* Department of Immunology, and Department of Cell Biology, The Scripps Research Institute, La Jolla, California 92037; and § Department of Medicine, University of California San Diego, La Jolla, California 92037
The mechanisms through which the small GTPases Rac1 and Cdc42 regulate the formation of membrane ruffles, lamellipodia, and filopodia are currently unknown. The p21-activated kinases (PAKs) are direct targets of active Rac and Cdc42 which can induce the assembly of polarized cytoskeletal structures when expressed in fibroblasts, suggesting that they may play a role in mediating the effects of these GTPases on cytoskeletal dynamics.
We have examined the subcellular localization of endogenous PAK1 in fibroblast cell lines using specific PAK1 antibodies. PAK1 is detected in submembranous vesicles in both unstimulated and stimulated fibroblasts that colocalize with a marker for fluid-phase uptake. In cells stimulated with PDGF, in v-Src-transformed fibroblasts, and in wounded cells, PAK1 redistributed into dorsal and membrane ruffles and into the edges of lamellipodia, where it colocalizes with polymerized actin. PAK1 was also colocalized with F-actin in membrane ruffles extended as a response to constitutive activation of Rac1. PAK1 appears to precede F-actin in translocating to cytoskeletal structures formed at the cell periphery. The association of PAK1 with the actin cytoskeleton is prevented by the actin filament-disrupting agent cytochalasin D and by the phosphatidylinositol 3-kinase inhibitor wortmannin. Co-immunoprecipitation experiments demonstrate an in vivo interaction of PAK1 with filamentous (F)-actin in stimulated cells. Microinjection of a constitutively active PAK1 mutant into Rat-1 fibroblasts overexpressing the insulin receptor (HIRcB cells) induced the formation of F-actin- and PAK1-containing structures reminiscent of dorsal ruffles. These data indicate a close correlation between the subcellular distribution of endogenous PAK1 and the formation of Rac/Cdc42-dependent cytoskeletal structures and support an active role for PAK1 in regulating cortical actin rearrangements.
A variety of growth factors, oncogenes, chemokines,
and extracellular matrix components induce dramatic morphological and cytoskeletal changes in
cells. The polymerization of cortical actin and the associated production of membrane ruffles and lamellipodia are
important components of cellular motile responses and
may regulate other aspects of cellular signaling as well
(Stossel, 1993 The mechanisms by which Rac and Cdc42 initiate and
regulate the formation of cytoskeletal structures are not
currently understood. Evidence has been obtained that in
some systems Rac and related GTPases can regulate actin
polymerization through their ability to modulate cellular
levels of phosphatidylinositol 4-monophosphate via phosphatidylinositol (PI)1 5-kinase (Chong et al., 1994 The ability of PAKs to regulate MAP kinase activities is
analogous to the role of the PAK homolog Ste20 in the
pheromone response pathway of Saccharomyces cerevisiae
(Herskowitz, 1995 Cell Culture and Preparation for Analysis
Cell culture and maintenance techniques were identical to those described
in Ridley (1995) Cells were prepared for adhesion assays as described in Hotchin and
Hall (1995) Transfection of Swiss 3T3 Cells with Semliki
Forest Virus
The cDNA fragment encoding Rac1 wild-type and mutations Q61L and
T17N were amplified by PCR using primers that contain a BamHI restriction enzyme site and a myc tag at the 5 Affinity Purification of Antibodies
PAK1 was purified as a glutathione-S-transferase fusion protein from Escherichia coli as described in Knaus et al. (1995)
Immunofluorescence Microscopy
Cells were prepared and fixed according to Ridley (1995) Cells were examined using a confocal scanning microscope (MRC600;
Bio Rad, Hercules, CA) equipped with an argon-krypton mixed gas laser.
The confocal unit was attached to an inverted microscope (IM35M; Zeiss,
Inc., Thornwood, NY) and the data collected using a 63× oil immersion
lens. Side by side images were collected using the K1/K2 block combination, merged together, and processed with the COMOS software (Bio
Rad). Images were photographed on a slave monitor, the Focus Imagecorder Plus (Focus Graphics, Inc.) with a 35-mm camera back and Kodak
print film (Royal Gold ASA25).
Cellular controls treated with anti-PAK1 alone or fluorescein-labeled
goat anti-rabbit antibody alone did not show significant fluorescence in
either the fluorescein or rhodamine channels. Cells treated with either
rhodamine phalloidin or primary antibody, followed by either fluorescein-
or rhodamine-conjugated secondary antibody, did not exhibit any crossover fluorescence between the fluorescein and rhodamine channels.
Subcellular Fractionation
Quiescent, serum-starved Swiss 3T3 cells were incubated with ±5 ng/ml
PDGF for 6, 9, or 10 min before fractionation by the method of Krek et al.
(1992) Wounding Technique
The wound healing model (Todaro et al., 1967 Coimmunoprecipitation Assay
Quiescent, serum-starved Swiss 3T3 cells were stimulated with 5 ng/ml
PDGF for 10 min and harvested from plates by addition of 500 µl ice-cold
lysis buffer containing 1% NP40/100-mm plate. The clarified cell lysates
were incubated with 25 µl of either anti-PAK1 or preimmune serum. Both
batches of antibody were preadsorbed against an actin/CNBr/Sepharose
resin, as described in Dharmawardhane et al. (1991) Analysis of Triton-Insoluble Cytoskeletons
Triton-insoluble cytoskeletons were isolated from quiescent- or PDGF-stimulated Swiss 3T3 cells using procedures modified from Dharmawardhane et al. (1991) Kinase Assays
PAK1 activity was assayed as described in Knaus et al. (1995) Microinjection of PAK1 Mutants into HIRcB Cells
Microinjections were performed as described in Martin et al. (1996) PAK1 Is Activated by PDGF in Swiss 3T3 Cells
We examined the effect of PDGF on the kinase activity of
endogenous PAK1 in Swiss 3T3 cells. PAK1 autocatalytic
activity was rapidly stimulated by PDGF (Fig. 1), with enhanced activity detected within 1 min and maximum stimulation seen between 2 and 5 min in various experiments.
