P21-activated kinase 4 interacts with integrin alpha v beta 5 and regulates alpha v beta 5-mediated cell migration
p21-activated kinase 1 (PAK1) can affect cell migration (Price et al., 1998; del Pozo et al., 2000) and modulate myosin light chain kinase and LIM kinase, which are components of the cellular motility machinery (Edwards, D.C., L.C. Sanders, G.M. Bokoch, and G.N. Gill. 1999. Nature Cell Biol. 1:253-259; Sanders, L.C., F. Matsumura, G.M. Bokoch, and P. de Lanerolle. 1999. SCIENCE: 283:2083-2085). We here present a novel cell motility pathway by demonstrating that PAK4 directly interacts with an integrin intracellular domain and regulates carcinoma cell motility in an integrin-specific manner. Yeast two-hybrid screening identified PAK4 binding to the cytoplasmic domain of the integrin beta 5 subunit, an association that was also found in mammalian cells between endogenous PAK4 and integrin alpha v beta 5. Furthermore, we mapped the PAK4 binding to the membrane-proximal region of integrin beta 5, and identified an integrin-binding domain at aa 505-530 in the COOH terminus of PAK4. Importantly, engagement of integrin alpha v beta 5 by cell attachment to vitronectin led to a redistribution of PAK4 from the cytosol to dynamic lamellipodial structures where PAK4 colocalized with integrin alpha v beta 5. Functionally, PAK4 induced integrin alpha v beta 5-mediated, but not beta1-mediated, human breast carcinoma cell migration, while no changes in integrin cell surface expression levels were observed. In conclusion, our results demonstrate that PAK4 interacts with integrin alpha v beta 5 and selectively promotes integrin alpha v beta 5-mediated cell migration.
The Rockefeller University Press, 0021-9525/2002/09/1287/11 $5.00
The Journal of Cell Biology, Volume 158, Number 7, September 30, 2002 1287–1297
p21-activated kinase 4 interacts with integrin
5-mediated cell migration
and Staffan Strömblad
Karolinska Institutet, Department of Microbiology, Pathology, and Immunology, SE-141 86 Huddinge, Sweden
Södertörns Högskola, SE-141 89 Huddinge, Sweden
21-activated kinase 1 (PAK1) can affect cell migration
(Price et al., 1998; del Pozo et al., 2000) and modulate
myosin light chain kinase and LIM kinase, which are
components of the cellular motility machinery (Edwards,
D.C., L.C. Sanders, G.M. Bokoch, and G.N. Gill. 1999.
. 1:253–259; Sanders, L.C., F. Matsumura,
G.M. Bokoch, and P. de Lanerolle. 1999.
2083–2085). We here present a novel cell motility pathway
by demonstrating that PAK4 directly interacts with an integrin
intracellular domain and regulates carcinoma cell motility
in an integrin-speciﬁc manner. Yeast two-hybrid screening
identiﬁed PAK4 binding to the cytoplasmic domain of the
5 subunit, an association that was also found in
mammalian cells between endogenous PAK4 and integrin
5. Furthermore, we mapped the PAK4 binding to the
membrane-proximal region of integrin
5, and identiﬁed
an integrin-binding domain at aa 505–530 in the COOH
terminus of PAK4. Importantly, engagement of integrin
5 by cell attachment to vitronectin led to a redistribution
of PAK4 from the cytosol to dynamic lamellipodial structures
where PAK4 colocalized with integrin
PAK4 induced integrin
5–mediated, but not
human breast carcinoma cell migration, while no changes
in integrin cell surface expression levels were observed. In
conclusion, our results demonstrate that PAK4 interacts
5 and selectively promotes integrin
mediated cell migration.
The p21-activated kinase (PAK)* family contains homologous
serine/threonine protein kinases that can act as downstream
effectors of the small GTPases Cdc42 and Rac (Lim et al.,
1996; Bagrodia and Cerione, 1999; Bar-Sagi and Hall, 2000).
So far, six human PAKs (hPAKs) have been identified, and
based on homology, they can be classified into two groups:
group I including PAK1–3 and group II including PAK4–6
(Dan et al., 2001b). A marked difference between the two
PAK groups is the autologous inhibitory sequence in the
-terminal regulatory domain found in group I PAKs,
with no obvious homologous sequence in group II (Dan et
al., 2001b). Due to the presence of an inhibitory sequence
that binds to the COOH terminus of group I PAKs, PAK1
displays little or no endogenous kinase activity, but can be
activated by the presence of GTP-bound active Cdc42 or
Rac, which opens up the folded structure of PAK1 (Lei et
al., 2000). PAK1 is known to regulate cell morphology and
cytoskeletal reorganization (Sells et al., 1997). Further-
more, membrane-targeted PAK1 has been found to induce
neurite outgrowth from PC12 cells (Daniels et al., 1998).
