From the Department of Molecular Biology, Osaka
Bioscience Institute, Suita, Osaka 565-0874, Japan and the
§ Graduate School of Biostudies, Kyoto University, Sakyoku,
Kyoto 606-8502, Japan
Received for publication, July 5, 2000, and in revised form, October 24, 2000
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ABSTRACT |
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p21-activated kinases (PAKs) are implicated in
integrin signalings, and have been proposed to associate with paxillin
indirectly. We show here that paxillin can bind directly to PAK3. We
examined several representative focal adhesion proteins, and found that paxillin is the sole protein that associates with PAK3. PAK3 associated with the p21-activated kinases
(PAKs)1 are localized to
focal adhesions and implicated in integrin signalings, regulating cell
morphology, polarity, and motility (1). PAKs were identified as
serine/threonine kinases whose activity is regulated by the small
GTPases, Rac and Cdc42 (2-9). Mammalian PAKs consist of at least three
isoforms (PAK1, PAK2, and PAK3), whose size and sequence are highly
homologous to one another (reviewed in Ref. 10). Mammalian PAKs show
significant sequence homology to Ste20p/PAK-like kinases in lower
eukaryotes, which play a role in regulating actin-based morphogenic
events (reviewed in Refs. 1 and 11). In mammalians, PAKs have been implicated in a number of different intracellular processes, including cytoskeletal regulation (12-17), stimulation of mitogen-activated protein kinase cascades (7, 8, 18-20), and control of the phagocyte
NADPH oxidase (4).
The small GTPases Rac and Cdc42 are not the sole activators of the PAK
kinase activity. It has been shown that several lipids, particularly
sphingolipids, activate PAK1 independent of the action of Rac or Cdc42
(21). The kinase activity of PAKs is also regulated through interaction
with the guanine-nucleotide exchange factor PIX/Cool family proteins
(reviewed in Ref. 22). It has been shown that PAK1 is activated through
interaction with Paxillin acts as a scaffolding adaptor protein in integrin signaling,
by binding to several other integrin-assembly proteins including
vinculin, integrin It has been shown that PAK1 is well co-localized with paxillin at
Cdc42- and Rac1-driven small substrate adhesions at the cell periphery,
called focal complexes (23); or co-localized with integrin at focal
adhesions (43). Turner et al. (44) have shown the
association of PAK3 with paxillin. They reported that this association
is not mediated by the direct interaction of these two proteins, but
mediated through the aid of two other proteins. They showed that
paxillin directly binds to p95PKL, which also binds to To analyze the mechanism involved in the recruitment and activation of
the PAK kinases in integrin signaling, we here examined the binding of
PAKs to focal adhesion proteins. Consistent with a previous report by
Turner et al. (44), we found that paxillin associated with
PAK3. On the other hand, other focal adhesion proteins, including Fak,
Pyk2, vinculin, talin, tensin, and p130Cas, did not
exhibit a significant association with PAK3. Among paxillin isoforms,
the Cells and Tissue--
3Y1 and COS7 cells were cultured with
Dulbecco's modified Eagle's medium (with 4.5 g of glucose/liter)
supplemented with 5 and 10% fetal calf serum (Hyclone Laboratories), respectively.
For preparation of cell extracts from neonatal rat brain (NRB),
neonatal Wistar strain rats (postnatal days 1-3) purchased from Nippon
Doubutsu (Osaka, Japan) were killed by decapitation, and whole brains
were removed quickly and frozen at cDNA Clones and Expressions--
Rat PAK1 cDNA was a
gift from K. Mizuno (Tohoku University), and a polymerase chain
reaction (PCR) amplified using oligonucleotides 5'-ATGGATCCTCAAATAACGGCTTAGACGTCCAGGAC-3' and
5'-ATGCGGCCGCCTAGTGATTGTTCTTGGTTGCCTCTTTTGC-3'. The resulting
BamHI-NotI fragment was subcloned into pEBG (46) vector to be fused in-frame to the COOH terminus of glutathione S-transferase (GST) protein. Mouse PAK3 cDNA was
isolated from first strand cDNAs, prepared from adult male BALB/c
mouse brain (CLONTECH), by the PCR amplification
method using oligonucleotides 5'-ATGGATCCATGTCTGACAGCTTGGATAACGAAGAAA-3' and
5'-ATGCGGCCGCTTAACGGCTACTGTTCTTAATTGCTTCCTTTG-3'. The resulting
BamHI-NotI PAK3 cDNA fragment was subcloned
into each of pEBG to be fused in-frame to the COOH terminus of GST protein, or pEFBOS-HA (47) to be tagged with the HA-tag at its NH2 terminus. Deletion mutants for PAK3 were then
constructed using the following oligonucleotides:
5'-ATGGATCCATGTCTGACAGCTTGGATAACGAAGAAA-3' and
5'-ATGCGGCCGCCTATTTCTTCTTTGGGTCCCCAACACTCAC-3' for the M1 mutant
encompassing amino acids (1-267 aa),
5'-ATGGATCCAAAGAGAAAGAGCGCCCAGAGATCTCTCTTCC-3' and
5'-ATGCGGCCGCCTAGGCACTTTTATCTCCTGACGTAAAGCTC-3' for M2 (63-147 aa),
5'-ATGGATCCGACAATGAACCTCCGCCTGTCATTGC-3' and
5'-ATGCGGCCGCCTAGGAGCGAGTATAGATTGATTTTG-3' for M3 (184-205 aa),
5'-ATGGATCCTATACGAGATTGGAAAAAATTGGCCAAGGG-3' and
5'-ATGCGGCCGCTTAACGGCTACTGTTCTTAATTGCTTCCTTTG-3' for M4 (268-544 aa),
5'-ATGCGGCCGCCTAGGAGCGAGTATAGATTGATTTTG-3' and
5'-ATGCGGCCGCCTATTTCTTCTTTGGGTCCCCAACACTCAC-3' for M5 (184-267 aa),
5'-ATGGATCCATGTCTGACAGCTTGGATAACGAAGAAA-3' and
5'-ATGCGGCCGCCTACTTCTTATTGGTTTTATCCCCTCCTCCTGG-3' for M6 (1-62 aa),
and 5'-ATGGATCCCATGGATACATAGCAGCACATCAGTCG-3' and
5'-ATGCGGCCGCCTATTTCTTCTTTGGGTCCCCAACACTCAC-3' for M7 (148-267 aa).
The resulting BamHI-NotI PCR fragment for the M1
mutant was ligated into pEBG, and other
BamHI-NotI PCR fragments for the M2-M7 mutants
were each ligated into pGEX4T-3 (Amersham Pharmacia Biotech)
vector, to be fused in-frame to the COOH terminus of GST protein. The
autoinhibitory domain of PAK3 (78-146 aa), corresponding to the
similar region of PAK1 (83-149 aa) that has been shown to act as
inhibitor for PAK1 in vivo (16, 48), was synthesized using
oligonucleotides 5'-ATGGATCCCATACGATTCATGTGGGTTTTGATGCAGTC-3' and
5'-ATGCGGCCGCCTAACTTTTATCTCCTGACGTAAAGCTC-3', and the resulting PCR BamHI-NotI fragment was ligated into pEBG
vector to be fused in-frame to the COOH terminus of GST protein.
