 5'-CTCACAACCAATGAGGAAttcTTGGATCTCACCCAGCC-3' and
5'-GGCTGGGTGAGATCCaagAATTCCTCATTGGTTGTGAG-3'. Plasmids for the
mammalian expression of HA-tagged full-length mPAK4 (pHA-mPAK4) and its
amino-terminal (pHA-mPAK4/NT) domains were constructed by subcloning
full-length or truncated cDNA encoding the amino-terminal domain of
PAK4 into the NotI/Asp718 sites of the
appropriate pCruz-HA vector for cloning in the correct reading frame
(Santa Cruz Biotechnology).
Immunoprecipitation and Western Blotting--
To express the
protein(s) of interest, the constructs were either transfected
individually or co-transfected into HEK293 cells. 36 h
post-transfection, cells were starved in serum-free Dulbecco's modified Eagle's medium for 3 h and then stimulated with or
without KGF (100 ng/ml, Roche Molecular Biochemicals) or NGF (100 ng/ml, Roche Molecular Biochemicals), depending on whether we wished to
stimulate the endogenous receptor or the chimeric receptor in
trk-KGFR-transfected cells. Cells were then lysed in
immunoprecipitation (IP) buffer containing protease inhibitors (20 mM Tris, pH 7.5, 150 mM NaCl, 1 mM
EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM
sodium pyrophosphate, 1 mM Cloning of Murine PAK4--
To identify proteins that interact
with FGFR2III-b/KGFR, we used the yeast two-hybrid system to screen for
proteins that interact with the kinase (cytoplasmic) domain of KGFR. A
murine embryonic cDNA library was screened for possible
interactions with a chimeric bait comprising LexA and the cytoplasmic
domain of KGFR. Among the several positive clones identified in this
assay, three independent clones contained DNA fragments with open
reading frames that were highly homologous to the carboxyl terminus of
human PAK4. The corresponding full-length cDNA was cloned using
5'-RACE. Fig. 1A shows the
predicted amino acid sequence of the full-length cDNA. Domain
search revealed the presence of a p21 Rho-binding domain (PBD) and a
protein kinase domain (PKD) in hPAK4 (GenBankTM Accession
number NM_005884). The amino acid sequence was found to share 86%
identity with that of hPAK4 (Fig. 1B) and less than 50%
identity, only in the PKD domain, with the corresponding domain present
in murine PAK1-3. We therefore consider this to be murine PAK4 (mPAK4;
GenBankTM Accession number AY217016).
PAK4 Associates with KGFR in Mammalian Cells--
We investigated
whether endogenous KGFR and PAK4 associate with each other in HEK293
epithelial cells. HEK293 cell extracts were immunoprecipitated with
anti-KGFR antibody or control immunoglobulin, and analysis of the
immunoprecipitates by immunoblotting with anti-human PAK4 revealed the
presence of PAK4 in immunoprecipitates obtained with the anti-PAK4 but
not the control Ig (Fig. 2A). The association between the two proteins was detected in the absence of
KGF and was not appreciably augmented upon treatment of the cells with
KGF. Interaction between these two proteins was also detected when
HA-tagged murine PAK4 was used (Fig. 2B). To further explore
interactions between KGFR and PAK4, we expressed a chimeric receptor
containing the extracellular ligand-binding domain of NGF (Trk) and the
cytoplasmic domain of KGFR, which we termed trk-KGFR. The rationale for
using the chimeric receptor was that, because epithelial cells do not
express the NGF receptor, treatment of cells with NGF would allow us to
specifically monitor the activity of the hybrid receptor without
interference from the endogenous KGF receptor. Also, in the absence of
an effective anti-KGFR antibody that could be used for
immunoprecipitation, the anti-TrkA antibody was particularly useful. As
shown in Fig. 2C, at a lower level of trk-KGFR expression,
NGF-stimulation was required for co-immunoprecipitation of trk-KGFR and
PAK4 (Fig. 2C). However, at higher levels of expression of
the chimeric receptor, the association was readily detectable with
(Fig. 2C) or without (not shown) NGF stimulation. Because receptor tyrosine kinases are known to undergo ligand-independent activation (autophosphorylation) due to dimerization when the protein
