1 Ludwig Institute for Cancer Research, Royal Free and University College
Medical School Branch, 91 Riding House Street, London WIW 7BS, UK
2 Onyx Pharmaceuticals, 3031 Research Drive, Richmond, CA 94806, USA
3 Department of Biochemistry and Molecular Biology, University College London,
Gower Street, London, UK
* Present address: PPD Discovery, 1505 O'Brien Dr Menlo Park, CA 94025,
USA
Address for correspondence (e-mail:
anne{at}ludwig.ucl.ac.uk)
Accepted 1 August 2002
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Summary |
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Key words: p21-activated kinase, Phosphoinositide 3-kinase, PAK4, HGF, Actin, Cytoskeleton, Focal complexes
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Introduction |
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Both Group I and Group II PAKs can induce actin rearrangement in cells
(Daniels and Bokoch, 1999;
Jaffer and Chernoff, 2002
;
Sells and Chernoff, 1997
). For
example, PAK1 can induce lamellipodium formation and has been localised to
sites of membrane ruffling in fibroblasts
(Dharmawardhane et al., 1997
;
Sells et al., 1997
). PAK4 has
been shown to be involved in Cdc42-induced filopodium extension in endothelial
cells (Abo et al., 1998
) and
fibroblasts (Qu et al., 2001
),
and PAK5 can induce neurite extension (Dan
et al., 2002
). PAKs are also implicated in the regulation of
cell:substrate adhesion: expression of constitutively activated PAK1, 2 and 4
has been reported to induce cell rounding
(Manser et al., 1997
;
Qu et al., 2001
;
Zeng et al., 2000
). In
addition, PAK1 has been localised to focal adhesions
(Manser et al., 1997
;
Zhao et al., 1998
) and
co-immunoprecipitated with paxillin, a focal adhesion component
(Turner et al., 1999
). Effects
of PAK1 are cell-type-dependent. However, as in fibroblasts, expression of an
activated PAK1 mutant enhances the cell migration rate
(Sells et al., 1999
), whereas
in endothelial cells the same mutant increases cell contractility and reduces
cell migration (Kiosses et al.,
1999
).
HGF is a multifunctional cytokine that can act as a motility-inducing
factor for epithelial cells (Stoker et
al., 1987; Weidner et al.,
1993
) through the c-Met tyrosine kinase receptor
(Bottaro et al., 1991
;
Naldini et al., 1991
).
HGF-stimulated epithelial cells lose cell:cell contacts, take on a more
fibroblastic-like morphology and become more migratory. PAK1 is activated by
HGF (Royal et al., 2000
), as
well as by other pro-migratory stimuli including PDGF
(Dechert et al., 2001
) and
heregulin (Adam et al., 1998
).
In contrast, the activation of group II PAKs by growth factors has not been
studied. We show here that PAK4 kinase activity is stimulated by HGF in MDCK
cells, and that activated PAK4 induces a loss of cell:substratum adhesion in
an HGF-dependent manner. Furthermore, both activity and subcellular
localisation of PAK4 during HGF signalling are regulated by PI3K.
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Materials and Methods |
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Time-lapse video microscopy
MDCK cells were microinjected with pSR-HA-PAK4
GBD at a
concentration of 100 ng/µl. After 3 hours, cells were transferred to DMEM
with 0.2% FCS and 10 ng/ml HGF and then placed in an incubator mounted on the
stage of a Zeiss Axiovert 135 microscope, maintained at 37°C in a
humidified atmosphere containing 10% CO2. Cell images were
collected by a KPM1E/K-S10 CCD camera (Hitachi Denshi, Japan) every 2 minutes
for 20 hours using Tempus software (Kinetic Imaging, Liverpool, UK). Cells
expressing PAK4 were subsequently identified by immunofluorescence.
Immunofluorescence
Mouse anti-HA antibody was obtained from Berkeley Antibody Company
(Richmond, CA), rabbit anti-HA antibody from Santa Cruz Biotechnology (Santa
Cruz, CA), mouse anti-paxillin antibody from Transduction Laboratories
(Lexington, KY) and mouse anti-flag antibody from Sigma. FITC-conjugated goat
anti-mouse IgG secondary antibody was purchased from Jackson ImmunoResearch
(West Grove, PS) and TRITC-conjugated goat anti-rabbit IgG from Southern
Biotechnology (Birmingham, AL). TRITC-conjugated phalloidin was obtained from
Sigma. Cells were fixed with 4% paraformaldehyde in PBS for 20 minutes at room
temperature and then permeabilised with 0.2% Triton X-100 in PBS for 5
minutes. Primary and secondary antibodies were diluted in PBS containing 0.5%
bovine serum albumin and all incubations were for 1 hour at room temperature.
For detection of expressed proteins, cells were incubated with a 1:100
dilution of mouse anti-HA antibody, a 1:200 dilution of rabbit anti-HA
antibody, or a 1:200 dilution of mouse anti-flag antibody. Following
incubation with the primary antibody, cells were washed six times in PBS and
then incubated with a 1:400 dilution of FITC-conjugated goat anti-mouse IgG, a
1:200 dilution of TRITC-conjugated goat anti-rabbit IgG and/or
TRITC-conjugated phalloidin. Images of cells were obtained using a Zeiss
LSM510 confocal laser-scanning microscope (Welwyn Garden City, UK), using the
accompanying LSM 510 software, and were processed in Adobe PhotoShop 4.0.