Human
DNA fragment encoding the 1-7-amino acid portion of paxillin fused
in-frame to the COOH terminus of His-tag was made by hybridizing oligonucleotides of
5'-GATCTCCGGCCATGCATCACCATCACCATCACGACGACCTCGACGCCCTGC-3' and
5'-GGGCGTCGAGGTCGTCGTGATGGTGATGGTGATGCATGGCCGGA-3', and the resulting fragment was ligated into the
BglII-BglI site of pVL1392/paxillin
Mouse Nck cDNA was a gift from H. Hanafusa (Osaka Bioscience
Institute), and amplified using oligonucleotides
5'-ATGGATCCATGGCTGAAGGAGTGGTGGTGGTGGCC-3' and
5'-ATGGATCCTCAAGACAAATGCTTGACGAG-3'. The resulting
BamHI-BamHI fragment was ligated into pGEX2TK
vector, to be fused in-frame to the COOH terminus of GST protein.
Nucleotide sequences were confirmed with all these plasmids after the
construction. pEFBOS-HA-Cdc42 G12V was a gift from S. Kuroda and K. Kaibuchi (Nara Institute of Science and Technology).
Proteins encoded by pGEX vectors were expressed in Escherichia
coli by induction with
isopropyl-
COS7 cells were transfected with plasmid DNAs using DEAE-dextran
method, and cell extracts were prepared with 1% Nonidet P-40 buffer
(1% Nonidet P-40 (Nonidet P-40), 150 mM NaCl, 20 mM Tris-HCl (pH 7.4), 5 mM EDTA, 1 mM Na3VO4, 1 mM
phenylmethylsulfonyl fluoride, 1% aprotinin, 2 µg/ml leupeptin and 3 µg/ml pepstatin A), as previously described (50).
Antibodies--
Anti- Protein Binding and Immunoblotting Analyses--
For in
vitro protein binding analysis, cell lysates prepared by 0.5%
Triton X-100 buffer, or purified protein preparations which were
dialyzed against 0.5% Triton X-100 buffer, were mixed with GST fusion
proteins bound to glutathione-Sepharose beads, incubated for 2 h
at 4 °C and then washed four times with 0.5% Triton X-100 buffer.
For in vivo protein binding analysis, 300 µg of COS7 cell
lysates prepared by 1% Nonidet P-40 buffer were mixed with 5 µl of
glutathione-Sepharose beads, incubated for 2 h at 4 °C. Beads
were then washed four times with 1% Nonidet P-40 buffer. For
immunoblotting, protein samples retained on glutathione beads were
boiled in Laemmli's SDS sample buffer, separated on 8% SDS-PAGE,
transferred to membrane filters (Immobilon P, Millipore), and subjected
to immunoblotting analysis as previously described (50). Antibodies
retained on the filter membranes were visualized by horseradish
peroxidase-conjugated secondary antibodies (Jackson ImmunoResearch)
coupled with an enzyme-linked chemiluminescence method according to the
manufacturer's instructions (Amersham Pharmacia Biotech).
In Vitro Kinase Assays--
Protein precipitants with
glutathione-Sepharose beads were washed four times in 1% Nonidet P-40
buffer and then twice in a kinase buffer (50 mM Tris-HCl
(pH 7.4), 10 mM MgCl2, 10 mM
MnCl2, 0.2 mM dithiothreitol). The beads were
then resuspended in 20 µl of the kinase buffer containing 2 µg of
myelin basic protein (MBP) (Sigma), and incubated for 20 min at
30 °C in the presence of 10 µCi of [ In Vitro Phosphorylation and Phosphoamino Acid Analysis--
To
prepare paxillin Association of PAKs with Paxillin, but Not Other Focal Adhesion
Proteins--
The PAK kinases are localized to focal complexes and
focal adhesions, and stimulated upon integrin activation (23, 28, 29,
43). To understand the mechanism for the regulation of the PAK kinases
at focal adhesions, we examined several known focal adhesion proteins
for their binding to PAKs. We prepared GST fusion forms of PAK1 and
PAK3 proteins produced in COS7 cells; and proteins co-precipitated from
mammalian cell lysates with these GST fusion proteins were analyzed. As
shown in Fig. 1A, paxillin was
clearly co-precipitated with PAK1 and PAK3 from extracts of 3Y1 cells
and NRB tissues, both expressed PAK1 and PAK3 endogenously (see Fig.
1B). On the other hand, other focal adhesion proteins we
examined, including tensin, talin, p130Cas, Fak,
Pyk2, and vinculin, were not detected to be co-precipitated with these
recombinant PAK proteins from either cell lysate (Fig. 1A).
Paxillin consists of two isoforms ( Direct Binding of Paxillin
These observations prompted us to investigate whether paxillin The NH2 Terminus Domain of PAK3 Is Responsible for the
Binding to Paxillin Paxillin
Nck has also been shown to bind to the P1 motif of PAK1, via the SH3
domain of Nck (30, 34). The amino acid sequence of the P1 motif is
highly conserved in all three PAK isoforms, and we thus examined
whether paxillin Paxillin
It has been shown that paxillin is associated with some cellular
serine/threonine kinases whose identities were unknown (54). The
autoinhibitory domain of PAK1 has been shown to act as a specific inhibitor for the PAK1 kinase activity (16, 48). To confirm that the
kinase activities co-precipitated with GST-paxillin Paxillin We showed in this paper that among representative focal adhesion
proteins, paxillin is the sole protein that can associate with PAK1 and
PAK3. Moreover, our results revealed that the These biochemical properties of paxillin Turner et al. (44) were the first to show the association of
paxillin with PAK3. However, they could not detect direct binding of
paxillin with PAK3 in vitro, and instead proposed that PAK3 associates indirectly with paxillin through the aid of other proteins, p95PKL and Paxillin consists of three isoforms ( Three isoforms (PAK1, PAK2, and PAK3) of PAKs have been identified in
mammals. PAK1, the first isolated PAK (2), has been most throughly
studied. Because size and sequence are highly conserved among PAK
isoforms, most of the biochemical properties as well as the possible
physiological functions ascribed to PAK1 pertain to the other isoforms
(reviewed in Refs. 1, 10, and 22), although there may be also some
important differences. For example, PAK3 is released from Rac1
following activation, whereas PAK1 and PAK2 remain bound to this GTPase
(2, 3, 6). Our analysis was performed mostly with PAK3, but we also
showed that paxillin The results described in this paper, however, do not preclude a complex
formation of the four proteins, paxillin Additional signaling seems to be required for the activation of the
paxillin-bound PAK3. We showed that the catalytically active form of
PAK3 can stay associated with paxillin and
isoforms of paxillin, but not with
. We also show that paxillin
associated with both the kinase-inactive and the
Cdc42-activated forms of PAK3 in vivo, without affecting the activation states of the kinase. A number of different functions have been ascribed to PAKs; and PAKs can bind directly to growth factor
signaling-adaptor molecule, Nck, and a guanine nucleotide exchanger,
PIX. Our results revealed that paxillin
can compete with Nck and
PIX in the binding of PAK3. Moreover, paxillin
can be
phosphorylated by PAK3 at serine. Therefore, paxillin
, but not
,
appears to be capable of linking both the kinase-inactive and activated
forms of PAK3 to integrins independent of Nck and
PIX, as Nck links
PAK1 to growth factor receptors. Our results also revealed that
paxillin is involved in highly complexed protein-protein interactions
in integrin signaling.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
PIX/p85Cool-1 even in the absence of direct binding
to Cdc42 (23).