is overexpressed, it is possible that phosphorylation augments or
stabilizes association between PAK4 and KGFR. We also found that the
tyrosine residues 542 and 543, corresponding to tyrosine residues 653 and 654 in FGFR1 (which are autophosphorylation sites of KGFR), are
critical for the association with PAK4 (Fig 3A). However, mutations of
tyrosine residues 352 and 655 corresponding to tyrosine residues 463 and 766 in FGF receptor 1, the putative binding sites for Shc and
PLC- Grb2 Participates in the Association between mPAK4 and
KGFR--
Grb2 is an adapter protein that associates with receptor
tyrosine kinases and couples the activated receptor to downstream signaling molecules such as Ras (28, 29). The PAK family members have
been shown to associate with the adapter protein Nck, which has
similarities to Grb2 (30, 31). However, PAK4, the sequence of which is
the most divergent of all PAK proteins, does not associate with Nck
(16). We therefore investigated whether PAK4 has the ability to
associate with Grb2. Because Nck and PAK1 have been shown to form a
complex in the absence of growth factor stimulation (32), we
investigated whether PAK4 exists in a complex with Grb2 in unstimulated
cells. As shown in Fig. 4A,
anti-Grb2 antibody-immunoprecipitated PAK4 and KGF had no effect on
Grb2-PAK4 association. We also investigated whether endogenous KGFR
associates with Grb2. Immunoprecipitation of HEK293 cell extracts with
anti-Bek but not control Ig led to co-immunoprecipitation of Grb2 as
revealed by immunoblotting of the immunoprecipitates with anti-Grb2
(Fig. 4B). If anti-Bek and anti-Grb2 have comparable
affinities for their respective proteins, then a comparison of the
amount of Grb2 co-immunoprecipitated with KGFR with the net Grb2
obtained by immunoprecipitation with monoclonal anti-Grb2 shows that a
small fraction of Grb2 is complexed with KGFR in the cells, which
probably reflects the level of KGFR expression in the cells. Also, we
detected similar levels of Grb2 in the immunoprecipitate whether cells
were left with or without KGF. To determine the importance of specific
phosphorylation sites for KGFR-Grb2 association, HEK293 cells were
transfected with expression vectors for trk-KGFR (wild type or
individual mutants). We uniformly treated all cells with NGF to achieve
maximum receptor activation, because overexpression of the wild type
receptor consistently resulted in basal autophosphorylation and,
therefore, only small differences between stimulated and unstimulated
cells were expected (Fig. 3). As shown in Fig. 4C, the wild
type receptor associated with Grb2 and the autophosphorylation site of
the receptor was critical for this association. Again, the other
tyrosine residues, Tyr352 and Tyr655, did not
affect association with Grb2 and, similar to what was observed with
PAK4, the mutation of Tyr352 actually augmented association
of Grb2 with the receptor. The receptor-Grb2 complex also contained
PAK4 (Fig. 4D). Because the endogenous KGFR was found to
associate with both PAK4 and Grb2 in the absence of KGF stimulation of
cells, and because for both associations the autophosphorylation sites
in the KGFR were found to be critical for association, it is possible
that the endogenous KGFR (at least a fraction of the receptor) in
HEK293 cells exists in an autophosphorylated state. Also, because Grb2
is widely expressed in many species, including yeasts, it is possible
that the association between KGFR and PAK4 is indirect and mediated by
Grb2, because Grb2 has been shown to bind to receptor tyrosine kinases
in yeast two-hybrid assays. Also, we were unable to
co-immunoprecipitate KGFR and PAK4 when the recombinant proteins were
expressed in bacteria, which reinforces our suspicion that Grb2
mediates association between the receptor and PAK4.
PAK4 Is Phosphorylated by Ligand-activated
trk/KGFR--
The association between PAK4 and KGFR (Fig.
1) prompted us to investigate whether PAK4 undergoes tyrosine
phosphorylation in response to receptor activation. As shown in Fig.