Antibody production and immunoblotting
A rabbit polyclonal anti-PAK4 antibody was raised against a peptide derived
from the PAK4 kinase domain, PRRK[SL]VGTPYMAPE. The antibody was then
affinity-purified against this peptide. An anti-PAK1 (C19) antibody was
obtained from Santa Cruz Biotechnology (Santa Cruz, CA); C19 is known to
crossreact with PAK2 and PAK3. Mouse anti-HA antibody was obtained from
Berkeley Antibody Company (Richmond, CA). MDCK cells were lysed for 10 minutes
in lysis buffer (0.5% NP-40, 30 mM sodium pyrophosphate, 50 mM Tris-HCl pH
7.6, 150 mM NaCl, 0.1 mM EDTA, 50 mM NaF, 1 mM Na3VO4, 1
mM PMSF, 10 µg/ml leupeptin and 1 µg/ml aprotinin). Lysates were
clarified by centrifugation at 14,000 g for 10 minutes. Equal amounts
of protein were electrophoresed on 10% SDS-polyacrylamide gels then
transferred to nitrocellulose membranes (Schleicher and Schell). Membranes
were blocked in 5% nonfat dried milk in PBS then incubated for 16 hours at
4°C with either a 1:1000 dilution of rabbit anti-PAK4 antibody, a 1:1000
dilution of C19 anti-PAK1 antibody or a 1:1000 dilution of mouse anti-HA in
0.5% nonfat dried milk/PBS. Membranes were then incubated for 1 hour at room
temperature with a 1:2000 dilution of horseradish-peroxidase-conjugated donkey
anti-rabbit or anti-mouse antibody in 0.5% nonfat milk: PBS (Amersham
Pharmacia, Little Chalfont, UK). Blots were developed by enhanced
chemiluminescence (ECL, Amersham Pharmacia).
Kinase assay
MDCK cells were transiently transfected with HA-PAK4wt, HA-PAK4GBD
or PAK4
using Fugene transfection reagent (Roche, Roche Molecular
Biochemicals, IN). Cells were incubated for 24 hours in DMEM with 10% FCS
prior to 4 hours starvation in DMEM with 0.2% FCS. Cells were then stimulated
for up to 30 minutes with HGF (10-100 ng/ml; R and D systems, Abingdon, UK).
For LY294002 experiments cells were pre-incubated (20 µM, Calbiochem,
Nottingham, UK) for 30 minutes prior to stimulation with HGF. Following
stimulation, cells were harvested in lysis buffer (0.5% NP-40, 30 mM sodium
pyrophosphate, 50 mM Tris-HCl pH 7.6, 150 mM NaCl, 0.1 mM EDTA, 50 mM NaF, 1
mM Na3VO4, 1 mM PMSF, 10 µg/ml leupeptin and 1
µg/ml aprotinin). Lysates were clarified by centrifugation at 14,000
g for 10 minutes. Cell lysates were then pre-cleared twice with mouse
anti-myc antibody 9E10 (Santa Cruz Biotechnology, Santa Cruz, CA), and protein
G-Sepharose (Amersham Pharmacia, Little Chalfont, UK). A small amount of
lysate was removed from each sample for SDS-PAGE anaylsis. The pre-cleared
lysates were then mixed with anti-HA antibody (Berkeley Antibody Company,
Richmond, CA) overnight at 4°C followed by a 1 hour incubation with
protein G-Sepharose at 4°C. The immune complexes were washed twice with
lysis buffer, once with Wash buffer 1 (0.5 M LiCl and 20 mM Tris pH 8.0) and
twice with kinase buffer (50 mM Tris-HCl pH 7.5, 10 mM MgCl2 and 1
mM DTT) then incubated in kinase buffer containing 30 µM ATP and 3 µCi
of [
-32P]ATP together with Histone H1 (Roche) for 30 minutes
at 30°C. The reaction was stopped by adding 6x SDS loading buffer.
As a control a kinase assay was also performed on cell lysates from
untransfected cells, which had been immunoprecipitated with protein
G-Sepharose beads coupled to anti-HA antibody. Proteins were resolved by
SDS-PAGE and any phosphorylation was visualised by autoradiography.
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Results |
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|
Activated PAK4 induces cell rounding in HGF-stimulated MDCK
cells
To analyse the morphological responses induced by PAK4, we transiently
expressed wild-type PAK4 (PAK4wt) and various PAK4 mutants in MDCK cells. PAK4
lacking the GBD domain (PAK4GBD) has increased kinase activity compared
with wild-type PAK4 (Abo et al.,
1998
) (Fig. 1B).
The kinase domain alone of PAK4 also has elevated kinase activity when
expressed in MDCK cells (Fig.
1B). Exogenous PAK4 protein expression was readily detectable by
immunofluorescence up to 24 hours after microinjection of expression vectors
(Fig. 2 and data not shown),
indicating that PAK4 overexpression is not toxic to the cells.
|
Expression of PAKGBD for up to 23 hours in the absence of HGF had no
discernible effect on cell morphology (Fig.
2ii, A). In contrast, in the presence of HGF the majority of cells
expressing PAK4
GBD rounded up (Fig.
2ii, B). This response was titratable, and at lower levels of
expression PAK4
GBD was unable to induce cell rounding (data not shown).
For PAK4
GBD to induce cell rounding, it was not essential for HGF to be
present for the whole timecourse, but cells needed to be stimulated with HGF
for a minimum of 15-30 minutes. This is similar to the length of HGF
stimulation required to induce discernible scattering (data not shown).
PAK4-induced cell rounding required an activated protein, as expression of
PAK4wt at comparable levels had no effect on cell morphology either in the
absence or presence of HGF (data not shown). This is consistent with previous
reports where overexpression of PAK4wt had no effect on the actin cytoskeleton
or cell morphology (Dan et al.,
2001
; Qu et al.,
2001
). MDCK cells expressing activated PAK1 or PAK3 were able to
round up in the absence of HGF stimulation, thus cell rounding was not
strictly dependent on the morphological changes that occur during HGF
stimulation (data not shown). Although it has been previously reported that an
activated PAK4 mutant can induce cell rounding in fibroblasts
(Qu et al., 2001
), this is the
first demonstration of a growth-factor-dependent response to PAK4.