PDGF also stimulated phosphorylation of the exogenous
substrate, MBP, in the PAK1 immunoprecipitates (not shown).
PAK1 Localizes to Pinocytic Vesicles in Unstimulated
Swiss 3T3 Cells
Since stimulation of cells by PDGF induces the formation
of Rac-dependent cytoskeletal structures (Ridley et al.,
1992
Confocal imaging (see Fig. 4) indicated that these assemblies did not colocalize with F-actin and were found
closely apposed to the plasma membrane. The vesicular
structures did not appear to be artifacts of the method of
fixation, as they were detected using either formaldehyde,
glutaraldehyde, or methanol as fixative. They were not
disrupted by treatment with cytochalasin D, an inhibitor of
actin polymerization, but pretreatment of cells with Triton X-100 before fixation disrupted the vesicles to which
PAK1 localized, suggesting they are indeed membranous
in nature. These vesicles did not represent mitochondria,
as shown by costaining with PAK1 antibody and an antibody to cytochrome oxidase, a mitochondrial membrane
component (Fig. 3 A). Using various organelle markers, we were able to establish that PAK1 was not colocalized
with endoplasmic reticulum (anti-Bip-2, Fig. 3 C) or with
lysosomes and late endosomes (anti-Lamp-2, Fig. 3 B).
The distribution of PAK1 was also distinct from Golgi
(anti-
PAK1 Localizes to Sites of Actin Assembly in
PDGF-stimulated Swiss 3T3 Cells
Very little PAK1 was detected in direct association with
the plasma membrane in the unstimulated cells, which also
contained a minimal cytoskeleton (Fig. 2, A and B). When
cells were treated with 10% fetal calf serum, we observed
extensive formation of actin stress fibers (Fig. 2 F). However, PAK1 did not localize to the stress fibers (Fig. 2, E
and F), nor did it accumulate at the plasma membrane under these conditions. In contrast to the distribution of
PAK1 in resting cells, when cells were stimulated with
PDGF we observed that PAK1 now became evident in areas of active cytoskeletal rearrangement. At 10 min after
stimulation, PAK1 was detected in membrane ruffles and
at the edges of lamellipodia (Figs. 2, C and D, and 4). We
observed that endogenous Rac1 was also localized to areas
of active membrane ruffling (not shown). Such changes in
cortical actin induced by PDGF are known to be Rac dependent and are inhibited by the PI 3-kinase inhibitor
wortmannin (Wennström et al., 1994 The apparent colocalization of PAK1 with F-actin in
membrane ruffles was verified by confocal microscopy
(Fig. 4). PAK1 was detected in large membrane ruffles
that often appeared at one end of the cell (Fig. 4, A and
D). In some cells we observed ruffles that contained PAK1
primarily at the very edge of the active ruffle, while
smaller ruffles deficient in PAK1 could be seen on the
same cell (e.g., see cell in Fig. 4 A). In cells expressing dorsal ruffles the overlap of PAK1 with F-actin was particularly evident (Fig. 4, B and C). In data obtained from three
representative experiments (481 total cells counted), we
detected PAK1 associated with 117 of 166 membrane ruffles (70%), in 86 of 88 lamellipodia (97%), and in 199 of
216 dorsal ruffles (92%). While few in number in Swiss
3T3 cells, in areas where actin microspikes or filopodial extensions were evident, PAK1 was found to colocalize
with F-actin in 18 of 20 (90%) of these structures as well.
Because the localization of PAK1 in the polarized fibroblasts appeared to be primarily at the leading edge, we examined the localization of PAK1 in Swiss 3T3 cells during
a wound healing response. About 3 h after the initial
wounding, fibroblasts display a polarization response at
the edge of the wound and begin to migrate into the
wound itself (Conrad et al., 1993 PAK1 Localizes to Cortical Actin Structures in
v-src-transformed 10 T1/2 Fibroblasts
To determine whether PAK1 was associated with similar
cytoskeletal structures in cells in which actin rearrangements were induced by means other than PDGF, we examined v-src-transformed 10 T1/2 cells. As compared to a
control cell line, the v-src-transformed cells were characterized by loss of actin stress fibers, extension of numerous
lamellipodia, and areas of active membrane ruffling (Fig.
2, G and H), as previously described (Luttrell et al., 1988 Rac Induces Relocalization of PAK1 into
Membrane Ruffles
We examined whether induction of membrane ruffling by
expression of a constitutively active Rac1 was sufficient to
induce the redistribution of PAK1 into the ruffles. Quiescent Swiss 3T3 cells were infected using Semliki Forest virus containing the cDNAs for wild-type and mutant Rac1.
Expression of lac Z gene control, wild-type Rac1, or inactive Rac1 T17N had no effect on cell morphology or PAK1
distribution (not shown). Cells expressing constitutively
active Rac1 Q61L formed extensive peripheral membrane ruffles. Fig. 5 shows that PAK1 becomes relocalized into
the area of membrane ruffling induced by the activated
Rac1. Thus, Rac-mediated cytoskeletal rearrangement is
associated with the movement of PAK1 into the induced
structures.
PAK1 Localization Precedes F-Actin Assembly in
PDGF-induced Ruffles
To examine the redistribution of PAK1 in response to
PDGF in more detail, we analyzed cells at various times
after stimulation with PDGF (Fig. 6). At early times up to
3 min after stimulation there was little F-actin staining, and
PAK1 was found in the pinocytic vesicles only. By 6 min after PDGF addition, we observed PAK1 staining in what
appeared to be early dorsal ruffles at the cell surface. Surprisingly, these structures did not contain F-actin. However, at longer times (9 min) polymerized actin appeared
in dorsal ruffles, where they colocalized with PAK1 by
confocal microscopy, as in Fig. 4. These data suggest that
translocation of PAK1 either precedes recruitment of
F-actin into the forming ruffles, or that PAK1 may be involved in the polymerization of F-actin at these sites.