5, activation of the hybrid receptor with
NGF induced tyrosine phosphorylation of PAK4, which was clearly evident
in cells that expressed higher levels of the chimeric receptor. The
phosphorylation status paralleled that of the chimeric receptor. The
bottom panel of Fig. 5 demonstrates equivalent
expression of HA-PAK4 in all cells.
Dominant Negative Mutant of PAK4 (PAK4/NT) Prevents
KGF-mediated Inhibition of Oxidant-induced Poly(ADP-ribose) Polymerase
(PARP) Cleavage--
Because our data suggested an important role for
PAK4 in cellular protection by KGF, we investigated whether
interference of PAK4 function would influence anti-apoptotic functions
induced by KGF in HEK293 cells. For this purpose, we treated HEK293
cells with H2O2, which has been shown to induce
pro-apoptotic pathways in these cells (33). Because apoptosis is
associated with caspase-3 activation, a focal point of different
pro-apoptotic pathways, we investigated the state of protein PARP, a
substrate of caspase-3, in the cells under different conditions of
treatment. Also, to inhibit the functions of endogenous PAK4, we
overexpressed the N-terminal fragment of PAK4 lacking the kinase
domain, the inactivation of which imparts dominant negative functions
to PAK4 (16, 34). As shown in Fig. 6,
treatment of cells with H2O2 promoted cleavage of the PARP protein. Cleavage of PARP was inhibited in the presence of
the caspase-3 inhibitor DEVD-fluoromethylketone or KGF. However, when
the PAK4 dominant negative mutant was expressed in the cells, KGF was
unable to inhibit PARP cleavage. Furthermore, Fig. 6C shows
that the PAK4 mutant is able to interact with the KGF receptor. By
binding to the receptor, the mutant is probably able to disrupt KGFR
signaling to downstream pathways.
In this study, we have identified PAK4 as a KGFR-interacting
protein using a yeast two-hybrid assay. We show that this interaction has functional relevance, because expression of a dominant negative mutant of PAK4 prevented the inhibition of oxidant-induced caspase-3 activation by KGF in epithelial cells. It is interesting to note that
just as mice deficient in the KGF receptor show organ malformation with
evidence of enhanced apoptosis of developing organs such as the lung
(9, 10), Drosophila lacking the protein Mushroom Body Tiny
(MBT), the closest known homolog of PAK4, also shows defects in organ
development with a reduced number of Kenyon cells in a structure in the
brain called the mushroom body (35).
Human PAK4 was identified as the most divergent member of the PAK
family of proteins (16). The mouse PAK4 we cloned shares 86% homology
with hPAK4, with greatest identity in the p21 Rho-binding domain and
the protein kinase domain. In addition to its role as a cytoskeletal
regulatory protein, PAK4 has been shown to induce anchorage-independent
growth and promote cell survival in response to different stimuli such
as tumor necrosis factor It has been reported previously that PAK1 is recruited to growth factor
receptors such as the epidermal growth factor (EGF) and the
platelet-derived growth factor (PDGF) receptors through association
with Nck (37, 38). However, unlike other PAK proteins, PAK4 does not
interact with Nck. We show that Grb2, an adaptor protein similar to
Nck, associates with PAK4. Grb2 is recruited to ligand-activated growth
factors, which results in targeting of the Ras guanylnucleotide
exchange factor SOS to the plasma membrane location of Ras, which, in
turn, promotes Raf-1 recruitment and activation of the MAP kinase
pathway (28, 29, 39-41). In our studies, we show that Grb2 associates
with PAK4 and the chimeric trk-KGFR. The association between Grb2 and
PAK4 may involve the SH3 domains in Grb2 and the proline-rich
PXXP residues in the p21-binding domain in PAK4. Thus,
although independent studies have implicated both molecules, KGF and
PAK4, in protection from cell death, our study connects them in a novel
signaling axis that may have critical protective functions during organ
development in the embryonic stage and during injury in adults.