PIX/Cool-2 also binds to activate PAK3 kinase (24).
On the other hand, binding of p50Cool-1, a splice variant of
PIX/p85Cool-1, to PAK3 represses the activation of PAK3 by upstream
activators such as Dbl (25). Accordingly, the kinase activity of
cellular PAKs has been shown to be regulated by a variety of external
stimuli that act through cell surface receptors, including G
protein-coupled receptors (4), growth factor receptor tyrosine kinases
(26), proinflammatory cytokine receptors (18), Fc receptors (27), and
integrins (28, 29). However, PAK kinase may not be constantly associated with these cell surface molecules. It has been shown that
growth factor stimulations, such as by epidermal growth factor and
platelet-derived growth factor, recruit PAK1 to the activated receptors, whereby PAK kinase appears to be activated (26, 30). Nck, an
adaptor molecule recruited to the activated growth factor receptors
(31, 32), has been shown to bind to PAK1, and implicated to serve as a
link connecting the kinase activity of PAK1 to the receptor signaling
pathways (30, 34, 35). A similar scenario has been proposed in
photoreceptor guidance receptor signaling in Drosophila
(36). However, the molecular mechanisms involved in the recruitment and
activation of PAKs in most of the other receptors have not been elucidated.
1, Fak, and c-Src (reviewed in Refs. 37 and 38). Paxillin is tyrosine phosphorylated upon integrin activation (39), and thus creates binding sites for several Src
homology 2 domain-containing proteins such as Crk-I, Crk-II, Crk-L, and
Csk (reviewed in Ref. 38). Tyrosine phosphorylation of paxillin has
been shown to be important for cell spreading on the extracellular
matrix (40), cell cycle progression (39), as well as
adhesion-dependent function of leukocytes (41). Paxillin is
composed of multiple isoforms,
,
, and
(42). These isoforms exhibit different biochemical properties in their binding to other proteins such as vinculin and Fak. For example, both the
and
isoforms bind to Fak whereas the
isoform does not (42).
PIX; and
proposed a model in which paxillin associates with PAK3 through three
steps: paxillin binds to p95PKL, p95PKL then binds to
PIX, and
PIX finally binds to PAK3 (44).
and
isoforms significantly associated with PAK3, whereas
the association with PAK3 was only marginally detectable with the
isoform. In contrast to the model previously proposed (44), however, we
found that paxillin
could bind directly to PAK3. We also showed
that paxillin
could be phosphorylated by the Cdc42-activated PAK3.
Our results collectively indicates that paxillin
fulfills several
biochemical properties required to serve to link PAK3 to integrin
signaling, which have been ascribed to Nck in linking PAK1 to growth
factor receptor signaling (30, 34, 35, 45). Moreover, we found that
paxillin
associates with both the kinase-inactive and the
Cdc42-activated forms of PAK3 in vivo; and that the paxillin
binding to PAK3 could be competitive with the
PIX binding to
PAK3 in vivo and in vitro. It has been shown that
PIX binding to PAK3 activates the kinase activity of PAK3 (23);
thus, according to the previous model for the association of PAK3 with
paxillin (44), only the
PIX-bound and thereby activated form of PAK3
seems to be associated with paxillin. Therefore, our results indicate
that the association of paxillin with PAK3 through the aid of p95PKL
and
PIX (44) may not be the sole way of the interaction of paxillin
with PAK3. On the other hand, our model of the direct binding of PAK3
to paxillin
even in the absence of
PIX is consistent with the recent finding that the kinase-inactive and/or nonautophosphorylated form of PAK1 is preferentially localized to focal adhesions (45).
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
70 °C until use. Tissue lysates
were then prepared by homogenization of NRB in 5 volumes of an ice-cold
0.5% Triton X-100 buffer (50 mM Tris-HCl (pH 7.6), 50 mM NaCl, 1 mM EGTA, 2 mM
MgCl2, 0.1% mercaptoethanol, 0.5% Triton X-100)
containing protease inhibitors (1% aprotinin and 1 mM
phenylmethylsulfonyl fluoride) using a Dounce-type glass homogenizer,
followed by clarification by 15,000 × g for 10 min at
4 °C.
PIX cDNA (KIAA0142) was a gift from T. Nagase (Kazusa DNA
Research Institute), and amplified using oligonucleotides 5'-ATGGATCCATGACCGACAATAGCAACAATCAACTGGTAG-3' and
5'-ATGGATCCCGTCCCTTATAGATTGGTCTC-3'. Resulting
BamHI-BamHI fragment was ligated into pQE-30
(Qiagen) and pcDNA3.1 His-C (Invitrogen) vectors, to be tagged with
six consecutive histidines (His-tag) at its NH2 terminus.
and
pVL1392/paxillin
plasmids (49), to make paxillin
and paxillin
proteins each tagged with the His-tag at their NH2 termini, respectively. PCR-amplified DNA fragments which each corresponds to the LD1 (1-20 aa), LD2 (141-160 aa), LD3 (212-231 aa), LD4 (263-282 aa), and LD5 (299-317 aa) motifs of human paxillin
were subcloned into the BamHI-EcoRI site of
pGEX-2TK vector, each to be fused in-frame to the COOH terminus of the
GST protein, as previously reported (44). The LD4 motif-deletion mutant
of paxillin
was synthesized using oligonucleotides
5'-ATGGATCCATGGACGACCTCGACGCCCTGCTGGCGGAC-3', and
5'-ATGCCGGCAGAGGAGGCCGAGATGCGTGTCTGCTG-3'; and
5'-ATGCCGGCAAGACAGGGAGCAGCTCACCCCCTGGG-3', and
5'-ATGCGGCCGCCTAGCAGAAGAGCTTGAGGAAGCAGTTCTG-3'. Resulting BamHI-NaeI and NaeI-NotI
PCR fragments were simultaneously ligated into pEBG vector to be fused
in-frame to the COOH terminus of the GST protein. pAcG2T (Pharmingen)
plasmid containing human paxillin isoform cDNAs to be fused
in-frame to the COOH terminus of GST were described previously
(49).
-D-thiogalactopyranoside, and proteins encoded
by the pEBG vector were expressed in COS7 cells, as described
previously (49). These GST-tagged proteins were then subjected to
purification using glutathione-Sepharose beads, as previously described
(50). Proteins encoded by the pQE-30 vector were expressed in E. coli by induction with
isopropyl-
-D-thiogalactopyranoside. These His-tagged
proteins were then subjected to purification using nickel beads
according to the manufacturer's protocol (Qiagen) and further
fractionated on Mono Q column using Biologic System (Bio-Rad), if
necessary. Nck was purified as followed; GST-Nck was cleaved by
thrombin protease at room temperature for 2 h and then purified by
Mono Q column chromatography using the Biologic System. Proteins
encoded by the pAcG2T and pVL1392 vectors were expressed in Sf9
cells using the baculovirus system according to the manufacturer's
instructions (Pharmingen).