The removal of the GBD domain of PAK4 is presumably required to make the
kinase sufficiently active to stimulate this response. Cdc42 binding is unable
to mimic the effect of GBD domain deletion, as co-expression of PAK4wt and
V12Cdc42 did not induce cell rounding (data not shown). This is consistent
with the observation that Cdc42 binding does not elevate the kinase activity
of PAK4 (Abo et al., 1998).
PAK4-induced MDCK cell rounding is kinase dependent
PAK1-induced changes in cell morphology and actin organisation can be
either kinase-dependent (Frost et al.,
1998; Zhao et al.,
1998
) or kinase-independent
(Sells et al., 1999
;
Sells et al., 1997
).
PAK1-induced fibroblast cell rounding requires a functional kinase domain
(Frost et al., 1998
), and so
we investigated whether PAK4-induced rounding of MDCK cells also required a
functional kinase domain. Expression of the kinase domain alone of PAK4
(PAK4
) induced cell rounding in the presence of HGF
(Fig. 2ii, A), but expression
of the inactive kinase domain PAK4
M350
[(Abo et al., 1998
) and data
not shown] did not (Fig. 2ii,
B). This confirms that PAK4-induced cell rounding also requires
kinase activity. Interestingly, expression of PAK4
induced cell
rounding in the absence of HGF (Fig. 2ii,
C). This indicates that the N-terminal region of PAK4 negatively
regulates PAK4 activity and suggests that this region normally negatively
regulates the kinase domain of PAK4wt and PAK4
GBD in unstimulated MDCK
cells.
Activated PAK4 expression reduces cell-substratum adhesion
To investigate the time course of PAK4-induced responses, PAK4-expressing
cells were followed by time-lapse video microscopy. As expected,
PAK4GBD-expressing cells in the absence of HGF did not round up (unless
entering mitotic division) or detach from the substratum, and cells injected
with control IgG or expressing PAK4wt exhibited a normal motile response to
HGF and did not round up (data not shown). In addition, we found no evidence
of HGF-dependent cell rounding in uninjected cells. However, cells expressing
PAK4
GBD (Fig. 2iii;
Movie 1, see
http://jcs.biologists.org/supplemental)
started to round up as early as 5 hours post injection, after 2 hours in the
presence of HGF. By 4 hours, in the presence of HGF, the majority of cells
were almost completely rounded and some had begun to detach from the
substratum. Interestingly, although cells detached from the substratum they
retained cell-cell contacts with each other and with uninjected cells in the
surrounding colony. This observation accounts for the detection of rounded
cells up to 23 hours after injection (Fig.
2i, B). PAK4
GBD-expressing cells remained rounded but did
not bleb for up to 24 hours, indicating that they were not apoptotic.
The video time-lapse observations indicated that cell-substratum adhesions
are altered by PAK4GBD as early as 2 hours after HGF stimulation. HGF
normally induces a decrease in actin stress fibres and large focal adhesions
and an increase in smaller peripheral focal complexes in MDCK cells
[(Dowrick et al., 1991
) data
not shown]. This change in the nature of cell-substratum adhesions can be
detected by following paxillin localisation. Paxillin-containing peripheral
focal complexes were clearly observed in PAK4wt-expressing cells
(Fig. 3D) at 3 hours after HGF
stimulation. However, in PAK4
GBD-expressing cells
(Fig. 3C) there was a
significant reduction in the number of paxillin-containing peripheral focal
complexes. This could only be visualised in cells expressing very low levels
of PAK4
GBD where the spreading edge had not been completely retracted.
These results suggest that PAK4-induced cell rounding is either due to an
increase in focal complex turnover or to a decrease in focal complex
formation.
|
Activated PAK4 is localised to the cell periphery
Time-lapse microscopy and paxillin localisation in
PAK4GBD-expressing cells suggests that induction of cell rounding
occurs approximately 2-3 hours after HGF stimulation. We therefore
investigated the localisation of PAK4
GBD and PAK4wt at this time point
after microinjection, with or without short-term HGF stimulation. PAK1 is
known to be localised in lamellipodia and focal adhesions
(Dharmawardhane et al., 1997
;
Sells et al., 2000
). However,
in the majority of both unstimulated and HGF-stimulated cells, PAK4
GBD
was localised at the periphery (Fig.
4A,B; Table 1) 5.5
hours post-injection, but localisation was not restricted to the
lamellipodium. In a small number of cells the distribution was diffusely
cytoplasmic and PAK4
GBD was occasionally localised to the nucleus (data
not shown), as observed in PAE cells (Abo
et al., 1998
). Whether PAK4 plays a role in the nucleus is not
known, but it is interesting that PAK6 can also localise to the nucleus
(Yang et al., 2001
). However,
we found no evidence for PAK4 localisation in focal adhesions.
|
|
HGF induced relocalisation of PAK4wt to the cell periphery
(Fig. 4D) in a proportion of
cells, which was particularly significant in cells at the edge of the colonies
(Table 1). At higher expression
levels, PAK4wt exhibited a diffuse cytoplasmic staining in the absence or
presence of HGF (data not shown; there are currently no antibodies available
that detect endogenous PAK4 by immunofluorescence). PAK4 exhibited a
peripheral localisation similar to that of PAK4
GBD either in the
absence (Fig. 4E) or presence
of HGF (data not shown), whereas PAK
M350 was diffusely localised
(Fig. 2ii, B and data not
shown). This indicates that the region of the protein involved in localisation
of PAK4 to the cell periphery is within the kinase domain and not the
regulatory domain and that this localisation is dependent on the kinase
activity. Our results suggest that PAK4 subcellular localisation is
growth-factor-regulated. This may explain why PAK4wt and PAK4
GBD were
reported to have a diffuse cytoplasmic localisation in PAE cells and
fibroblasts (Abo et al., 1998
;
Callow et al., 2002
;
Qu et al., 2001
).