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-subunits, which suggests that these PAK proteins may be
regulated by heterotrimeric G proteins in mammalian cells. In Group II
are PAK4 (16), PAK5, and PAK6 (19). Although overall human PAK4
resembles other PAK members in that it also contains an amino-terminal
GTPase-binding domain and a carboxyl-terminal kinase domain, it
lacks a G protein
-binding domain or the ability to bind to Nck
(16). Even within the GTPase-binding domain and the kinase domain, it
shares only 50% similarity with other PAK members (16). Although a
role for PAK proteins in the regulation of cytoskeletal organization is
well described, PAK proteins, particularly those belonging to Group II,
have been implicated in other distinct cellular processes (19). For
example, PAK6 has been shown to bind to the androgen receptor and
repress androgen receptor-mediated gene transcription in the presence
of the ligand androgen (20). The different members of the PAK family
have been shown to have different roles in the apoptotic response of cells. Although PAK2 promotes apoptosis (21, 22), PAK1 has been shown
to protect cells from apoptosis induced by growth factor withdrawal
(23, 24). Recently, PAK4 was shown to protect cells from apoptosis
induced by multiple stimuli, including serum withdrawal, tumor necrosis
factor-
(TNF
) treatment, or UV irradiation (25). Although PAK4
has been shown to be a target for Cdc42, extracellular stimuli that
regulate endogenous PAK4 activity have not been identified. Here, using
the yeast two-hybrid assay, we have identified an association between
the KGFR and PAK4. Our studies suggest that this association may play
an important role in the protective effects of KGF on epithelial cells.
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-strain) and used in the mating test. Appropriate
controls were used in the mating assay. Among the positive clones,
three independent clones contained sequences that matched with the
sequence of human PAK4. Full-length cDNA of mPAK4 was cloned by
5'-RACE (Clontech). The mouse lung cDNA library
(Clontech) was applied as a template in touchdown PCR using PfuTurbo (Stratagene), which amplified the
full-length mPAK4 gene. The PCR product was subcloned into a TOPO
vector (Invitrogen). SP6 and T7 primers were used for sequencing and,
after sequence confirmation, the full-length cDNA was named mPAK4.
-glycerolphosphate, 1 mM Na3VO3, and the complete
protease-inhibitor mixture from Roche Molecular Biochemicals, catalog
number 1697498). Protein concentration in the cell extracts was
detected and measured using the DCTM protein assay kit
(Bio-Rad, catalog number 500-0112). In general, the appropriate
polyclonal antibody coupled with Sepharose was used for
immunoprecipitation, and either unconjugated mouse monoclonal antibody
or enzyme-conjugated antibody from the same species was used for
immunoblotting. Cell extracts and the appropriate antibody coupled to
Sepharose beads were incubated together at 4 °C for 12-16 h.
Immunoprecipitates were collected by centrifugation and washed fours
times with IP buffer. The immunoprecipitates were resolved by SDS-PAGE,
transferred to polyvinylidene difluoride membrane (Millipore), and
probed with the appropriate antibodies. The antibodies used were
anti-HA affinity matrix and anti-HA-peroxidase high affinity
antibodies, catalog numbers 1815016 and 2013819 respectively (Roche
Molecular Biochemicals), anti-Bek (KGFR) antibody (SC122, Santa Cruz
Biotechnology), anti-Grb2 rabbit polyclonal antibody (SC-255AC, Santa
Cruz Biotechnology), anti-Grb2 monoclonal antibody (SC8034, Santa Cruz
Biotechnology), anti-phosphotyrosine (pY) antibody pY99 (SC7020 AC,
Santa Cruz Biotechnology), and anti-TrkA antibody against the
extracellular domain of TrkA (06574, Upstate Biotechnology). Blots were
visualized by enhanced chemiluminescence.
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Fig. 1.
Sequence of murine PAK4 and homology with
human PAK4. A, nucleotide and putative amino acid
sequence of murine PAK4. The p21 Rho-binding domain (amino acids
11-47) and the kinase domain (serine/threonine and tyrosine kinase
catalytic domain, amino acids 327-578) are underlined.
B, alignment of the amino acid sequences of murine and human
PAK4.
, respectively, with FGF receptor 1 (26-28), did not affect
association with PAK4. It is to be noted that, compared with the wild
type receptor, these mutant receptors displayed reduced basal
autophosphorylation, which may be due to reduced receptor dimerization
when these residues are mutated.