PIX antibody was prepared by immunizing
rabbit with the GST fusion form of the NH2-terminal half of
human
PIX/p85Cool-1, which corresponds to the entire region of
p50Cool-1. Anti-paxillin antibody was previously described (42).
Antibodies against the following proteins were purchased from
commercial sources: PAK1 and PAK3 from StressGen; talin and vinculin
from Sigma; Nck, Fak, and Pyk2 from Transduction Labs; tensin from
Chemicon; p130Cas from Upstate Biotechnology; and
HA-tag sequence and GST-tag sequence from BAbCO.
-32P]ATP
(6,000 Ci/mmol; Amersham Pharmacia Biotech). The reactions were
terminated by boiling in Laemmli's SDS sample buffer, and samples were
visualized by separating on 12% SDS-PAGE followed by autoradiography.
protein which was not contaminated with
serine/threonine kinase activities, His-paxillin
bound to nickel
beads was incubated in a denaturing buffer (6 M guanidine HCl, 0.1 M Na phosphate (pH 8.0)) at room temperature for
1 h, which was then followed by sequential incubation with a
renaturing buffer A (3 M guanidine HCl, 0.1 M
Na phosphate (pH 8.0), 5 mM dithiothreitol), a renaturing
buffer B (1.5 M guanidine HCl, 0.1 M Na
phosphate (pH 8.0), 5 mM dithiothreitol), and a
renaturing buffer C (100 mM NaCl, 0.1 M Na
phosphate (pH 8.0), 5 mM dithiothreitol). Each incubation
with renaturing buffers was carried out at 4 °C for 1 h.
His-paxillin
was then subjected to elution from the nickel beads
according to the manufacturer's instruction (Qiagen), and dialyzed
against 1% Nonidet P-40 buffer. GST-PAK3 bound to glutathione beads
was washed four times in 1% Nonidet P-40 buffer and then twice in the
kinase buffer. The beads were then resuspended in 20 µl of the kinase
buffer containing the "kinase-free" His-paxillin
and incubated
for 20 min at 30 °C in the presence of 10 µCi of
[
-32P]ATP (6,000 Ci/mmol). The reactions were
terminated by boiling the sample in Laemmli's SDS sample buffer, and
samples were then separated on 8% SDS-PAGE. After excision of the
radiolabeled band from the gel, phosphoamino acid analysis was
performed as described (51), where 1000 cpm of the radioactivity was
separated on cellulose thin-layer plates electrophoresis with pH 3.5 buffer (10:1:189 mixture of glacial acetic acid, pyridine, and
water) at 1000 V for 60 min, after being mixed with phosphoamino acid
standards (Sigma). 32P-Labeled phosphoamino acids were then
identified by autoradiography and phosphoamino acid standards were
identified by spraying the place with ninhydrin as described (51).
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (57K):
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Fig. 1.
Association of PAK1 and PAK3 with the
isoforms of paxillin, but not other representative
focal adhesion proteins in vitro. A,
PAK1 and PAK3 associate with paxillin but not other representative
focal adhesion proteins. 600 µg of 3Y1 (lanes 2-4) or NRB
(lanes 6-8) lysates, prepared in 0.5% Triton X-100 buffer,
were incubated with 5 µg of GST-PAK1 (lanes 2 and
6), GST-PAK3 (lanes 3 and 7), or GST
alone (lanes 4 and 8), each coupled with
glutathione-Sepharose beads. After washing, proteins bound to the beads
were separated on SDS-PAGE and subjected to sequential immunoblotting
analysis using antibodies against focal adhesion proteins, as
indicated. Antibodies retained on filter membranes were visualized by
the enzyme-linked chemiluminescence method. 20 µg of total cell
lysates of 3Y1 (lane 1) and NRB (lane 5) were
also included (total). Controls were without cell lysates
(lanes 9-11). GST-PAK1 and GST-PAK3 were produced in COS7
cells, and GST was in E. coli; and purified on glutathione
beads before use, as described under "Experimental Procedures."
B, association of paxillin isoforms with PAK1 and PAK3. 600 µg of 3Y1 (lanes 2-5) or NRB (lanes 7-10)
lysates as above were incubated with 5 µg of GST-paxillin
(lanes 2 and 7), GST-paxillin
(lanes
3 and 8), GST-paxillin
(lanes 4 and
9), or GST alone (lanes 5 and 10),
each coupled with glutathione-Sepharose beads. After washing, proteins
bound to the beads were subjected to separation on SDS-PAGE and
sequential immunoblotting analysis as above using antibodies against
PAK1 and PAK3. 20 µg of total cell lysates of 3Y1 (lane 1)
and NRB (lane 6) were also included (total).
Controls were without cell lysate (lanes 11-14). All GST
proteins of paxillin isoforms were produced in the baculovirus system
and purified on glutathione beads before use, as described under
"Experimental Procedures."
and
) in rodents and three
isoforms (
,
, and
) in human (42, 52). Using GST fusion proteins of each paxillin isoform produced in the baculovirus system,
we found that the
and
isoforms exhibited significant associations with PAK1 and PAK3, whereas the association with the PAKs
was very weak and only marginally detectable with the
isoform (Fig.
1B). We could not find cell extracts of human origin
expressing both PAK1 and PAK3, and thus used rat 3Y1 cell extracts for
this analysis.
to PAK3--
It has already been
reported that paxillin is associated with PAK3 (44). This association,
however, has been proposed to be not mediated by the direct interaction
of these two proteins, but mediated with the aid of p95PKL and
PIX
proteins: paxillin binds to p95PKL, p95PKL binds to
PIX, and
PIX
binds to PAK3 (44). It has been shown that the LD4 motif of paxillin is
responsible for the direct binding of paxillin to p95PKL, and therefore
also responsible for the association of paxillin with PAK3 and
PIX (44). Using GST fusion proteins of the full-length paxillin
and the
LD4 motif, we compared amounts of PAK3 and
PIX co-precipitated with
these fusion proteins. We found that almost equal amounts of PAK3 were
precipitated with the full-length paxillin
and with the LD4 motif,
under the experimental conditions shown in Fig.
2A. However, reblotting the
same membrane filter with an anti-
PIX antibody revealed that the
amount of
PIX co-precipitated with the full-length paxillin
was
much higher than that with the LD4 motif. The same results were
obtained with both NRB and 3Y1 cell lysates (Fig. 2A) in
repeated experiments; indicating a possible independent behavior of
PAK3 and
PIX in their association with paxillin
.
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Fig. 2.