In fibroblasts, activated PAK1 has been shown to localize
in the leading edge of motile cells (Sells et al., 1999), and
in human endothelial cells, PAK1 has been suggested to
coordinate the formation of new substrate adhesions at the
front of the cell with contraction and detachment at the rear
(Kiosses et al., 1999), indicating that PAK1 may be involved in
the regulation of cell motility.
PAK4 is the first identified member of the group II
PAKs, and is implicated in cytoskeletal reorganization and
filopodia formation (Abo et al., 1998). Importantly, PAK4
was recently found to be overexpressed in 78% of a variety
of human cancer cell lines, an overexpression that might be
mediated by gene amplification and may play a role in Ras-
mediated transformation (Callow et al., 2002). In addition,
overexpression of a hyperactive PAK4 mutant can protect
cells from apoptosis induced by TNF
(Gnesutta et al.,
2001). The capability of PAK4 to promote cell survival is
Address correspondence to Staffan Strömblad, Karolinska Institutet,
Department of Microbiology, Pathology, and Immunology, Huddinge
University Hospital F46, SE-141 86 Huddinge, Sweden. Tel.: 46-8-585-
81032. Fax: 46-8-585-81020. E-mail: email@example.com
*Abbreviations used in this paper: hPAK, human PAK; IBD, integrin-
binding domain; IP, immunoprecipitation; KD, kinase domain; LIMK,
LIM kinase; MBT, mushroom body tiny gene product; MLCK, myosin
light chain kinase; mPAK, mouse PAK; PAK, p21-activated kinase; PLL,
-lysine; VN, vitronectin; wt, wild-type.
Key words: cell motility; cell signaling; integrin; lamellipodia; p21-activated
1288 The Journal of Cell Biology
Volume 158, Number 7, 2002
shared with PAK1, but the anti-apoptotic properties of
PAK1 and PAK4 may be mediated by distinct mechanisms
(Schurmann et al., 2000; Gnesutta et al., 2001). In addi-
tion, hyperactive PAK4 is able to transform fibroblasts to
grow in soft agar in an anchorage-independent manner
(Qu et al., 2001), perhaps in part due to it’s ability to pro-
mote cell survival.
Integrins are heterodimeric transmembrane receptors and
the major group of receptors for ECM proteins. Integrins are
essential during development, in tissue homeostasis, and in
the progression of various diseases (Hynes, 1992; Giancotti
and Ruoslahti, 1999). By mediating cellular attachment to
ECM, integrins are also a central part of the cellular motility
machinery, where they are regulated by intracellular signaling
molecules, which influence integrin localization, clustering,
and binding to the ECM. In addition, integrin engagement
to the ECM initiates various signaling events, e.g., activation
of the ERK1/2 pathway, which is also important for the reg-
ulation of cell motility (Giancotti and Ruoslahti, 1999).
Previous studies have shown that
v integrins are up-
regulated or activated in migratory and invasive mecha-
nisms in vivo, including wound healing, angiogenesis,
and metastasis (Felding-Habermann and Cheresh, 1993;
Brooks et al., 1994; Friedlander et al., 1995; Strömblad et
al., 1996; Brooks et al., 1997). Integrin
5 is the pre-
dominant vitronectin (VN) receptor for carcinoma cells in
vivo, because most carcinoma specimens from patients ex-
5 but not
3 (Lehmann et al., 1994; Jones et
al., 1997). Importantly, integrin
5 has been found to
be functionally involved in growth factor–induced carci-
noma cell migration in vitro and metastasis in vivo (Klemke
et al., 1994; Yebra et al., 1996; Brooks et al., 1997).
Furthermore, activation of integrin
5 is implicated in
VEGF-induced angiogenesis (Friedlander et al., 1995). In
this report, we present a novel role for PAK in cell motility.
We found that PAK4 directly interacts with the integrin
5 subunit and specifically regulates
Identified interactors with human integrin
5 subunit cytoplasmic domain by yeast two-hybrid screening
Interacting cDNA clones Number of clones GenBank/EMBL/DDBJ accession no.