Constitutively Active PAK1 Mutant Induces Formation
of Dorsal Ruffles
We have demonstrated that a dominant active PAK1
(H83L,H86L) mutant is capable of inducing the formation
of polarized lamellipodia and ruffles in Swiss 3T3 cells
(Sells et al., 1997
PAK1 and Focal Complexes
Swiss 3T3 cells trypsinized from quiescent cultures were
plated on fibronectin immediately after PDGF addition.
In contrast to the cells shown in Figs. 2-6, these cells are
just forming cell-substrate attachments and exhibit a
spreading phenotype at early times. We observed that
PAK1 is found at the cell periphery at 15 min after addition of PDGF and plating on fibronectin, while there is little rhodamine-phalloidin staining evident at the edges of the cells (Fig. 8). Staining of cells at this stage with an anti-actin antibody to detect both F- and G-actin confirmed
this observation, as there was very little actin detected at
the cell edge (data not shown). By 20 min after PDGF and
plating on fibronectin, a wave of F-actin begins to move
toward the cell periphery. Similar actin "rings" or peripheral "arcs" have been described previously in human
erythroleukemia cells spreading on fibronectin (Niu and Nachmias, 1994
Focal complexes have been shown to be triggered by the
combined effect of extracellular matrix, via activation of
integrin receptors, and activity of Rac and Cdc42 (Hotchin
and Hall, 1995 PAK1 Translocates to a Membrane Fraction and Is
Associated with F-actin in PDGF-stimulated Cells
The PDGF-stimulated redistribution of PAK1 to the
plasma membrane in areas of membrane ruffling was verified by preparing subcellular fractions from cells in the
presence or absence of PDGF. As shown in Fig. 9 A
(right), in unstimulated Swiss 3T3 cells, PAK1 was located
primarily in the cytosolic fraction, with very little PAK1 in
the membrane fraction. Isolation of a highly enriched nuclear fraction demonstrated the absence of significant immunodetectable PAK1 associated with nuclei, in either the
presence or absence of PDGF (Fig. 9 A, left). Treatment of
cells with PDGF caused a substantial time-dependent increase in the amount of PAK1 that became associated
with the membrane fraction, peaking at 9-10 min (Fig. 9
B). The increase of PAK1 in the membrane fraction at 6 min
corresponds to the appearance of PAK1 as discrete circular rings on the cell surface, which precede the formation
of dorsal ruffles at 9 min after PDGF addition (Fig. 6).
This membrane fraction represents primarily plasma membrane (Thom et al., 1977 The physical association of PAK1 with F-actin was
probed in PAK1 immunoprecipitates (Fig. 10). We observed a band reactive with a specific actin antibody in the
PAK1 immunoprecipitates from PDGF-stimulated cells
(Fig. 10, inset). Scanning densitometry of the actin band
detected in PAK1 immunoprecipitates shows that a statistically significant amount of actin is associated with PAK1
in cell lysates treated with PDGF when compared to the
association of actin in the absence of PDGF. The amount
of actin associated with anti-PAK1 immunoprecipitates
from control cells was similar to the amount of actin associated nonspecifically with immunoprecipitates using pre-immune serum. It cannot be determined at this point whether the association of F-actin with PAK1 immunoprecipitates represents a direct interaction or whether it involves indirect association mediated by other protein components.
Although PAK1 has been identified as a direct target for
the small GTPases Rac and Cdc42, its role in mediating
the biochemical responses of these GTPases remains unclear. The present data indicate that PAK1 is closely physically associated with the formation of Rac- and Cdc42-dependent actin structures. Stimuli that induce membrane
ruffling and the formation of lamellipodia and filopodia, including PDGF, transformation by v-src and constitutive
activation of Rac1, cause PAK1 to associate with these dynamic cytoskeletal structures, where it is closely localized
with F-actin.
We observed that PAK1 kinase activity is stimulated
within 1 min after addition of PDGF (Fig. 1), and changes
in the intracellular distribution of PAK1 occur within 5 min
of PDGF stimulation. PAK1 localizes in ring-like structures at the cell surface at early times (6-9 min after stimulation) which ultimately evolve into F-actin-containing
dorsal ruffles (Fig. 6). Introduction of a cytoskeletally active PAK1(H83L,H86L) into the insulin-responsive Rat-1
cell line, HIRcB, induced the formation of very similar ring-like structures containing F-actin (Fig. 7). PAK1 colocalizes with F-actin in these dorsal ruffles and in membrane ruffles subsequently formed at later times. When
Swiss 3T3 cells were plated on fibronectin-coated surfaces
and stimulated with PDGF, we observed that PAK1
clearly preceded the appearance of F-actin in the cell cortex (Fig. 8). Taken together with the results of Sells et al.