Direct binding of PAK3 to paxillin
in vitro, in the absence of
PIX. A, independent behavior of
PAK3 and
PIX in their association with paxillin
and its LD4
motif. 600 µg of NRB (lanes 2-8) or 3Y1 (lanes
10-12) lysates were incubated with 5 µg of GST fusion proteins
of the full-length paxillin
(FL; lanes 7 and
11) or its LD motifs (lane 2, LD1 (1-20 aa); lane
3, LD2 (141-160 aa); lane 4, LD3 (212-231 aa);
lanes 5 and 10, LD4 (263-282 aa); and lane
6, LD5 (299-317 aa)) or GST alone (lanes 8 and
12), each bound to glutathione beads. After washing,
proteins retained on the beads were separated on SDS-PAGE and subjected
to sequential immunoblotting analysis using antibodies against PAK3 and
PIX. Lanes 1 and 9 included 20 µg each of
the total cell lysates of NRB and 3Y1, respectively (total).
Controls were without cell lysate (lanes 13-19). The GST
fusion form of the full-length paxillin
was produced in the
baculovirus system, and the GST fusion forms of the LD motifs were
produced in E. coli; and purified on glutathione beads
before use. Note that
PIX appeared as multiple bands in NRB lysates
as previously shown (23, 44), while it showed a single band in 3Y1
lysates, as indicated by arrowheads. B, direct
binding of PAK3 to paxillin
in vitro. His-paxillin
(100 ng), which was produced in the baculovirus system, purified on
nickel beads and then eluted, was incubated with 500 ng of GST-PAK3
(lane 3), expressed in COS7 cells and purified on
glutathione-Sepharose beads, or GST alone (lane 4), each
bound to glutathione beads. After washing, proteins bound to the beads
were separated on SDS-PAGE and subjected to immunoblotting analysis
using anti-paxillin antibody. Lane 2 included 10 ng of the
purified His-paxillin
. Immunoblotting of the same membrane filter
with anti-
PIX antibody was also performed to show that the binding
was conducted essentially free from the contamination of
PIX
(lower panel); where 10 ng of purified His-
PIX (see Fig.
3A) was included in lane 1. Controls were without
His-paxillin
(lanes 5 and 6). Coomassie
Brilliant Blue (CBB) stainings of each purified preparation
of GST-PAK3 (lane 7) and His-paxillin
(lane
8) are also shown, and protein bands corresponding to each
purified protein are indicated by asterisks. Molecular sizes
are shown on the left.
could bind to PAK3, independent of
PIX. We prepared His-tagged paxillin
, which was expressed in the baculovirus system, purified on nickel beads, and then eluted; and GST-PAK3, which was expressed in
COS7 cells and purified on glutathione-Sepharose beads (Fig. 2B). Using these purified preparations of 100 ng of
His-paxillin
and 500 ng of GST-PAK3, we tested whether paxillin
and PAK3 bound directly. As shown in Fig. 2B, more than 10%
of His-paxillin
was recovered with the GST-PAK3 bound to
glutathione-Sepharose beads. Contamination of
PIX in these protein
preparations as well as in the GST-PAK3 pull-down fraction was minimal
(Fig. 2B, lower lanes). Therefore, paxillin
appears to
be capable of binding to PAK3 directly in vitro, even in the
absence of
PIX. Such direct binding was also detected between
GST-PAK1 and His-paxillin
(data not shown).
--
We next determined the structural basis
for the direct binding of paxillin
to PAK3. The coding sequence of
PAK3 contains four potential Src homology 3 (SH3) binding
Pro-X-X-Pro motifs (where X is any
amino acid; P1 to P4), the Cdc42/Rac-binding domain termed the
p21-binding domain (65-128 aa), an autoinhibitory domain overlapping with the p21-binding domain, an acidic region, the PIX/Cool-binding region (184-205 aa), and the kinase domain (268-523 aa) (7, 23) (see Fig. 3). We prepared
various deletion mutants of PAK3 as shown in Fig. 3, each fused to GST,
and expressed in COS7 cells (full-length and the M1 mutant) or in
E. coli (the M2-M7 mutants), and purified on
glutathione-Sepharose beads. Each 5 µg of these PAK3 peptides bound
to the beads was then incubated with 500 ng of purified His-paxillin
or with 200 ng of purified His-
PIX, which were produced in the
baculovirus system and in E. coli, respectively. As shown in
Fig. 3, upper lanes, the M1 and M6 mutants, but not other
mutants, bound to His-paxillin
, with almost equal affinities seen
with the full-length PAK3; suggesting that the NH2-terminal
domain of PAK3 containing the P1 and P2 motifs is primarily responsible
for the binding to paxillin
. Contamination of
PIX in these
protein preparations as well as in the GST-PAK3 pull-down fraction was
minimal (Fig. 3, middle lanes). On the other hand, the M1,
M3, M5, and M7 mutants, but not the M2, M4, and M6 mutants, bound to
His-
PIX, confirming that our preparation of recombinant His-
PIX
binds to the 184-205 aa region of PAK3 (Fig. 3, lower
lanes), as has been previously reported (23). Each 10 ng of
PIX
and paxillin
proteins was included in Fig. 3, lanes 1 and 2, respectively; and judging from the intensities of the
chemiluminescence signals from paxillin
and
PIX co-precipitated
with PAK3, the binding affinity of
PIX toward PAK3 seemed to be at
least severalfold higher than that of paxillin
toward PAK3.
View larger version (31K):
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Fig. 3.
Paxillin binds to
the NH2-terminal region of PAK3. His-paxillin
(500 ng) or His-
PIX (200 ng) was incubated with 5 µg each of GST fusion
forms of the full-length PAK3 (FL; lane 3), the deletion
mutants (M1-M7 in lanes 4-10), or GST alone
(lane 11), each bound to glutathione beads. After washing,
His-paxillin
proteins bound to the beads were subjected to
immunoblotting analysis using anti-paxillin (upper lanes),
and His-
PIX proteins bound to the beads were subjected to
immunoblotting analysis using anti-
PIX antibody (lower
lanes). Possible contamination of
PIX in these protein
preparations as well as in the GST-PAK3 pull-down fraction was assessed
(middle lanes). Lanes 1 and 2 included
10 ng each of His-
PIX and His-paxillin
, respectively
(authentic control). Controls were also GST fusion proteins
only (lanes 12-20). Each of the deletion mutants of PAK3 is
shown at the bottom panel, where each number
corresponds to amino acid residues of mouse PAK3 (7) (see
"Experimental Procedures" and text for details). GST fusion forms
of the full-length PAK3 and the M1 mutant were expressed in COS7 cells,
and all the other mutants (M2-M7) were produced in E. coli;
and all purified on glutathione beads before use. His-paxillin
was
the same purified preparation as in Fig. 2. His-
PIX was expressed in
E. coli, and purified on nickel beads. Coomassie Brilliant
Blue (CBB) staining of His-
PIX is shown in the
middle panel (lane 21), where a protein band
corresponding to His-
PIX is indicated by an asterisk.
Molecular sizes are shown on the left.