Activated PAK4 causes stress fibre disassembly
HGF stimulation of MDCK cells is known to induce initially a loss of actin
stress fibres and associated focal adhesions [data not shown
(Dowrick et al., 1991)].
Expression of PAK4
GBD caused a decrease in actin stress fibres in the
presence or absence of HGF (Fig.
4F), with 75% of PAK4
GBD-expressing cells lacking actin
stress fibres, 5.5 hours post-injection, compared with 12.5% of uninjected
cells. Furthermore, expression of PAK4
induced an even more dramatic
loss: 91.3% of PAK4
-expressing cells had no actin stress fibres 5.5
hours post-injection (Fig. 4J).
Consistent with these data, it has recently been reported that expression of a
different highly activated PAK4 mutant in fibroblasts causes disruption of
actin stress fibres and cell rounding (Qu
et al., 2001
).
However after 23 hours of expression in the absence of HGF,
PAK4GBD-expressing cells appeared similar to uninfected cells and
contained normal levels of actin stress fibres
(Fig. 2i and data not shown).
In addition, after prolonged PAK4
GBD expression in the absence of HGF,
PAK4
GBD was no longer localised to the cell periphery suggesting that
peripheral localisation of activated PAK4 is a transient event
(Fig. 2i). Indeed, after 23
hours in the presence of HGF, PAK4wt expressed at low levels assumed a diffuse
cytoplasmic localisation (data not shown). As peripheral localisation and
stress fibre disassembly are both transient events and correlate with kinase
activity, these results suggest that PAK4
GBD activity is downregulated
in the absence of HGF.
PAK4 is activated by HGF
Our results suggest that PAK4 kinase activity is required for cell rounding
and localisation and that it is specifically regulated by HGF. A previous
report indicated that PAK1 kinase activity could be stimulated by HGF in MDCK
cells (Royal and Park, 1995).
To determine whether HGF can activate PAK4 kinase activity, MDCK cells were
transiently transfected with an expression vector encoding HA-tagged PAK4wt,
at levels where exogenous PAK4wt expression were no more than two-fold higher
than endogenous PAK. PAK4wt was immunopurified from stimulated and
unstimulated cell lysates, and kinase activity was measured using Histone H1
as a substrate. PAK4 kinase activity was very low in starved cells, which
suggests that in MDCK cells PAK4 is not constitutively activated. PAK4 became
autophosphorylated and had elevated kinase activity 5 minutes after
stimulation with HGF. Kinase activity was maximal (2.5-fold) after 15 minutes,
and then decreased at 30 minutes (Fig.
5A). A similar 2.5-fold activation of kinase activity by HGF has
also been reported for SGK1 (serum- and glucocorticoid-inducible kinase 1) and
PAK1 (Royal et al., 2000
;
Shelly and Herrera, 2002
).
This relatively low fold-activation of kinases is probably due to only a
proportion of cells in epithelial colonies responding to HGF stimulation. As
the HGF receptor Met is localised on the basolateral surface of polarized MDCK
cells (Crepaldi et al., 1994
),
polarised cells at the centre of colonies would be expected to respond very
weakly, if at all, to apically applied HGF.
|
It is unlikely that a co-precipitating kinase is responsible for the
phosphorylation of Histone H1, as no other proteins in the anti-HA
immunoprecipitates became detectably labelled with 32P. In
addition, Histone H1 phosphorylation was reduced in kinase assays with
kinase-defective HA-tagged PAK4M350. Consistent with a previous report
(Callow et al., 2002
), this
mutant retained a low level of kinase activity, but this was lower than that
of PAK4wt (no HGF) or the control (Fig.
5D). Immunoprecipitates of endogenous PAK4 also demonstrated
HGF-stimulated kinase activity towards Histone H1; however, in this case we
cannot rule out the presence of another kinase in the immunoprecipitates (data
not shown).
PAK4-induced rounding is prevented by PI3K inhibitors
PI3K is required for the response of MDCK cells to HGF
(Potempa and Ridley, 1998;
Royal and Park, 1995
).
Pre-incubation with LY294002 (a PI3K inhibitor) inhibits HGF-induced changes
to the actin cytoskeleton and intercellular junctions that are required for
cell migration (Potempa and Ridley,
1998
; Royal and Park,
1995
). PAK4
GBD-induced cell rounding was inhibited by
LY294002 (Fig. 6A) and 20 nM
wortmanin (data not shown). During long-term stimulation with HGF, in the
presence of LY294002, PAK4
GBD-expressing cells remained well spread,
their morphology was indistinguishable from uninjected cells and stress fibres
were readily detected. Furthermore the diffuse cytoplasmic localisation of
PAK4
GBD was similar to that observed for long-term expression of
PAK4
GBD in the absence of HGF (Fig.
2i). To determine when PI3K activity was required during the
response to HGF/PAK4
GBD, we added LY294002 at different time points. If
LY294002 was added 5 minutes after HGF stimulation the scattering response was
reduced but not completely inhibited and PAK4
GBD expression induced a
lower level of cell rounding, whereas LY294002 addition 15 minutes after HGF
had no inhibitory effect on either response (data not shown). As PI3K
inhibition requires time to allow the LY294002 not only to enter cells but
also to bind to the ATP-binding site, these results imply that PI3K activity
is required during the first 30 minutes of HGF stimulation for PAK4
GBD
to induce a morphological response. This is consistent with PI3K being
required for PAK4 activation, which peaks during the first 30 minutes after
HGF addition (Fig. 5A).
|
In contrast to its effect on PAK4GBD-induced rounding, LY294002 was
unable to block PAK4
-induced cell rounding
(Fig. 6B). The LY294002 was
clearly active as it inhibited HGF-induced scattering (data not shown). As
PAK4
is constitutively active and lacks the N-terminal regulatory
region of PAK4, these results indicate that PI3K acts upstream of PAK4 to
regulate its activity, rather than downstream or on a parallel pathway to
PAK4.