PAK4 6 AJ011855
Myosin X 5 AJ249706
Bodenin (ICAP) 3 AJ001373
Miz-1 9 AF039567
Mouse cDNA clone 1 AA445617
Mouse myotubes MPLRB5 1 AA816006
Six clones were identical to mPAK4. Five clones represented mouse myosin-X (Yonezawa et al., 2000) and bodenin is the mouse version of human integrin
cytoplasmic domain–associated protein (ICAP) (Faisst and Gruss, 1998). Miz-1 is a Myc-interacting zinc finger protein (Seoane et al., 2001). The other two
clones have unknown functions.
Figure 1. PAK4 interacts with the
integrin 5 subunit. (A) Amino acid
sequence comparison of mPAK4 KD,
found by the yeast two-hybrid screening
with hPAK4, Drosophila MBT (MBT), and
hPAK1 KDs. Dashes stand for identical
amino acids and the variations of amino
acids are indicated by letters in hPAK4,
MBT, and hPAK1 sequences. The
mPAK4 KD is numbered according to
that of hPAK4 (Abo et al., 1998). (B)
PAK4 association with integrin 5 in
GST pull-down assays. GST–PAK4 KD
fusion protein pulled down endogenous
integrin 5 in cell lysates (top panel).
GST–5 cytoplasmic domain fusion
protein pulled down overexpressed HA–
PAK4 (bottom). The positive controls
represent Western blot of the input lysates
for marking the size and showing the
presence of integrin 5 and PAK4. (C) PAK4 association with integrins in living cells. (C, top) Anti–integrin
3 mAb AP3 and anti–integrin v5 mAb P1F6 coimmunoprecipitation of PAK4 in COS-7 cells expressing
HA–PAK4. (C, middle and bottom) An anti-HA mAb coimmunoprecipitated HA–PAK4 with both v and 5
integrin subunits. The positive controls represent Western blot of the input lysates for marking the size and
showing the presence of integrins and PAK4. (D) Association of endogenous PAK4 with endogenous integrin
v5 in MCF-7 cells analyzed by IP. IP using rabbit IgG or -Rab1 are negative controls.
PAK4 interacts with and modulates integrin
5 function |
Zhang et al. 1289
PAK4 directly interacts with the integrin
By means of yeast two-hybrid screening of a 19-d mouse
embryo cDNA library and following remating tests, we
identified six known or hypothetic proteins specifically in-
teracting with the human integrin
5 cytoplasmic domain
(Table I). 25 clones were found to interact with the integrin
5 cytoplasmic domain. Sequence analysis revealed that six
of these mouse cDNA clones encoded a sequence highly ho-
mologous to hPAK4 kinase domain (KD), and were there-
fore identified as mouse PAK4 (mPAK4), which strongly
and specifically interacted with integrin
5 cytoplasmic do-
main in the repeated yeast mating tests (unpublished data).
An aa sequence comparison of the KD of mPAK4 with
hPAK4, hPAK1, and the
PAK homologue mush-
room body tiny gene product (MBT) (Melzig et al., 1998) is
shown in Fig. 1 A. mPAK4 KD shares 98% homology in aa
sequence with hPAK4 KD, 83% with MBT KD, and 56%
with hPAK1. The interaction of PAK4 with integrin
then further analyzed by independent biochemical methods
both in vitro and in living cells
In GST pull-down assays,
we found an association of integrin
5 from cell lysates to a
purified GST-fused PAK4 KD and of PAK4 to a purified
5 cytoplasmic domain (Fig. 1 B). In addition,
HA-tagged PAK4 was coimmunoprecipitated with integrins
5 in living cells (Fig. 1 C, top). In Fig. 1 C, the
3 immunoprecipitation (IP) appears to bring down more
PAK4 than the IP for
5. However, the expression levels
of the two integrins are different (unpublished data) and the
two antibodies used may be differently efficient for IP.
Therefore, differences in PAK4 amounts in this IP cannot
be used to indicate relative binding strengths. Furthermore,
the reverse IP of HA–PAK4 brought down both integrin
5 subunits from a cell lysate (Fig. 1 C, middle and bot-
tom), whereas IP of an irrelevant HA-tagged p21
(unpublished data). Importantly, by IP we also found an as-
sociation of endogenous PAK4 with endogenous integrin
5 in living cells (Fig. 1 D).