Binding to PAK3 Competes with
PIX and Nck Binding
to PAK3--
We next examined the interaction among paxillin
,
PAK3, and
PIX. GST-PAK3 (250 ng) bound to glutathione-Sepharose
beads was incubated with His-paxillin
(250 ng) in the presence of increasing amounts of His-
PIX (0-800 ng). All of these protein preparations were purified ones, as used above. As shown in Fig. 4A,
PIX binding to PAK3
appeared to compete with paxillin
binding to PAK3. On the other
hand, the
isoform of paxillin, which did not exhibit significant
binding affinity to PAK3, did not interfere with the
PIX binding to
PAK3 (Fig. 4A). The interaction of these three proteins was
also examined in vivo by expressing GST-paxillin
,
HA-PAK3, and increasing amounts of His-
PIX in COS7 cells. Consistent
with the notion that
PIX competes with paxillin
in binding to
PAK3, increasing expression of His-
PIX reduced the amounts of
HA-PAK3 co-precipitated with GST-paxillin
(Fig. 4B). We
confirmed that
PIX does not bind directly to paxillin
(data not
shown), as reported previously (44).
View larger version (27K):
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Fig. 4.
Paxillin competes
with
PIX and Nck in their binding to
PAK3. A, paxillin
, but not the
isoform,
competes with
PIX in its binding to PAK3 in vitro.
Top panel, GST-PAK3 (250 ng) bound to glutathione-Sepharose
beads was incubated with His-paxillin
(250 ng) in the presence of
increasing amounts of His-
PIX (0, 80, 200, and 800 ng in lanes
3-6, respectively), or only with His-
PIX (800 ng in lane
7). Middle panel, GST-PAK3 (250 ng) bound to
glutathione-Sepharose beads was incubated with His-
PIX (250 ng) in
the presence of increasing amounts of His-paxillin
(0, 80, 200, and
800 ng in lanes 10-13, respectively), or only with
His-paxillin
(800 ng in lane 14). Bottom
panel, GST-PAK3 (250 ng) bound to glutathione-Sepharose beads was
incubated with His-
PIX (250 ng) in the presence of increasing
amounts of His-paxillin
(0, 80, 200, and 800 ng in lanes
17-20, respectively), or only with His-paxillin
(800 ng in
lane 21). After washing, proteins bound to the beads were
separated on SDS-PAGE and subjected to sequential immunoblotting
analysis using antibodies against paxillin and
PIX. Lanes
1, 8, and 16 included 10 ng each of
His-
PIX; lanes 2 and 9, 10 ng each of
His-paxillin
; and lane 15, 10 ng of His-paxillin
.
GST-PAK3, His-paxillin
, and His-
PIX proteins were the same
purified preparations as in Figs. 2 and 3. His-paxillin
was
produced in the baculovirus system, purified on nickel beads; and its
Coomassie Brilliant Blue (CBB) staining is shown on the
right of the bottom panel (lane 22). A
protein band corresponding to His-paxillin
is indicated by an
asterisk. Molecular sizes are shown on the left.
B, competition of paxillin
and
PIX in their
association with PAK3 in vivo. COS7 cells were
co-transfected with 2.5 µg each of pEBG-paxillin
(encoding
GST-paxillin
) and pEFBOS-HA-PAK3, in the presence of increasing
amounts of pcDNA-His-
PIX (0, 3, and 10 µg in lanes
1-3, respectively). Controls included transfection only with
pEBG-paxillin
only (lane 4). At 36 h after transfection, GST-paxillin
was
precipitated with glutathione beads from 300 µg of each cell lysate;
and proteins retained on the beads were separated on SDS-PAGE and
subjected to sequential immunoblotting analysis using antibodies
against HA-tag and GST, and shown in the lower panel
(pull down with GST-pax
). In the upper panel,
20 µg of each total cell lysate was analyzed by immunoblotting to
show the expression levels of each exogenous protein
(total). Each arrow indicates positions of each
corresponding protein. C, competitive binding of paxillin
and Nck toward PAK3 in vitro. Purified GST-PAK3 (250 ng)
bound to glutathione beads was incubated with purified His-paxillin
(250 ng) in the presence of increasing amounts of purified Nck (0, 200, 400, and 1000 ng in lanes 3-6, respectively), or only with
Nck (1000 ng in lane 7). After washing, proteins bound to
the beads were processed and analyzed as above using antibodies against
paxillin and Nck. Lanes 1 and 2 included 10 ng of
His-paxillin
and Nck, respectively. GST-PAK3 and His-paxillin
were the same purified preparations as in Fig. 2. Coomassie Brilliant
Blue (CBB) staining of the Nck preparation, which was
produced as a GST fusion form in E. coli, then cleaved from
the tag and purified as described under "Experimental Procedures,"
is shown on the right (lane 8). A protein band
corresponding to Nck is shown by an asterisk. Molecular
sizes are shown on the left.
binding to PAK3 competed with Nck binding to PAK3.
GST-PAK3 (250 ng) bound to glutathione-Sepharose beads was incubated
with His-paxillin
(250 ng) in the presence of increasing amounts of
Nck (0-1000 ng). Using purified preparations of these proteins, we
found that Nck binding to PAK3 was competitive with paxillin
binding to PAK3 (Fig. 4C). Again, 10 ng each of paxillin
and Nck proteins was included in Fig. 4C; and judging from
the intensities of the chemiluminescence signals from paxillin
and
Nck bands co-precipitated with PAK3, the apparent binding affinities of
these two proteins toward PAK3 seemed to be similar.
Associates with Both the Kinase-inactive and the
Cdc42-activated Forms of PAK3--
The association between PAK1 and
Nck has been shown to be constitutive, irrespective of the PAK's
activation state (34, 53). We thus examined paxillin
binding to
PAK3 with regard to the activation state of PAK3. HA-PAK3 was
coexpressed with GST-paxillin
in COS7 cells in the presence or
absence of the dominant-active (DA) form of HA-tagged Cdc42. As shown
in Fig. 5A, GST-paxillin
co-precipitated almost equal amounts of HA-PAK3 irrespective of the
presence or the absence of HA-Cdc42 DA. Co-precipitation of HA-Cdc42 DA
together with HA-PAK3 and GST-paxillin
was also observed (Fig.
5A). On the other hand, the LD4-deletion mutant of
GST-paxillin
failed to co-precipitate HA-PAK3 (Fig. 5A). In vitro kinase assay using MBP as a substrate was performed
simultaneously. As shown in Fig. 5A, HA-PAK3 bound to
GST-paxillin
in the absence of HA-Cdc42 DA showed a very low
activity in phosphorylating MBP, whereas it showed a high activity in
the presence of HA-Cdc42 DA.
View larger version (34K):
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Fig. 5.
Paxillin associates
with PAK3 in vivo without changing its activation
states. A, paxillin
associates with the
kinase-inactive and the Cdc42-activated forms of PAK3 in
vivo. COS7 cells were transfected with 2.5 µg of pEFBOS-HA-PAK3
in the presence or absence of 2.5 µg each of pEFBOS-HA-Cdc42 G12V,
pEBG-paxillin
, or the pEBG-paxillin
(
LD4), as indicated. At 36 h after
transfection, GST-paxillin
or the LD4-deletion mutant was
precipitated with glutathione beads from 300 µg of each cell lysate.