(1997) At later times after cell activation by PDGF, some of the
cells assumed a polarized phenotype in which PAK1 became colocalized with F-actin in lamellipodia and associated membrane ruffles at the leading edge or "front" of
the cell. PAK1 was clearly absent in the smaller ruffles
near the "rear" of the cell (see Fig. 4 A), indicating that it
is not a required component for all types of ruffling. A
similar distribution of PAK1 at the leading edge was observed in polarized cells during the wound healing response (Fig. 2, I and J). This data suggests that PAK1 may
play a role in directed cellular movement by regulating actin dynamics at the leading edge of cells. Alternatively, association of PAK1 with F-actin may be transient, with
PAK1 dissociating from the older ruffles at the back of the
cell. However, in support of the former, we have observed
that the cytoskeletal assemblies induced by PAK1 in fibroblasts appear polarized in nature and are very reminiscent
of motile cells (Sells et al., 1997 The ability of exogenously introduced PAK1 to regulate
actin assembly is dependent upon an amino-terminal, proline-rich domain in PAK1 which has a predicted PXXP
SH3-binding motif (Sells et al., 1997 Whether PAK1 amino-terminal binding proteins actually regulate localized actin assembly at appropriate sites
or whether this interaction mediates the recruitment of
PAK1-target protein complexes to membrane-associated
sites of actin assembly is still undetermined. It is of particular interest that we observed PAK1 to precede the
appearance of F-actin in the cell cortex of adherent PDGF-stimulated Swiss 3T3 cells (Fig. 6 A). A transient association with focal complexes is suggested by the appearance
of PAK1 in focal complex-like structures in stimulated cells,
although confocal imaging reveals that there is only partial
overlap with the majority of phosphotyrosine-containing focal complexes (Fig. 8 B). As observed with formation of
dorsal and membrane ruffles, where PAK1 precedes the
apparent recruitment of F-actin, PAK1 may be transiently
required during an early stage of focal complex formation
and/or during their dissolution as well (Manser et al., 1997 Recently, POR1 (partner of Rac) was identified as a potential regulator of Rac-mediated membrane ruffling (Van
Aelst et al., 1996 We have demonstrated in the current study that PAK1
translocates into Rac- and Cdc42-dependent actin structures in Swiss 3T3, 10 t1/2, and Rat-1 (HIRcB) cells, where
PAK1 is closely associated with F-actin. This conclusion is
supported by immunofluorescence and confocal microscopy, as well as biochemical data. In light of recent evidence that introduction of constitutively active PAK1 proteins into cells is able to modulate the actin cytoskeleton, the current results establish that endogenous PAK1 does
translocate to areas of actin polymerization in response to
physiological Rac-dependent signals. Moreover, these
studies provide additional insights into the role of PAK1
in modulating various aspects of cytoskeletal dynamics,
suggesting it may participate in an early stage of actin recruitment and/or polymerization. The molecular mechanisms that regulate the association of PAK1 with F-actin and the protein targets through which PAK1 regulates the
cytoskeleton in motile cells remain to be established.
; Mitchison and Cramer, 1996
). Recent work
has implicated members of the Rho family of GTPases as
mediators of cytoskeletal changes (Ridley et al., 1992
; Hall,
1994
; Kozma et al., 1995
; Nobes and Hall, 1995
). Rac1 mediates the effects of many hormones and oncogenes on
formation of cortical actin structures (Hall, 1994
). Thus,
introduction of dominant negative forms of Rac into cells
inhibits, while active Rac mutants effectively induce,
membrane ruffling, lamellipod formation, and pinocytosis
(Ridley et al., 1992
). Similarly, the related GTPase Cdc42
regulates the extension of actin filament bundles into filopodia (Kozma et al., 1995
; Nobes and Hall, 1995
). Both Rac
and Cdc42 also induce the formation of multimolecular focal complexes distinct from the focal adhesions induced by
Rho (Nobes and Hall, 1995
).
; Hartwig
et al., 1995
) and/or arachidonic acid release via regulation
of PLA2 (Peppelenbosch et al., 1995
). Recently, a direct
target for active Rac has been identified as a family of
serine/thrionine kinases known as p21-activated kinases or
PAKs (Manser et al., 1994
, 1995
; Bagrodia et al., 1995b
;
Knaus et al., 1995
; Martin et al., 1995
). The activity of
PAKs is stimulated by the binding of GTP-bound Rac or
Cdc42. Reports show that both G protein-coupled receptors and cytokine receptors regulate PAK activity (Knaus
et al., 1995
; Zhang et al., 1995
). PAKs have been implicated in phosphorylation of the p47phox component of the Rac-regulated NADPH oxidase (Knaus et al., 1995
) and in
the activation of a Rac/Cdc42-controlled kinase cascade
leading to stimulation of the stress-activated MAP kinases,
p38 and JNK (Bagrodia et al., 1995a
; Zhang et al., 1995
).
), where it regulates a MAP kinase signaling cascade. Ste20 also plays important roles in regulating polarized cell growth, presumably through effects on
the actin cytoskeleton (Chant and Stowers, 1995
; Cvrckova et al., 1995
; Leeuw et al., 1995
; Zarzov et al., 1996
), as
does pak1+ in fission yeast (Ottilie et al., 1995
). Recently,
we have demonstrated that mammalian PAK1 can initiate
cytoskeletal rearrangements reminiscent of those produced by Rac and/or Cdc42 (Sells et al., 1997
). Introduction of activated forms of PAK1 into Swiss 3T3 fibroblasts
causes the formation of membrane ruffles, lamellipodia,
and filopodia. In this paper, we provide further evidence
in support of a role for PAK1 in regulating actin assembly
by demonstrating that endogenous PAK1 becomes co-localized with filamentous (F)-actin at the edge of lamellipodia and in dorsal and membrane ruffles induced by distinct upstream stimuli. PAK1 physically associates with
polymerized actin in PDGF-stimulated cells and may play
a role in initiating and/or integrating the formation of
F-actin-containing cytoskeletal structures.
Materials and Methods
. Swiss 3T3 cells were grown in DME containing 10% fetal bovine serum. v-src-transformed NIH3T3 cells and their control cells
were grown in DME containing 10 µg/ml G418 (Luttrell et al., 1988
). 5-7 d
after seeding, the confluent cells were incubated overnight (16 h) in DME
to obtain serum-free conditions. Immediately before fixation, cells were
treated with 3-5 ng/ml PDGF BB (Upstate Biotechnology, Inc., Lake
Placid, NY). In some experiments, cells were pretreated with 10 µM cytochalasin D or 100 nM wortmannin for 10 min before stimulation with
PDGF.
. Briefly, confluent Swiss 3T3 cells were serum starved overnight and then trypsinized and washed in serum-free DME. The cells were
resuspended in the same buffer and stimulated with 5 ng/ml PDGF and
plated onto coverslips coated with 50 µg/ml fibronectin. Cells were fixed
at the indicated times after plating.
end. The myc-tagged Rac constructs were subcloned into the BamHI site of pSFV3 (Life Technologies,
Gaithersburg, MD) and recombinant virus generated. Swiss 3T3 cells
were infected with Rac or Lac Z containing virus in serum-free media and
allowed to grow 13-15 h before experiment. Gene expression was confirmed by immunofluorescence using anti-myc (9E10).
and was coupled to cyanogen bromide-activated Sepharose 4B (Pharmacia Fine Chemicals, Piscataway, NJ). Anti-PAK1 antibody R626, prepared as detailed in Knaus et
al. (1995)
, and 2124/3, directed against PAK1 residues 174-306, were affinity-purified using this resin as described in Schneider et al. (1982)
. The
monospecificity of the affinity-purified polyclonal anti-PAK1 antibodies
was confirmed by Western blotting against Swiss 3T3 subcellular fractions
(see Fig. 9).