PAK4wt localisation is regulated by PI3K
We have shown that localisation of PAK4wt at the cell periphery is
dependent on HGF (Fig. 6D)
whilst PAK4GBD is localised at the cell periphery independently of HGF
(Fig. 6A,B). Although LY294002
inhibits PAK4
GBD-induced cell rounding, the transient peripheral
localisation of PAK4
GBD was not significantly inhibited by LY294002
(Fig. 6C). There was a slight
reduction in the percentage of PAK4
GBD-expressing cells with a
peripheral localisation (Table
1), but no inhibition of stress fibre disassembly (data not
shown). In contrast, LY294002 inhibited the HGF-dependent localisation of
PAK4wt at the cell periphery (Table
1; Fig. 6D). This
suggests that HGF-stimulated responses are PI3K-dependent, whereas the
transient responses to PAK4
GBD in unstimulated cells are
PI3K-independent.
PAK4 kinase activity is inhibited by LY294002
We have demonstrated that PAK4GBD-induced cell rounding requires an
active kinase domain and can be inhibited by the PI3K inhibitor, LY294002. We
therefore investigated the effect of LY294002 on PAK4 kinase activity. HGF
stimulated PAK4
GBD kinase activity was partially inhibited by LY294002
(Fig. 5B). Similarly, in the
presence of LY294002 there was a reduction in the activation of PAK4wt kinase
activity by HGF (Fig. 5C).
![]() |
Discussion |
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Although it has previously been shown that PAK1 kinase activity is
regulated by PI3K downstream of heregulin
(Adam et al., 1998), and
downstream of Ras during cell transformation
(Tang et al., 1999
), this is
the first time that PI3K has been shown to influence not only kinase activity
but also subcellular localisation of a group II PAK. Rounding induced by the
PAK4 kinase domain alone (PAK4
) is insensitive to PI3K inhibitors,
implying that the N-terminal region of PAK4 acts as an autoinhibitor of kinase
activity.
The fact that inhibition of PI3K did not perturb the transient
HGF-independent localisation of PAK4GBD at the cell periphery and loss
of stress fibres, but did inhibit the longer-term HGF-regulated
PAK4
GBD-induced cell rounding, suggests that PAK4 is regulated at
multiple levels. As described for group I PAKs, the N-terminal region of PAK4
may be subject to regulation both through protein-protein interaction and
phosphorylation (Daniels and Bokoch,
1999
; Zhao et al.,
2000a
). We predict that, like group I PAKs, PAK4 normally exists
in a folded inactive state, mediated in part by the GBD region of the protein
binding to the kinase domain. PAK4
GBD presumably exists in a partially
unfolded active conformation, as it lacks the GBD domain. However, it can
still be stimulated by HGF through PI3K, leading to further activation and
thereby cell rounding. This stimulation via PI3K could further reduce
autoinhibition by the N-terminal region, or directly increase the kinase
activity. In addition, we propose that PAK4
GBD is downregulated
following long-term expression in unstimulated cells, which explains the
reappearance of stress fibres and loss of peripheral localisation, despite
continuous expression of PAK4
GBD. PAK4
is insensitive to this
downregulation as it lacks the regulatory N-terminal domain.
It is possible that PAK4 is regulated by phosphorylation downstream of
PI3K. Little is known about the regulation of mammalian PAK kinase activity by
phosphorylation, although PAK1 has multiple autophosphorylation sites
(Chong et al., 2001). However,
recent data suggest that PAK1 is a substrate for 3-phosphoinositide-dependent
kinase-1 (PDK1) phosphorylation (King et
al., 2000
) and that Akt can stimulate its kinase activity
independently of Rac and Cdc42 (Tang et
al., 2000
). In addition, Dictyostelium PAKa
phosphorylation by Akt is thought to mediate kinase activation in response to
cAMP stimulation (Chung and Firtel,
1999
).
We, like others (Abo et al.,
1998; Dan et al.,
2001
; Qu et al.,
2001
), were unable to elicit any morphological effects by
expressing PAK4wt alone. Indeed, most of the reported morphological effects of
PAK1 have been identified through expression of an activated protein
(Daniels et al., 1999
;
Manser et al., 1997
), probably
because wild-type proteins are rapidly turned off after stimulation. However,
by using a mildly activated protein we have been able to study activation of
PAK4 by HGF. Expression of PAK4
GBD in fibroblasts and macrophages fails
to cause cell rounding in either unstimulated or growth-factor-stimulated
cells (C.M.W. and A.J.R., unpublished), although in endothelial (PAE) cells
wild-type PAK4 cooperates with Cdc42 to induce filopodium extension
(Abo et al., 1998
;
Qu et al., 2001
), and in
fibroblasts constitutively active PAK4 transiently induces filopodia
(Qu et al., 2001
). Together,
these results suggest that the response to PAK4 is cell-type specific.
Our data indicate that activated Cdc42-V12 does not stimulate PAK4wt to
induce cell rounding, consistent with previous observations that binding of
Cdc42 to PAK4 does not stimulate kinase activity
(Abo et al., 1998;
Lee et al., 2002
). However,
recent evidence suggests that expression of Cdc42-V12 leads to increased PAK4
phosphorylation (Callow et al.,
2002
). It is possible that Cdc42 affects PAK4 phosphorylation in
cells by bringing it into the vicinity of other kinases. Indeed, in
endothelial cells re-localisation of PAK4 to the Golgi by Cdc42-V12 is
required for induction of filopodia (Abo et
al., 1998
). It will therefore be interesting to investigate
whether Cdc42 affects the localization of PAK4 in MDCK cells.