After washing, proteins retained on the beads were separated on
SDS-PAGE and subjected to sequential immunoblotting analysis using
antibodies against HA-tag and GST, and shown in the middle
panel (pull-down with GST-pax
or GST-pax
(
LD4)). Each
half-amount of the precipitants was simultaneously subjected to
in vitro kinase assay using [
-32P]ATP and
MBP as a substrate as described under "Experimental Procedures,"
followed by SDS-PAGE and autoradiography; and shown in the bottom
panel (in vitro kinase assay); where molecular sizes
are shown on the left. In the top panel, 20 µg
of each total cell lysate was analyzed by immunoblotting to show the
expression levels of exogenous proteins (total). Each
arrow indicates positions of each corresponding protein.
Protein bands corresponding to HA-PAK3 and HA-Cdc42, both detected by
an antibody against the HA-tag, were distinguished by their molecular
sizes. B, assessment of kinase activities bound to the
GST-paxillin
by use of the PAK3 kinase inhibitor. COS7 cells were
transfected with 2.5 µg each of pEFBOS-HA-PAK3, pEFBOS-HA-Cdc42 G12V,
and pEBG-paxillin
, in the absence (lane 1) or presence
(lane 2) of 2.5 µg of pEBG-PAK3 KI, which encoded the
autoinhibitory domain for the kinase activity (see "Experimental
Procedure"). At 36 h after transfection, GST-paxillin
was
precipitated using glutathione beads from 300 µg of each cell lysate
and subjected to in vitro kinase assay as above, and the
resulting autoradiograph is shown (in vitro kinase assay). Each
half-amount of the precipitants was separated on SDS-PAGE and analyzed
using antibodies against HA-tag and GST, to examine the amounts of
HA-PAK3 co-precipitated with GST-paxillin
and that of GST-PAK3 KI,
respectively; and shown below (pull-down with GST-pax
). Each
arrow indicates positions of each corresponding
protein.
in the presence
of HA-Cdc42 DA was primarily due to the kinase activity of PAK3, the
PAK3 autoinhibitory domain was coexpressed. As shown in Fig.
5B, coexpression of the autoinhibitory domain of PAK3 with
HA-PAK3 and HA-Cdc42 DA in COS7 cells largely reduced the kinase
activity co-precipitated with GST-paxillin
, although the amounts of
HA-PAK3 co-precipitated with GST-paxillin
were almost unchanged.
Thus, PAK3 accounts for the major kinase activity that is associated
with GST-paxillin
in the presence of HA-Cdc42 DA. The results shown
in Fig. 5, A and B, collectively indicate that
the binding of paxillin
to PAK3 per se does not
significantly activate its kinase activity, or interfere with the
Cdc42-mediated activation of the PAK3 kinase activity.
Is Directly Phosphorylated by PAK3 at
Serine--
Finally, we examined whether paxillin
could be
phosphorylated by PAK3, again with the analogy to Nck phosphorylation
by PAK1 (34, 45). Paxillin is well phosphorylated on serine in vivo (54-56), and is known to associate with some cellular
serine/threonine kinase activities (54). Likewise, we found that the
His-paxillin
protein preparation, which was produced in the
baculovirus system and collected on nickel beads, was still highly
contaminated with serine/threonine kinase activities, although the
purity of the protein seemed to be more than 95% as assessed by
Coomassie staining (data not shown). To remove the contaminating kinase
activities, His-paxillin
protein bound to nickel beads was treated
with guanidine HCl, and then subjected to a renaturing process. GST fusion form of PAK3 has been known to be constitutively active in the
kinase activity (4). The kinase-free His-paxillin
(see Fig.
6, lane 1) was then
incubated with a purified preparation of GST-PAK3 in the presence of
ATP and divalent cations; and found to be well phosphorylated by
GST-PAK3 in vitro (Fig. 6A). Phosphoamino acid
analysis revealed that the phosphorylation had exclusively taken place
at the serine residue(s) (Fig. 6B). Note that
phosphorylation of paxillin
in vitro in GST-PAK3
precipitants was already observed in Fig. 5, A and
B, where coexpression of the PAK3 autoinhibitory domain
substantially, but not completely, suppressed the phosphorylation.
View larger version (34K):
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Fig. 6.
Paxillin is
phosphorylated by PAK3 on serine in vitro.
A, purified kinase-free preparation of His-paxillin
(0.5 µg; see text and "Experimental Procedures") was incubated with
purified GST-PAK3 (0.5 µg) in the presence of
[
-32P]ATP (lane 3). GST-PAK3 was the same
preparation as in Fig. 2. Control included His-paxillin
only
(lane 1) or GST-PAK3 only (lane 2), incubated
with [
-32P]ATP. B, phosphorylated
His-paxillin
was then excised from the gel, and subjected to
phosphoamino acid analysis. Phosphoamino acid standards were visualized
by ninhydrin to identify the position of each phosphoamino acid. 1000 cpm of the radioactivity was loaded for thin layer chromatography. In
A and B, resulting autoradiographies are shown;
and each arrow indicates positions of each corresponding
protein, phosphoamino acids, free phosphate, and the origin of the
sample spot. Note that the His-tag is composed only of six consecutive
histidine residues; thus the serine phosphorylation occurred at
His-paxillin
is within the paxillin moiety.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
isoform of paxillin
can bind directly to PAK3. In vivo association of paxillin
with PAK3 took place both with the kinase-inactive and the
Cdc42-activated forms of PAK3; and this association did not seem to
change or interfere with the activation states of the kinase. We
furthermore, demonstrated in vitro that paxillin
can be
phosphorylated by PAK3 at serine.
are similar to those
ascribed to Nck in its binding to PAK1 (30, 34, 35, 45). Nck binding to
PAK1 had no stimulatory effect on PAK1 kinase activity, and it did not
alter the ability of PAK1 to be stimulated by Rac- or Cdc42-GTP
S;
and Nck can be phosphorylated by PAK1. Nck has been shown to associate
with several growth factor receptors, and is thus thought to link PAK1
to growth factor receptor signalings. Nck has been shown to bind to
PAK1 at its NH2-terminal proline-rich sequence. We showed
that paxillin binds to the NH2-terminal region of PAK3, and
the binding competes with the Nck binding to PAK3. Our results also
indicated that the binding affinities of paxillin
and Nck in their
direct interaction with PAK3 seem to be similar. It has been well
documented that PAKs are activated by integrin-mediated cell adhesion
to the extracellular matrixes (28, 29). Therefore, we collectively
propose that paxillin
is capable of linking both the kinase-active
and -inactive forms of PAK3 to integrins, as Nck serves to link PAK1 to
growth factor receptors. However, since our results revealed that
paxillin
and Nck compete with each other in their binding to PAK3,
a single molecule of PAK3 does not appear to bridge these two proteins.