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Fig. 2.
Association of mPAK4 with endogenous KGFR and
trk/KGFR. A, association between endogenous KGFR and
PAK4 proteins in HEK293 cells. HEK293 cells were serum starved for
3 h and left with or without KGF (100 ng/ml; Roche Molecular
Biochemicals) for 30 min. Cell extracts were immunoprecipitated with
control Ig (con. IG) or anti-KGFR, and immunoprecipitates
were analyzed by immunoblotting with anti-human PAK4 (Cell Signaling).
The blot was stripped and reprobed with anti-KGFR. Cell extracts were
also directly analyzed with anti-PAK4 to assess PAK4 expression in the
cells. B, association of HA-murine PAK4 (HA-mPAK4) with
endogenous KGFR. HEK293 cells were transfected with HA-tagged mPAK4
(pHA-mPAK4). 36 h post-transfection, cells were stimulated with
KGF (100 ng/ml) for 30 min after 3 h of serum deprivation. Cells
were lysed, and 0.4 mg of protein in the lysates was used for IP with
anti-HA-coupled agarose (Roche Molecular Biochemicals). The
immunoprecipitates were analyzed by Western blotting techniques using
anti-KGFR (Santa Cruz Biotechnology). The membrane was stripped and
reprobed with anti-HA to demonstrate co-immunoprecipitation of
HA-mPAK4. C, Co-immunoprecipitation of trk/KGFR and mPAK4.
HEK293 cells were transiently co-transfected with ptrk/KGFR (1.25 or
2.5 µg) and pHA-mPAK4. 0.4 mg of cell extract protein was used for
immunoprecipitation with anti-HA (anti-HA was coupled to agarose beads,
Roche Molecular Biochemicals). The immunoprecipitates were analyzed by
immunoblotting with anti-trkA (Upstate Biotechnology). The membranes
were stripped and reprobed with anti-HA (Roche Molecular
Biochemicals).
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Fig. 3.
Association of mPAK4 with different mutant
forms of trk/KGFR. HEK293 cells were co-transfected with pHA-mPAK4
and ptrk/KGFR or the indicated mutants (mut). 36 h
post-transfection, cells were serum deprived and then left with or
without NGF (100 ng/ml; Roche Molecular Biochemicals) for 30 min. 0.4 mg of lysate protein was used for IP with anti-HA-coupled agarose
(Roche Molecular Biochemicals). The immunoprecipitates were analyzed by
immunoblotting with anti-Bek (Santa Cruz Biotechnology). The membrane
was stripped and reprobed with anti-HA to demonstrate expression of
HA-mPAK4. The bottom panel shows direct Western
blot analysis with anti-trkA (Upstate Biotechnology) demonstrating
expression of all of the mutant proteins in the cells. wt,
wild type.
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Fig. 4.
Grb2 associates with both mPAK4 and
trk/KGFR. A, HEK293 cells were transfected with
pHA-mPAK4 and stimulated with KGF as described in the legend to Fig. 2.
0.4 mg of protein present in the cell extracts was used for IP with
anti-Grb2-coupled agarose (Santa Cruz Biotechnology) and analyzed by
immunoblotting with anti-HA. The membrane was stripped and reprobed
with anti-Grb2 (Santa Cruz Biotechnology). B, association
between KGFR and endogenous Grb2 in HEK293 cells. Cells were serum
starved, left with or without KGF, and cell extracts were
immunoprecipitated with control Ig, anti-KGFR, or anti-Grb2. The
immunoprecipitates were analyzed by immunoblotting (Iblot)
with anti-Grb2. C, association between KGFR mutants and
Grb2. HEK293 cells were transfected with ptrk/KGFR or its different
mutant forms and stimulated with NGF as described in the legend to Fig.