Fig. 9.
(A) Detection of PAK1 in Swiss 3T3 subcellular fractions. Serum-starved cells with either no addition (PDGF) or
with addition (+PDGF) of 5 ng/ml PDGF for 10 min were separated into membrane (M) and cytosolic (C) fractions and immunoblotted for PAK1 using affinity-purified anti-PAK1 antibody,
as described in Materials and Methods. Alternatively, highly purified nuclei (N) were prepared as described in Materials and
Methods for immunoblotting. PAK1 was detected as a single
band at 68-kD that comigrated with authentic human PAK1 expressed in Cos cells (last lane). (B) Detection of PAK1 in the
membrane fraction during PDGF stimulation. Membrane fractions of quiescent Swiss 3T3 cells with either no addition or at 6 and 9 min after the addition of 5 ng/ml of PDGF were immunoblotted for PAK1 using affinity-purified anti-PAK1 antibody.
Quantitation of the 68-kD band was by phosphoimager analysis.
The mean density of the 68-kD band from unstimulated membrane fractions was set at 100%. The data shown represent the
mean ± SEM of three separate experiments. The presence of 20-
40% of total PAK1 immunoreactivity in the isolated membrane
fraction from PDGF-stimulated but not unstimulated cells was
consistently observed. (Inset) Representative Western blot of membrane fractions blotted for PAK1. Arrow indicates 68-kD band.
[View Larger Versions of these Images (28 + 25K GIF file)]
. Briefly, quiescent Swiss 3T3 cells on coverslips were stimulated with 3 to 5 ng/ml PDGF
for 10 min and fixed in either 3% paraformaldehyde or 3.7% formaldehyde (Sigma Chemical Co., St. Louis, MO) for 15 min, or in 100% MeOH
at
20°C for 10 min. After fixation with formaldehyde, the cells were permeabilized for 5 min in 0.2% Triton X-100. Coverslips were then incubated with 0.5 µg/ml rhodamine phalloidin (Molecular Probes, Inc., Eugene, OR) and 20 µg/ml affinity-purified polyclonal anti-PAK1 R626; 20 µg/ml affinity-purified polyclonal anti-PAK1 2124/3; 1:50 polyclonal anti-Rac1 R785 (Quinn et al., 1993
); 1:100 anti-BiP (StressGen, Biotechnologies, Victoria, BC, Canada); 1:50 anti-cytochrome oxidase (Molecular
Probes, Inc.); or 1:10 anti-LAMP-2 (Granger et al., 1990
) for 1 h. After
extensive washing in 10 mM Hepes/0.5 M NaCl buffer, pH 7.4, containing
0.1% saponin, the coverslips were stained with 1:200 fluorescein-conjugated anti-rabbit IgG (Cappel Laboratories, Cochranville, PA) and 1:50 rhodamine-conjugated anti-mouse IgG, or 1:200 fluorescein-conjugated anti-mouse IgG and 1:50 rhodamine-conjugated anti-rabbit IgG for 1 h.
For localization of focal complexes, cells were incubated for 1 h with rabbit polyclonal anti-PAK1 and mouse monoclonal anti-phosphotyrosine
(4G10; Upstate Biotechnology, Inc.), mouse monoclonal anti-vinculin
(Sigma Chemical Co.), or mouse monoclonal anti-actin (Sigma Chemical
Co.). After washing, the coverslips were stained with fluorescein-conjugated goat anti-rabbit IgG and rhodamine-conjugated goat anti-mouse
IgG (Cappel). Washed coverslips were mounted in Slow Fade (Molecular
Probes, Inc.) and examined by light and confocal microscopy.
. Cells were then harvested in ice-cold trypsin-EDTA and washed
in 1 mM Hepes/NaCl buffer, pH 7.5. Approximately 107 cells were resuspended in 300 µl ice-cold hypotonic buffer containing 20 mM Hepes-KOH, pH 7.5, 5 mM KCl, 1.5 mM MgCl2, 1 mM dithiothreitol, and protease cocktail (1 µg/ml each of chymostatin, leupeptin, and pepstatin, 1 mM PMSF, 2 µg/ml aprotinin, 0.2 mM sodium vanadate). After incubation on ice for 10 min, cells were homogenized using 15 strokes in a
Dounce homogenizer and centrifuged at 2,000 rpm for 10 min. The resulting supernatant was spun at 10,000 g for 1 h in a centrifuge (TL-100; Beckman Instruments, Fullerton, CA) according to Thom et al. (1977)
. The
membrane pellet obtained was dissolved in 250 µl 1× Laemmli sample
buffer. 50 µl of 4× sample buffer was added to the supernatant (cytosolic)
fraction to bring the total volume to 250 µl. Highly purified Swiss 3T3 nuclei, prepared according to the procedure of Schreiber et al. (1989)
, were
generously provided by P. Maher, (The Scripps Research Institute, La
Jolla, CA); these nuclear preparations have been characterized and shown
to be free of contaminating organelles (Maher, 1996
). The samples were
analyzed on 10% SDS-polyacrylamide gels and either stained with Coomassie brilliant blue to analyze total protein or Western blotted using a
1:1,000 dilution of affinity-purified PAK1 antibody or a 1:500 dilution of
rabbit polyclonal anti-actin (Sigma Chemical Co.). Proteins were then detected either by autoradiography with I125 protein A or alkaline phosphatase-conjugated anti-rabbit IgG (Bio Rad). Bands of interest were
quantified by scanning densitometry using the phosphoimager system
from Molecular Dynamics (Sunnyvale, CA).