Previous reports of activated PAK1-induced cell rounding have inferred that
the loss of cell-substratum adhesions (and subsequent cell rounding) are a
consequence of de-regulated turnover of focal adhesions
(Manser et al., 1997). It is
probable that the progressive decrease in substratum adhesion induced by
PAK4
GBD also reflects a failure to make or retain focal complexes in
MDCK cells responding to HGF. There is considerable evidence to suggest that
PAKs are involved in cell-substratum adhesion. Adhesion to fibronectin
strongly stimulates PAK1 kinase activity
(Price et al., 1998
) and PAK3
can bind to and phosphorylate the focal adhesion/complex component paxillin
(Hashimoto et al., 2001
). It
has recently been suggested that PAK1-induced loss of focal complexes could be
achieved through its interaction with a complex of proteins including the
PAK-interacting exchange factor PIX and a paxillin-interacting GIT1 family
member (de Curtis, 2001
;
Zhao et al., 2000b
). However,
PAK4 does not interact with PIX (Abo et
al., 1998
), and we have been unable to demonstrate an association
between paxillin and PAK4 by co-immunoprecipitation (data not shown). In
addition, kinase-inactive PAK1 has been reported to localise to focal
complexes (Manser et al.,
1997
), but we did not observe any localisation to focal complexes
of PAK4wt, kinase-inactive PAK4 or PAK
GBD (data not shown). It is
therefore likely that PAK4 affects focal complexes via a different mechanism
to PAK1, and indeed the respective localisations of PAK1 and PAK4 in
HGF-stimulated cells imply that they play distinct roles. Whereas PAK1 was
reported to localise within lamellipodia
(Royal et al., 2000
),
activated PAK4 is localised to the cell periphery but not specifically to
lamellipodia. To complement our studies and those of others using exogenously
expressed PAKs, it will be important in the future to devise methods to
inhibit each PAK selectively, in order to elucidate their respective
contributions to morphological responses.
To date, the only substrate for PAK4 identified is LIMK1, which can also be
phosphorylated by PAK1 (Dan et al.,
2001). Activated LIMK1 in turn induces phosphorylation of cofilin,
an actin filament-depolymerising protein that is inactivated by LIMK1
phosphorylation (Arber et al.,
1998
). Increased cofilin phosphorylation should therefore lead to
an increase in polymerised actin in cells. However, expression of
dominant-negative LIMK1 and cofilin mutants affects the rounding response to
PAK4, suggesting that it might in part contribute to PAK4 morphological
changes (Dan et al., 2001
).
How cofilin phosphorylation contributes to PAK4-induced responses is unclear,
as cofilin is generally believed to act in the lamellipodia to promote actin
filament turnover (Carlier et al.,
1999
). PAK4 undoubtedly has additional substrates in addition to
LIMK1, which will contribute to PAK4 responses.
In conclusion, we have demonstrated for the first time growth-factor-induced activation of a group II PAK, and investigated the signalling pathways regulating PAK4 that lead to cell rounding. We have identified PI3K as a key regulator of PAK4 responses and localisation. As both PAK4 and PAK1 are activated by HGF but have different intracellular localisations, they may act together to mediate HGF-induced dissolution of stress fibres and reorganisation of cell-substratum adhesions, which are essential for initiation of cell migration.
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Acknowledgments |
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Footnotes |
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References |
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---|
Abo, A., Qu, J., Cammarano, M. S., Dan, C., Fritsch, A., Baud,
V., Belisle, B. and Minden, A. (1998). PAK4, a novel effector
for Cdc42Hs, is implicated in the reorganization of the actin cytoskeleton and
in the formation of filopodia. EMBO J.
17,6527
-6540.
Adam, L., Vadlamudi, R., Kondapaka, S. B., Chernoff, J.,
Mendelsohn, J. and Kumar, R. (1998). Heregulin regulates
cytoskeletal reorganization and cell migration through the p21-activated
kinase-1 via phosphatidylinositol-3 kinase. J. Biol.
Chem. 273,28238
-28246.
Arber, S., Barbayannis, F. A., Hanser, H., Schneider, C., Stanyon, C. A., Bernard, O. and Caroni, P. (1998). Regulation of actin dynamics through phosphorylation of cofilin by LIM-kinase. Nature 393,805 -809.[CrossRef][Medline]
Bokoch, G. M., Wang, Y., Bohl, B. P., Sells, M. A., Quilliam, L.
A. and Knaus, U. G. (1996). Interaction of the Nck adapter
protein with p21-activated kinase (PAK1). J. Biol.
Chem. 271,25746
-25749.
Bottaro, D. P., Rubin, J. S., Faletto, D. L., Chan, A. M., Kmiecik, T. E., Vande Woude, G. F. and Aaronson, S. A. (1991). Identification of the hepatocyte growth factor receptor as the c-met proto-oncogene product. Science 251,802 -804.[Medline]
Burbelo, P. D., Drechsel, D. and Hall, A.
(1995). A conserved binding motif defines numerous candidate
target proteins for both Cdc42 and Rac GTPases. J. Biol.
Chem. 270,29071
-29074.
Callow, M. G., Clairvoyant, F., Zhu, S., Schryver, B., Whyte, D.
B., Bischoff, J. R., Jallal, B. and Smeal, T. (2002).
Requirement for PAK4 in the anchorage-independent growth of human cancer cell
lines. J. Biol. Chem.
277,550
-558.
Carlier, M. F., Ressad, F. and Pantaloni, D.
(1999). Control of actin dynamics in cell motility. Role of
ADF/cofilin. J. Biol. Chem.
274,33827
-33830.
Chong, C., Tan, L., Lim, L. and Manser, E.