PIX, as already mentioned earlier. Contrary
to this, we could detect direct binding of PAK3 to paxillin
in vitro, in the absence of
PIX. For the in
vitro binding, we used PAK3 molecules produced in COS7 cells and
paxillin
molecules produced in the baculovirus system. On the other
hand, Turner et al. (44) used the 54-313 amino acid region
of paxillin produced in E. coli and PAK3 molecules
synthesized by in vitro transcription/translation system. We
also found that the in vitro translated product of PAK3 was
almost incapable of binding to GST-paxillin
.2 To examine the
bindability of the in vitro translated PAK3 molecules, Turner et al. (44) have confirmed that it could bind to
PIX, which we also detected. Interaction between PAKs and
PIX is, however, mediated through the proline-rich sequence of PAKs and the SH3
domain of
PIX; thus the binding of these two proteins can be
detected even with a protein overlay assay using
PIX as a probe,
where PAK1 protein was run on SDS-PAGE and thereby denatured (23).
Binding of the in vitro translated PAK3 to
PIX may
therefore have been able to be detected, even if the PAK3 may not have
preserved its native structure. We found that the in vitro
translated PAK3 protein ran as doublet bands on SDS-PAGE, as also seen
previously (Fig. 7 in Ref. 44). Some artificial events may have
occurred with PAK3 molecules in the in vitro
transcription/translation system, and might have hampered its capacity
to bind to paxillin.
,
, and
), which exhibit
different properties of protein binding (42). Association with PAK3 has
been reported to be primarily mediated through the LD4 motif (44),
which we also confirmed. The
and
isoforms are generated by the
insertion of each specific exon just after the LD4 motif of the
isoform. We have thus tested and found that the
and
isoforms of
paxillin exhibit significant affinities to PAK1 and PAK3, while the
isoform exhibits a very weak and only marginally detectable level of
association with these PAKs. It is interesting to note that this
property of PAKs in the association with paxillin isoforms is similar
to that of Fak (42), whose interaction with paxillin is also primarily
mediated by the LD4 motif of paxillin (44). Among the three isoforms of
paxillin, no significant difference in the subcellular localization is
detected (52). Thus, the
isoform of paxillin may act as a
dominant-negative form with regard to the PAK and Fak signalings
mediated by paxillin
.
can associate with PAK1. Our preliminary
experiment also suggested that PAK2 seemed to be co-precipitated with
GST-paxillin
from NRB lysates,2 but this has not been
able to be confirmed due to the insufficient quality of PAK2 antibody.
/p95PKL/
PIX/PAK3, in a
linear configuration as Turner et al. (44) have proposed. We
have isolated a cDNA for Git2 (33), which encodes a
paxillin-binding protein.3
Git2 is highly homologous to p95PKL; and we indeed detected ternary complexes of paxillin
/Git2/
PIX, and
Git2/
PIX/PAK3.2 However, since paxillin
itself can
bind to PAK3, we could not properly assess whether or not these four
proteins make the tetra-protein complex in a linear configuration. On
the other hand, we showed that both the kinase-inactive and the
Cdc42-activated forms of PAK3 can be co-precipitated with paxillin
from COS7 cells.
PIX binding to PAK3 activates the kinase activity
of PAK3 (23); thus, our results strongly suggest that the tetra-protein
complex formation proposed by Turner et al. (44), in which
the
PIX binding to PAK3 is required, is not the sole way of the
association of paxillin
with PAK3. Moreover, we showed that
paxillin
association with PAK3 can compete with
PIX association
with PAK3 in vitro and in vivo. PAK1 has been
shown to be colocalized with paxillin at focal complexes at the cell
periphery (23); and it has been shown recently that the kinase-inactive
and/or non-autophosphorylated form of PAK1 is preferentially localized
to focal adhesions (45). Consistent with this, we demonstrated that
paxillin
association with PAK3 per se did not activate
the kinase activity of PAK3. A fraction of PAK3 was detected at focal
adhesions, largely, but not completely co-localized with paxillin
.2 Collectively, it seems reasonable to suppose that
paxillin
can associate with PAK3 at focal adhesions, and at focal
complexes may as well, even in the absence of
PIX.
. However, our preliminary
results indicate that when paxillin
was highly phosphorylated by
PAK3 in vitro, these two phosphorylated proteins could no
longer stay bound to each other.2 Thus, it is possible that
when PAK3 is activated and phosphorylates paxillin
at high levels,
this activated PAK3 molecule is released from the paxillin
.
Similarly, PAK1 has been proposed to be dissociated from Nck, when PAK1
is highly activated and phosphorylated (45). However, it should be also
noted that the PAKs have two types of effects, one related to its
kinase activity and one that is kinase independent (12, 13, 15, 23).
Elucidation of the mechanism and timing of the recruitment of PAKs to
paxillin
, the regulation of the kinase activity as well as the
autophosphorylation of the paxillin-bound PAKs, and the phosphorylation
of paxillin
by PAKs, will be thus required for a precise
understanding of the PAK-mediated integrin signalings.
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ACKNOWLEDGEMENTS |
---|
We thank Mihoko Sato and Manami Hiraishi for
technical assistance, and Mayumi Yoneda for secretary work. We are also
grateful to Masato Okada (Osaka University) for the preparation of NRB lysates, Takahiro Nagase (Kazusa DNA Research Institute) for PIX (KIAA0142) cDNA, Hidesaburo Hanafusa (Osaka Bioscience Institute) for Nck cDNA, Kensaku Mizuno (Tohoku University) for PAK1 cDNA, and Shinya Kuroda and Kozo Kaibuchi (Nara Institute of Science and
Technology) for the dominant-active form of HA-tagged Cdc42 cDNA.
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FOOTNOTES |
---|
* This work was supported in part by a grant-in-aid from Ministry of Education, Science, Sports and Culture of Japan, grants from the Takeda Medical Foundation, The Mochida Memorial Foundation for Medical and Pharmaceutical Research, and Novartis Foundation for the Promotion of Science.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.
¶ To whom correspondence should be addressed: Dept. of Molecular Biology, Osaka Bioscience Institute, 6-2-4 Furuedai, Suita, Osaka 565-0874. Tel.: 81-6-6872-4814; Fax: 81-6-6871-6686; E-mail: sabe@obi.or.jp.
Published, JBC Papers in Press, November 28, 2000, DOI 10.1074/jbc.M005854200
2 S. Hashimoto, A. Tsubouchi, Y. Mazaki, and H. Sabe, unpublished results.
3 Mazaki, Y., Hashimoto, S. Okawa, K., Tsubouchi, A., Nakamura, K., Yagi, R., Yano, H., Kondo, A., Iwamatsu, A., Mizoguchi, and Sabe, H. (2001) Mol. Biol. Cell, in press.
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ABBREVIATIONS |
---|
The abbreviations used are:
PAK, p21-activated
kinase;
aa, amino acids;
Cas, Crk-associated substrate;
Fak, focal
adhesion kinase;
Git, G protein-coupled receptor kinase-interacting
protein;
GST, glutathione S-transferase;
HA-tag, hemagglutinin tag;
His-tag, six consecutive histidine tag;
MBP, myelin
basic protein;
NRB, neonatal rat brain;
PAGE, polyacrylamide gel
electrophoresis;
PCR, polymerase chain reaction;
PIX, PAK-interacting
exchanger factor;
PKL, paxillin-kinase linker;
Pyk2, proline-rich
tyrosine kinase 2;
SH3, Src homology 3;
DA, dominant active;
GTPS, guanosine 5'-3-O-(thio)triphosphate.
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