) was used to obtain polarized Swiss 3T3 cells. Confluent cells were wounded by a 1-2-mm-wide
slash through the cell monolayer using a sharp razor blade. Polarized cells
were fixed and stained for PAK1 and F-actin, as described, at 3 and 6 h after wounding.
, to eliminate IgG
molecules that may bind to actin. After 2 h at 4°C, 80 µl of 50% protein
A-Sepharose was added to the mixture and incubated for 1 h at 4°C. The
immunoprecipitates were recovered and washed twice in wash buffer with
1% NP40 containing 200 mM KCl and four times in the same buffer without NP40. The use of the high ionic strength buffer inhibits nonspecific
binding of actin to IgG (Fechheimer et al., 1979
). The washed immunoprecipitates were resuspended in 100 µl 1× Laemmli sample buffer and
analyzed by SDS-PAGE and Western blotting using anti-actin, as described above. The 42-kD actin band was quantified by scanning densitometry (Molecular Dynamics).
and Burgess et al. (1989)
. Confluent cells on 100-mm
Petri dishes were lysed in 1 ml of pH 7.5 buffer containing 75 mM KCl, 2 mM
MgCl2, 5 mM EDTA, 5 mM DTT, 20 mM PIPES, 1 mM ATP, protease inhibitors, and 0.5% Triton X-100. After extraction on ice for 5 min, the lysates were centrifuged at 14,000 rpm for 3 min at 4°C. The Triton-insoluble cytoskeletons recovered in the pellets were dissolved in 100 µl of 1×
Laemmli sample buffer and analyzed by SDS-PAGE and Western blotting for PAK1.
using both
autophosphorylation and phosphorylation of the exogenous substrate myelin basic protein (MBP) to assess activity. Unstimulated cells or cells
stimulated with 3 ng/ml PDGF for the indicated times were lysed and immunoprecipitated with PAK1 antibody as described above for actin analysis. The immunoprecipitates were washed twice with 1 ml of lysis buffer
with 1% NP40, twice in the same buffer without NP40, and twice with kinase buffer consisting of 50 mM Hepes, pH 7.5, 10 mM MgCl2, 2 mM
MnCl2, 0.2 mM DTT. Washed pellets were then subjected to in vitro kinase assays (Knaus et al., 1995
).
.
Briefly, HIRcB cells grown on glass coverslips were rendered quiescent by
starvation for 36-48 h in serum-free DME. Expression vectors (Sells et al.,
1997
) for PAK1 wild-type and PAK1 (H83L,H86L) suspended in microinjection buffer (5 mM NaPO4 and 100 mM KCl, pH 7.4) at a concentration
of 0.1 mg/ml, were injected directly into the nucleus using glass capillary
needles. 6-8 h after injection, cells were fixed with 3.7% formaldehyde in
PBS and fluorescently stained for protein expression and actin localization. Primary incubation with anti-myc (9E10) ascites fluid (1:500 in PBS
for 1 h at room temperature) was followed by incubation with a fluorescein-conjugated goat anti-mouse antibody (Jackson ImmunoResearch,
West Grove, PA) diluted 1:100 in PBS for 1 h at room temperature.
Rhodamine-phalloidin (Sigma Chemical Co.) was added to the secondary
incubation at a concentration of 0.5 µg/ml.
Results
Fig. 1.
Stimulation of PAK1 activity by PDGF in Swiss 3T3
cells. Swiss 3T3 cells were stimulated with 3 ng/ml PDGF for the
indicated times and PAK1 activity determined in immunoprecipitates by in vitro kinase assays as described in Materials and Methods. Activity at t = 0 was set as 100%. Results shown are representative of four similar experiments.
[View Larger Version of this Image (11K GIF file)]
; Hall, 1994
) and increases PAK1 activity, we examined the subcellular localization of endogenous PAK1 in
unstimulated and PDGF-stimulated Swiss 3T3 cells using
affinity-purified polyclonal antibody against full length
PAK1. In unstimulated cells, PAK1 was sparsely distributed throughout the cytoplasm but was particularly evident in structures that had the appearance of elongated
vesicles (Fig. 2, A and B). These structures were dispersed
throughout the cell, with increased abundance apparent
around the nucleus. An identical vesicular distribution was
observed with the PAK1 peptide antibody. Rac1 was
present in similar structures as determined using polyclonal anti-Rac1, R785 (Quinn et al., 1993
), and a commercial anti-Rac1 (Santa Cruz Biotechnology, Santa Cruz, CA).
Fig. 2.
PAK1 and F-actin
localization in Swiss 3T3 fibroblasts. (Left column)
Cells stained with affinity-purified anti-PAK1 antibody; (right column) cells stained
with rhodamine phalloidin.
(A and B) Serum-starved
cells with no addition or (C
and D) stimulated for 10 min
with 3 ng/ml PDGF. (E and
F) Cells in fetal bovine serum.
(G and H) v-Src-transformed 10 t1/2 cells; untransformed 10 t1/2 controls were
similar in appearance to the
serum-starved Swiss 3T3
controls in A and B and are
thus not shown here. I and J
show a polarized Swiss 3T3
cell migrating into the area of
a wound. All procedures
were as described in Materials and Methods. Arrows indicate areas where PAK1
and F-actin colocalize in
membrane ruffles. Micrographs shown are at 5,000×
(A-F, I, and J) and 7,000× (G and H).
[View Larger Version of this Image (90K GIF file)]
-mannosidase) or microtubules (anti-tubulin); data
not shown. However, when cells were allowed to take up
BSA-rhodamine as a marker for fluid phase uptake, the
BSA colocalized with the PAK1-containing vesicles as determined by confocal microscopy (Fig. 3 D). In support of
an association of PAK1 with pinocytic vesicles, we observed
that PAK1 became diffusely distributed throughout the
cytoplasm when the cells were treated with amiloride, a
potent inhibitor of Na+/H+ exchange, which is known to
block pinocytosis (West et al., 1989
; data not shown).
Fig. 4.