3. 0.4 mg of cell extract protein was used for IP with
anti-Grb2-coupled agarose and analyzed by immunoblotting with anti-trkA
(Upstate Biotechnology). The membrane was stripped and reprobed with
anti-Grb2. D, 0.3 mg of protein extract from cells
transfected with ptrk-KGFR and pHA-PAK4 was immunoprecipitated with
anti-Grb2. As shown, sequential probing with anti-trkA and anti-HA and
anti-Grb2 showed evidence of the presence of all of the three proteins
in the immunoprecipitate.
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Fig. 5.
mPAK4 is phosphorylated through binding to
trk/KGFR. Samples from the experiment shown in Fig. 2 were used
for IP with anti-HA, and the immunoprecipitates were analyzed for
phosphorylation by immunoblotting with anti-phospho Tyr (pY99) (Santa
Cruz Biotechnology). The bottom panel shows
expression of HA-mPAK4 in all samples.
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Fig. 6.
Dominant negative PAK4 (PAK4/NT) prevents
inhibition of PARP cleavage by KGF. HEK293 cells were incubated in
serum-containing medium with or without H2O2
(control). The caspase-3 inhibitor DEVD-fluoromethylketone was added at
a concentration of 200 nM 1 h before the addition of
H2O2 (1 mM). 24 h before
treatment with H2O2, some cells were
transfected with pHA-mPAK4/NT. KGF (50 ng/ml) was added to the cells
1 h prior to the addition of H2O2. Cell
extracts were made 4 h after H2O2
stimulation. A, PARP cleavage was assessed by Western
blotting as an indicator of cellular apoptosis. -actin expression
was used as a loading control. B, densitometric analysis of
PARP cleavage. C, interaction between KGFR and mPAK4/NT.
HEK293 cells were transfected with trk-KGFR together with empty vector
or expression constructs encoding HA-tagged full-length or mutant
mPAK4. Extracts were immunoprecipitated with anti-HA antibody, and the
immunoprecipitates were analyzed by sequential immunoblotting with
anti-trkA and anti-HA. The experiment was performed twice with similar
results.
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and UV irradiation. Although important
functions of PAK4 have been identified, factors that stimulate PAK4
activity have heretofore not been identified. The KGFR-PAK4
association, as identified in this study, provides a link between
extracellular stimuli and endogenous PAK4 activity. We also show that
the KGFR-PAK4 complex is dependent on the tyrosine kinase activity of
the KGFR and that PAK4 undergoes tyrosine phosphorylation upon receptor
activation. In previous studies, tyrosine phosphorylation of PAK1 was
reported in v-ErbB-transformed cells, and dephosphorylation of PAK led
to a reduction in PAK kinase activity (36). Interestingly, tyrosine
phosphorylation of PAK was not Ras-, Rac- or
Cdc42-dependent, which led to the suggestion that this
activity of PAK1 may be distinct from growth factor-induced mitogenic
activity (36). Similarly, PAK4 was also shown to promote
anchorage-independent growth (34), and both PAK1 and PAK4 have been
shown to possess anti-apoptotic functions and cause phosphorylation and
inactivation of the death-promoting protein Bad (23-25).
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ACKNOWLEDGEMENT |
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We thank Dr. A. Ray for helpful suggestions and critical reviewing of the manuscript.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grants HL 60207 and HL 69810 (to P. R.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AY217016.
§ These three authors contributed equally to this manuscript.
** To whom correspondence should be addressed: Dept. of Medicine, Pulmonary, Allergy, and Critical Care Medicine, University of Pittsburgh School of Medicine, 3459 Fifth Ave., MUH 628 NW, Pittsburgh, PA 15213. Tel.: 412-802-3192; Fax: 412-692-2260; E-mail: rayp@pitt.edu.
Published, JBC Papers in Press, January 15, 2003, DOI 10.1074/jbc.M205875200
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ABBREVIATIONS |
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The abbreviations used are: FGF, fibroblast growth factor; KGF, keratinocyte growth factor; PAK, p21-activated protein kinase; HA, hemagglutinin A; RACE, rapid amplification of cDNA ends; KGFR, KGF receptor; HEK293, human embryonic kidney 293; IP, immunoprecipitation; NGF, nerve growth factor; PARP, poly(ADP-ribose) polymerase; SH3, Src homology 3.
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