PAK4 interacts with the membrane-proximal region
of the integrin
5 cytoplasmic domain
To determine which region within the integrin cytoplasmic
domain interacts with PAK4, we generated cDNAs encoding
various regions of the cytoplasmic domain of integrin
PCR and cloned them into the bait vector pEG202. Yeast
mating experiments were performed using a prey vector that
contains PAK4 KD (aa 239–591) and the various bait vec-
tors. The PAK4-binding region was mapped to aa 759–767
within the integrin
5 cytoplasmic domain (Fig. 2 A). Fur-
thermore, association of endogenous PAK4 to the mem-
brane-proximal region of integrin
5 subunit was verified by
a GST pull-down assay (Fig. 2 B), in which PAK4 associated
5 cytoplasmic domain, but not with a GST–
deletion mutant lacking the PAK4-binding region identified
by yeast mating tests. Amino acid sequences of other integrin
subunits corresponding to the PAK4-binding region of in-
5 cytoplasmic domain were aligned (Fig. 2 C), dis-
playing a moderate sequence homology.
5 interacts with a PAK4
To determine the region within PAK4 that interacts with the
5 cytoplasmic domain, we generated cDNAs en-
coding various regions of the PAK4 KD that were amplified
by PCR and cloned into the prey vector pJG4-5. Yeast mat-
ing experiments were performed using a bait vector that con-
tains the integrin
5 cytoplasmic domain (aa 753–799) and
the various prey vectors. We mapped the integrin-binding
region to aa 505–530 within the PAK4 KD by yeast two-
hybrid mating tests (Fig. 3 A), and further confirmed the re-
quirement of this region of PAK4 for association with inte-
5 in mammalian cells by IP using a PAK4 deletion
mutant (Fig. 3 B). Therefore, we denote this region as the in-
tegrin-binding domain (IBD) in PAK4. The aa sequences of
other PAK family members, including the
homologue MBT, were aligned in comparison with the
PAK4 IBD (Fig. 3 C). The IBD region is highly homologous
among PAK family members, suggesting that family mem-
Figure 2. Mapping of the PAK4 binding region
within the integrin 5 cytoplasmic domain.
(A) Mapping of the PAK4-binding region in the
integrin 5 cytoplasmic domain. Various regions
of the integrin 5 cytoplasmic domain were
cloned into the bait vector pEG202 and then
mated with PAK4 KD in the prey vector in a
yeast two-hybrid assay. The PAK4 binding region
was mapped to aa 759–767 of the 5 cyto-
plasmic domain. (B) Association of endogenous
PAK4 with the membrane-proximal region
within integrin 5 cytoplasmic domain was
examined by a GST pull-down assay, including
GST fused to a 5 deletion mutant lacking the
PAK4-binding region mapped by yeast two-
hybrid analysis. (C) Sequence comparison of
the PAK4-binding region of integrin 5 with
other integrin cytoplasmic domains (top). The
Rack1- (Liliental and Chang, 1998) and PAK4-
binding regions within integrin 5 are indicated
1290 The Journal of Cell Biology | Volume 158, Number 7, 2002
bers other than PAK4 might also hold the capacity to bind to
integrin cytoplasmic domains. In addition, a schematic illus-
tration of known PAK4 functional motifs indicates the loca-
tion of IBD within the PAK4 KD (Fig. 3 D).
Translocation of PAK4 to lamellipodia by integrin
ligation to VN and colocalization with integrin v5
Given that PAK4 associates with integrin v5, we ana-
lyzed the effect on cellular distribution of endogenous
PAK4 by v5-mediated attachment to VN in MCF-7
cells, which exclusively use integrin v5 for attachment to
VN (unpublished data). Before replating, we observed a cy-
tosolic distribution of PAK4 in MCF-7 cells under normal
culture conditions (Fig. 4 A). We then examined the en-
dogenous PAK4 distribution after replating cells onto VN.