PAK1 and F-actin distribution after PDGF addition. Confocal microscopy was performed on Swiss 3T3 cells after PDGF stimulation for 10 min, as described in Materials and Methods. Red, rhodamine-phalloidin staining of F-actin; green, fluorescein staining of
PAK1; yellow, merged images indicating the areas of colocalization. PAK1 and F-actin are colocalized in dorsal ruffles (A and C), membrane ruffles (A and B), and lamellipodia (D). Bar, 15 µm.
[View Larger Version of this Image (80K GIF file)]
Fig. 3.
Confocal micrographs of cells immunostained for PAK1 and cytoplasmic organelle markers. Swiss 3T3 cells in serum were
fixed in 100% methanol and costained with anti-PAK1, and antibodies to organelle markers as described in Materials and Methods. (A, B, and D) Red, rhodamine staining of cytochrome oxidase (mitochondrial marker), LAMP-2 (lysosomal marker), and BSA, respectively; green, fluorescein staining of PAK1. (C) Green, fluorescein staining of BiP (endoplasmic reticulum marker); red, rhodamine
staining of PAK1; yellow, areas of colocalization.
[View Larger Version of this Image (33K GIF file)]
; Nobes et al., 1995
).
Treatment of the PDGF-stimulated Swiss 3T3 cells with
either the F-actin-disrupting agent cytochalasin D (10 µM) or the PI 3-kinase inhibitor wortmannin (100 nM)
caused the complete loss of membrane ruffles, and in the
presence of these inhibitors we no longer observed membrane-associated PAK1.
). As shown in Fig. 2 (I
and J), PAK1 is found within the leading lamellae of the
polarized cells migrating towards the wound.
). PAK1 was clearly localized in membrane ruffles and particularly at the ruffling tips of lamellipodial extensions.
Taken together, these data show that induction of cortical
actin structures by distinct stimuli in different cell types is
associated with the translocation of PAK1 into these structures.
Fig. 5.
PAK1 and F-actin
localization in Swiss 3T3 cells
expressing dominant active
Rac1. Swiss 3T3 cells infected with Rac1 (Q61L) virus were fixed after 14 h and
immunostained for endogenous PAK1 as described in
Materials and Methods.
(Left) PAK1; (right) staining for F-actin using rhodamine-phalloidin.
[View Larger Version of this Image (95K GIF file)]
Fig. 6.
Time course of PAK1 and F-actin localization after PDGF stimulation. (Left column) Cells stained with affinity-purified anti-Pak1 antibody; (right column) cells stained with rhodamine-phalloidin. Cells were fixed at 3, 6, and 9 min after stimulation with PDGF, as described in Materials and Methods. Bars, 15 µm.
[View Larger Version of this Image (154K GIF file)]
). We examined whether this activated
form of PAK1 could induce the formation of dorsal ruffles
in HIRcB cells, as these cells normally exhibit a dramatic dorsal ruffling response when stimulated with insulin (Martin
et al., 1996
). As shown in Fig. 7, PAK1(H83L,H86L) induced the formation of circular ruffles reminiscent of the
dorsal ruffles seen at early times in Swiss 3T3 cells (compare Figs. 4 C and 6). These dorsal ruffles contained both
F-actin and PAK1(H83L,H86L). Interestingly, insulin has
been shown to stimulate PAK activity (Tsakiridis et al.,
1996
), and we observed that endogenous PAK1 was localized in the insulin-induced dorsal ruffles of nontransfected HIRcB cells (data not shown).
Fig. 7.
Actin localization
in HIRcB cells injected with
PAK1. Quiescent HIRcB
cells were microinjected
with myc-tagged expression vectors carrying wild-type
(top row) or mutant (H83L,
H86L) PAK1 cDNA (bottom
row). (Left column) Cells
stained with anti-myc to localize injected PAK1 and to
identify expressing cells.
(Right column) Same cells
stained with rhodamine-phalloidin to detect F-actin.
[View Larger Version of this Image (69K GIF file)]
), as well as in fibroblasts after stimulation
with epidermal growth factor (Chang et al., 1995
) and during epithelial cell migration after wounding (Nusrat et al.,
1992
). At 25-30 min after plating, this spreading F-actin
colocalizes with the PAK1 in membrane ruffles at the edge
of the cell. Membrane ruffles containing both PAK1 and
F-actin are fully formed by 45 min.
Fig. 8.
Distribution of PAK1 and F-actin in Swiss 3T3 cells plated on fibronectin. (A) PAK1 and F-actin distribution after stimulation with PDGF and plating on a fibronectin matrix. Confocal micrographs of quiescent Swiss 3T3 cells at 15, 20, 25, and 30 min after stimulation are shown. Red, Rhodamine-phalloidin staining of F-actin; green, fluorescein staining of PAK1; yellow, merged images indicating the areas of colocalization. (B) PAK1 and phosphotyrosine localization after PDGF stimulation when plated on fibronectin. Confocal
micrographs of Swiss 3T3 cells at 30 and 90 min after stimulation are shown. (30 min) Red, Rhodamine staining of phosphotyrosine;
green, fluorescein staining of PAK1; yellow, merged images indicating the areas of colocalization. (90 min) Red, rhodamine staining of
PAK1: green, fluorescein staining of phosphotyrosine. Bars, 15 µm.
[View Larger Version of this Image (51K GIF file)]
). PAK1(H83L,H86L) can induce the formation of focal contacts when introduced into Swiss 3T3
fibroblasts attached to cell surfaces (Sells et al., 1997
), and
the ability of kinase-active PAK1 mutants to cause actin
stress fiber and focal complex dissolution has been reported (Manser et al., 1997
; Sells et al., 1997
). The PAK-induced focal complexes appear identical to focal complexes induced by Rac and Cdc42 (Nobes and Hall, 1995
)
in that they contain both vinculin and phosphotyrosine-containing proteins. We observed that PAK1 was detected
in the cell periphery of fibronectin-plated cells as a pattern
of discrete point contacts reminiscent of focal complexes
(Fig. 8 B, left). Staining with antibodies against phosphotyrosine (and vinculin; not shown) revealed only intermittent colocalization of PAK1 with the phosphotyrosine-containing focal complexes. There were clearly distinct
foci of phosphotyrosine staining that were devoid of PAK1
and vice versa. While overlap was noted in some complexes, these were only a fraction of the total. The relationship of the PAK1-containing foci to the phosphotyrosine- and vinculin-containing complexes is thus unclear.