(2001). The mechanism of PAK activation. Autophosphorylation
events in both regulatory and kinase domains control activity. J.
Biol. Chem. 276,17347
-17353.
Chung, C. Y. and Firtel, R. A. (1999). PAKa, a
putative PAK family member, is required for cytokinesis and the regulation of
the cytoskeleton in dictyostelium discoideum cells during chemotaxis.
J. Cell Biol. 147,559
-576.
Crepaldi, T., Pollack, A. L., Prat, M., Zborek, A., Mostov, K. and Comoglio, P. M. (1994). Targeting of the SF/HGF receptor to the basolateral domain of polarized epithelial cells. J. Cell Biol. 125,313 -320.[Abstract]
Dan, C., Kelly, A., Bernard, O. and Minden, A.
(2001). Cytoskeletal changes regulated by the PAK4
serine/threonine kinase are mediated by LIM kinase 1 and cofilin.
J. Biol. Chem. 276,32115
-32121.
Dan, C., Nath, N., Liberto, M. and Minden, A.
(2002). PAK5, a New Brain-Specific Kinase, Promotes Neurite
Outgrowth in N1E-115 Cells. Mol. Cell. Biol.
22,567
-577.
Daniels, R. H. and Bokoch, G. M. (1999). p21-activated protein kinase: a crucial component of morphological signaling? Trends Biochem. Sci 24,350 -355.[CrossRef][Medline]
Daniels, R. H., Zenke, F. T. and Bokoch, G. M.
(1999). alphaPix stimulates p21-activated kinase activity through
exchange factor-dependent and -independent mechanisms. J. Biol.
Chem. 274,6047
-6050.
de Curtis, I. (2001). Cell migration: GAPs
between membrane traffic and the cytoskeleton. EMBO
Rep. 2,277
-281.
Dechert, M. A., Holder, J. M. and Gerthoffer, W. T.
(2001). p21-activated kinase 1 participates in tracheal smooth
muscle cell migration by signaling to p38 Mapk. Am. J. Physiol.
Cell. Physiol. 281,C123
-C132.
Dharmawardhane, S., Sanders, L. C., Martin, S. S., Daniels, R.
H. and Bokoch, G. M. (1997). Localization of p21-activated
kinase 1 (PAK1) to pinocytic vesicles and cortical actin structures in
stimulated cells. J. Cell Biol.
138,1265
-1278.
Dowrick, P. G., Prescott, A. R. and Warn, R. M. (1991). Scatter factor affects major changes in the cytoskeletal organization of epithelial cells. Cytokine 3, 299-310.[Medline]
Frost, J. A., Khokhlatchev, A., Stippec, S., White, M. A. and
Cobb, M. H. (1998). Differential effects of PAK1-activating
mutations reveal activity-dependent and -independent effects on cytoskeletal
regulation. J. Biol. Chem.
273,28191
-28198.
Hashimoto, S., Tsubouchi, A., Mazaki, Y. and Sabe, H.
(2001). Interaction of paxillin with p21-activated Kinase (PAK).
Association of paxillin alpha with the kinase-inactive and the Cdc42-activated
forms of PAK3. J. Biol. Chem.
276,6037
-6045.
Jaffer, Z. M. and Chernoff, J. (2002). p21-Activated kinases: three more join the Pak. Int. J. Biochem. Cell Biol. 34,713 -717.[CrossRef][Medline]
King, C. C., Gardiner, E. M., Zenke, F. T., Bohl, B. P., Newton,
A. C., Hemmings, B. A. and Bokoch, G. M. (2000).
p21-activated kinase (PAK1) is phosphorylated and activated by 3-
phosphoinositide-dependent kinase-1 (PDK1). J. Biol.
Chem. 275,41201
-41209.
Kiosses, W. B., Daniels, R. H., Otey, C., Bokoch, G. M. and
Schwartz, M. A. (1999). A role for p21-activated kinase in
endothelial cell migration. J. Cell Biol.
147,831
-844.
Lee, S. R., Ramos, S. M., Ko, A., Masiello, D., Swanson, K. D.,
Lu, M. L. and Balk, S. P. (2002). AR and ER interaction with
a p21-activated kinase (PAK6). Mol. Endocrinol.
16, 85-99.
Manser, E., Chong, C., Zhao, Z. S., Leung, T., Michael, G.,
Hall, C. and Lim, L. (1995). Molecular cloning of a new
member of the p21-Cdc42/Rac-activated kinase (PAK) family. J. Biol.
Chem. 270,25070
-25078.
Manser, E., Huang, H. Y., Loo, T. H., Chen, X. Q., Dong, J. M., Leung, T. and Lim, L. (1997). Expression of constitutively active alpha-PAK reveals effects of the kinase on actin and focal complexes. Mol. Cell. Biol. 17,1129 -1143.[Abstract]
Manser, E., Loo, T. H., Koh, C. G., Zhao, Z. S., Chen, X. Q., Tan, L., Tan, I., Leung, T. and Lim, L. (1998). PAK kinases are directly coupled to the PIX family of nucleotide exchange factors. Mol. Cell 1,183 -192.[Medline]
Naldini, L., Weidner, K. M., Vigna, E., Gaudino, G., Bardelli, A., Ponzetto, C., Narsimhan, R. P., Hartmann, G., Zarnegar, R., Michalopoulos, G. K. et al. (1991). Scatter factor and hepatocyte growth factor are indistinguishable ligands for the MET receptor. EMBO J. 10,2867 -2878.[Abstract]
Pawson, T. and Scott, J. D. (1997). Signaling
through scaffold, anchoring, and adaptor proteins.
Science 278,2075
-2080.
Potempa, S. and Ridley, A. J. (1998).