It is possible that the points of staining observed represent
distinct stages in focal complex assembly/disassembly with
which PAK1 is transiently associated. PAK1 clearly was
not detected in classical Rho-dependent focal adhesions
(Fig. 8 B, right).
; Krek et al., 1992
), probably containing membrane-associated cytoskeletal elements. It is
unlikely that there is significant contamination with the pinocytic vesicles with which PAK1 is associated, as we detected very little PAK1 in this fraction from unstimulated cells. In other experiments, we detected reproducible
PDGF-induced increases in the amount of PAK1 found in
the Triton X-100-insoluble cytoskeletal fraction (not shown).
These findings are consistent with the immunolocalization
studies we have described herein.
Fig. 10.
Coprecipitation of PAK1 with F-actin in cellular immunoprecipitates. PAK1 was immunoprecipitated from quiescent Swiss 3T3 cell lysates before and after PDGF addition (5 ng/
ml) and immunoblotted with an anti-actin antibody, as described.
Quantitation of the 42-kD actin band (see inset below) was by
phosphoimager analysis (Molecular Probes, Inc.). The mean density of the 42-kD band from unstimulated pre-immune serum immunoprecipitates was set at 100%. The data shown represent the
mean ± SEM of duplicate determinations from three separate
experiments. (Inset) Representative Western blot of PAK1 immunoprecipitates immunostained for actin. Arrow indicates 42-kD actin band that comigrated with the purified rabbit actin
(Sigma Chemical Co.) standard (Std). The upper band in the immunoprecipitates is IgG heavy chain.
[View Larger Version of this Image (24K GIF file)]
Discussion
, these data suggest a role for PAK in initiating the
assembly of specific cytoskeletal elements. It is interesting
to speculate that PAK may play a role in regulating actin
assembly through effects on membrane flux. Bretscher
(1996)
has described a model by which regulated membrane flow into the leading edge of motile cells drives cell
locomotion. While envisioned as a polarized endocytic cycle involving coated pits, regulation of membrane flux via
a pinocytic mechanism could also account for the provisions of this model. The formation of dorsal ruffles has
been associated with the process of macropinocytosis
(Dowrick et al., 1993
), and it is of particular interest that
we observe PAK1 to colocalize with pinocytic vesicles
(Fig. 3). Formation of pinocytic vesicles has been shown to
be regulated by Rac (Ridley et al., 1992
) and we will direct
future studies at determining whether PAK1 is a mediator of the effects of Rac on fluid-phase pinocytosis and, potentially, cell movement.
).
). We have shown that
this domain does serve as a functional SH3-binding site, as
it effectively interacts with the second SH3 domain of the
Nck adapter protein (Bokoch et al., 1996
; Galisteo et al.,
1996
). It is possible that PAK binds an SH3 domain-containing protein that physically interacts with F-actin, as an
analogous situation has been described in S. cerevisiae
(Leeuw et al., 1995
). In this species, a protein known as
Bem1 binds to the yeast PAK homolog, Ste20 kinase,
through an SH3 domain present on Bem1. This interaction
with Bem1 mediates a physical association of Ste20 with
F-actin. In the present study, we have demonstrated that
increased amounts of F-actin are also associated with
mammalian PAK1 in immunoprecipitates from PDGF-stimulated cells. This interaction is likely to be mediated
by additional protein components as well, as we have been
unable to detect direct binding of F-actin to PAK1 in preliminary experiments. These data suggest a scenario in
which the formation of Rac-GTP induced by PDGF leads
to direct PAK1 activation. Binding of Rac-GTP to PAK1
will change the conformation of the amino terminus such
that the SH3-binding domain is exposed and/or has a
higher affinity for SH3-containing target proteins. These
putative target proteins, serving as the functional equivalent of Bem1 in yeast, would enable PAK1 to associate
with F-actin. In support of this model, we observed that
expression of the constitutively active Rac1(Q61L) mutant
in Swiss 3T3 cells induced the relocalization of PAK1 into
cortical actin structures (Fig. 5).
;
Sells et al., 1997
).
). Truncated versions of POR1 block both
Ras- and Rac-induced ruffling responses. Addition of
POR1 to cells microinjected with dominant active Ras enhanced subsequent ruffling, although POR1 itself could
not induce cytoskeletal changes. Therefore, other effectors may also be involved in regulation of the actin cytoskeleton by Rac, and it is possible that POR1 acts in concert
with PAK1 to regulate membrane cytoskeletal dynamics.
Similarly, Wiscott-Aldrich syndrome protein (WASP) is a
target for Cdc42 and has been shown to induce the formation of poorly characterized actin clusters when overexpressed in cells (Symons et al., 1996
). Both WASP (Rivero-Lezcan et al., 1995
) and PAK (Bokoch et al., 1996
;
Galisteo et al., 1996
) have been shown to interact with distinct SH3 domains on the adapter protein Nck. A complex
involving these two proteins could be important for regulation of filopodia by Cdc42.
Received for publication 22 October 1996 and in revised form 26 June 1997.
Please address all correspondence to Dr. Gary M. Bokoch, Department of Immunology-IMM14, The Scripps Research Institute, 10550 N. Torrey Pines Road, La Jolla, CA 92037. Tel.: (619) 784-8217; Fax: (619) 784-8218.This work was supported by National Institutes of Health grants GM39434 and GM44428 (to G.M. Bokoch). S.S. Martin was supported by grant DK33651 to Dr. Jerrold M. Olefsky. This is publication No. 10378-IMM from The Scripps Research Institute.
F-actin, filamentous actin; MBP, myelin basic protein; PI, phosphatidylinositol.
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