Activation of both MAP kinase and phosphatidylinositide 3-kinase by Ras is
required for hepatocyte growth factor/scatter factor-induced adherens junction
disassembly. Mol. Biol. Cell
9,2185
-2200.
Price, L. S., Leng, J., Schwartz, M. A. and Bokoch, G. M.
(1998). Activation of Rac and Cdc42 by integrins mediates cell
spreading. Mol. Biol. Cell
9,1863
-1871.
Qu, J., Cammarano, M. S., Shi, Q., Ha, K. C., de Lanerolle, P.
and Minden, A. (2001). Activated pak4 regulates cell adhesion
and anchorage-independent growth. Mol. Cell. Biol.
21,3523
-3533.
Ridley, A. J., Comoglio, P. M. and Hall, A. (1995). Regulation of scatter factor/hepatocyte growth factor responses by Ras, Rac, and Rho in MDCK cells. Mol. Cell. Biol. 15,1110 -1122.[Abstract]
Royal, I. and Park, M. (1995). Hepatocyte
growth factor-induced scatter of Madin-Darby canine kidney cells requires
phosphatidylinositol 3-kinase. J. Biol. Chem.
270,27780
-27787.
Royal, I., Lamarche-Vane, N., Lamorte, L., Kaibuchi, K. and
Park, M. (2000). Activation of cdc42, rac, PAK, and
rho-kinase in response to hepatocyte growth factor differentially regulates
epithelial cell colony spreading and dissociation. Mol. Biol.
Cell 11,1709
-1725.
Sells, M. A. and Chernoff, J. (1997). Emerging from the Pak: the p21-activated protein kinase family. Trends Cell Biol. 7,162 -167.[CrossRef]
Sells, M. A., Knaus, U. G., Bagrodia, S., Ambrose, D. M., Bokoch, G. M. and Chernoff, J. (1997). Human p21-activated kinase (Pak1) regulates actin organization in mammalian cells. Curr. Biol. 7,202 -210.[Medline]
Sells, M. A., Boyd, J. T. and Chernoff, J.
(1999). p21-activated kinase 1 (Pak1) regulates cell motility in
mammalian fibroblasts. J. Cell Biol.
145,837
-849.
Sells, M. A., Pfaff, A. and Chernoff, J.
(2000). Temporal and spatial distribution of activated Pak1 in
fibroblasts. J. Cell Biol.
151,1449
-1458.
Shelly, C. and Herrera, R. (2002). Activation
of SGK1 by HGF, Rac1 and integrin-mediated cell adhesion in MDCK cells:
PI-3K-dependent and -independent pathways. J. Cell
Sci. 115,1985
-1993.
Stoker, M., Gherardi, E., Perryman, M. and Gray, J. (1987). Scatter factor is a fibroblast-derived modulator of epithelial cell mobility. Nature 327,239 -242.[CrossRef][Medline]
Symons, M., Derry, J. M., Karlak, B., Jiang, S., Lemahieu, V., McCormick, F., Francke, U. and Abo, A. (1996). Wiskott-Aldrich syndrome protein, a novel effector for the GTPase CDC42Hs, is implicated in actin polymerization. Cell 84,723 -734.[Medline]
Tang, Y., Yu, J. and Field, J. (1999). Signals
from the Ras, Rac, and Rho GTPases converge on the Pak protein kinase in Rat-1
fibroblasts. Mol. Cell. Biol.
19,1881
-1891.
Tang, Y., Zhou, H., Chen, A., Pittman, R. N. and Field, J.
(2000). The Akt proto-oncogene links Ras to Pak and cell survival
signals. J. Biol. Chem.
275,9106
-9109.
Teo, M., Manser, E. and Lim, L. (1995).
Identification and molecular cloning of a p21cdc42/rac1-activated
serine/threonine kinase that is rapidly activated by thrombin in platelets.
J. Biol. Chem. 270,26690
-26697.
Turner, C. E., Brown, M. C., Perrotta, J. A., Riedy, M. C.,
Nikolopoulos, S. N., McDonald, A. R., Bagrodia, S., Thomas, S. and Leventhal,
P. S. (1999). Paxillin LD4 motif binds PAK and PIX through a
novel 95-kD ankyrin repeat, ARF-GAP protein: A role in cytoskeletal
remodeling. J. Cell Biol.
145,851
-863.
Weidner, K. M., Sachs, M. and Birchmeier, W. (1993). The Met receptor tyrosine kinase transduces motility, proliferation, and morphogenic signals of scatter factor/hepatocyte growth factor in epithelial cells. J. Cell Biol. 121,145 -154.[Abstract]
Yang, F., Li, X., Sharma, M., Zarnegar, M., Lim, B. and Sun,
Z. (2001). Androgen Receptor specifically interacts with a
novel p21-activated kinase, PAK6. J. Biol. Chem.
276,15345
-15353.
Zeng, Q., Lagunoff, D., Masaracchia, R., Goeckeler, Z., Cote, G.
and Wysolmerski, R. (2000). Endothelial cell retraction is
induced by PAK2 monophosphorylation of myosin II. J. Cell
Sci. 113,471
-482.
Zhao, Z. S., Manser, E., Chen, X. Q., Chong, C., Leung, T. and
Lim, L. (1998). A conserved negative regulatory region in
alphaPAK: inhibition of PAK kinases reveals their morphological roles
downstream of Cdc42 and Rac1. Mol. Cell. Biol.
18,2153
-2163.
Zhao, Z. S., Manser, E. and Lim, L. (2000a).
Interaction between PAK and nck: a template for Nck targets and role of PAK
autophosphorylation. Mol. Cell. Biol.
20,3906
-3917.
Zhao, Z. S., Manser, E., Loo, T. H. and Lim, L.
(2000b). Coupling of PAK-interacting exchange factor PIX to GIT1
promotes focal complex disassembly. Mol. Cell. Biol.
20,6354
-6363.