Interestingly, we found a remarkable redistribution of
PAK4 to forming lamellipodial structures in the cellular pe-
riphery as early as 10 min after replating on VN. With
longer cell attachment, PAK4 was distributed into mem-
brane ruffles and leading edges. However, cells replated
L-lysine (PLL) that are attached in an integrin-
independent manner remained unspread with PAK4 dis-
Figure 3. Mapping of the integrin 5–binding region within the
PAK4 KD. (A) Various regions of the PAK4 KD were cloned into the
prey vector pJG4-5 and then mated in a yeast two-hybrid assay with
the integrin 5 cytoplasmic domain in the bait vector. Amino acids
505–530 in the PAK4 KD were identified as the responsible region
for interaction with the integrin 5 cytoplasmic domain. Therefore,
PAK4 aa 505–530 is designated as the IBD. (B) PAK4-IBD with
deletion of aa 505–530 was incapable to associate with integrin
v5 in mammalian cells, as examined by an IP analysis. Lower
panel shows expression levels of PAK4 and PAK4-IBD in lysates
used for IP. (C) The corresponding sequences in other PAK family
members are compared with PAK4 IBD with identical amino acids
in bold. (D) Schematic illustration of the PAK4 structure, including
the Cdc42/Rac interactive domain ATP-binding domain, IBD, and
KD within PAK4.
Figure 4. Relocalization of PAK4 to the cell membrane and
colocalization of PAK4 and integrin v5 in MCF-7 cells.
(A) MCF-7 human breast carcinoma cells, under normal culture
conditions or replated onto VN or PLL for indicated times, were
stained for endogenous PAK4 using an anti-PAK4 pAb (green),
for actin using phalloidin–rhodamine (red), and for nuclei by
Hoechst (blue). Arrowheads indicate PAK4 localized to lamellipodia
or ruffles after replating onto VN. (B) MCF-7 cells were replated
onto VN and costained for endogenous PAK4 (green) and
endogenous integrin v5 (red). In the top panel (30 min after
replating), integrin v5 was found at lamellipodia (arrowheads).
In the bottom panel (60 min after replating), integrin v5 has
started to form focal complexes at lamellipodia where it colocalized
with PAK4 (arrowheads). Bars, 10 m.
PAK4 interacts with and modulates integrin v5 function | Zhang et al. 1291
tributed in the cytosol (Fig. 4 A). The fact that attachment
to VN is mediated by integrin v5, whereas attachment to
PLL is integrin independent, indicates that integrin ligation
may specifically stimulate relocalization of PAK4 to lamelli-
podia. Importantly, the redistribution of PAK4 upon cell
attachment may allow PAK4 to associate with integrins in
lamellipodia, which are sites of integrin attachment to the
underlaying ECM. In addition, we observed a similar relo-
calization of PAK4 in M21 human melanoma cells and
ECV 304 human bladder carcinoma cells (unpublished
data), suggesting that the relocalization of PAK4 upon re-
plating onto VN occurs in various cell types.
To examine whether the lamellipodia-localized PAK4
could physically meet with integrin v5 at the cell mem-
brane, we replated MCF-7 cells onto VN and costained the
cells for endogenous PAK4 and endogenous integrin v5.
As shown in Fig. 4 B, PAK4 and integrin v5 colocalized
in lamellipodia shortly after replating onto VN. PAK4 may
therefore be able to engage in integrin-mediated cellular
Relocalization of PAK4 to lamellipodia does not
require its kinase activity, integrin interaction,
or Cdc42/Rac binding
Given that PAK4 undergoes membrane relocalization upon
attachment onto VN, it was of interest to elucidate whether
the relocalization of PAK4 is dependent on its integrin or
Cdc42/Rac interaction and/or its kinase activity. Therefore,
Figure 5. PAK4 translocation to lamellipodia
by integrin ligation to VN does not depend on
Cdc42 binding, integrin interaction, or PAK4
kinase activity. (A) Flag-tagged PAK4 mutants
used for translocation studies. PAK4-L19, 22
lacks binding capacity to Cdc42/Rac. PAK4-
M350 and PAK4-IBD are both kinase dead,
and PAK4-IBD cannot bind integrin 5.
(B) M21 cells were transfected with Flag-tagged
wt PAK4, PAK4 mutants, or the control Flag-BAP
vector. Cells were stained using an anti-Flag
mAb (green), and stained for actin using
phalloidin–rhodamine (red) and for nuclei using
Hoechst (blue) before (B) or after (C) replating
onto VN for 1 h. Arrows indicate the distribution
in lamellipodia of PAK4 and PAK4 mutants.
Bars, 20 m. (D) Quantification of membrane-
localized wt PAK4 or PAK4 mutants before and
after replating onto VN for 1 h. Bars represent
percent of cells with membrane-localized wt
PAK4 or PAK4 mutants of the total cells
counted and are expressed as mean SD. In a
statistical evaluation comparing before and after
cell replating onto VN, all PAK4 variants gave
P 0.05 (*) or P 0.01 (**) by a paired t test.
PAK4-L19, 22 localization to the membrane
was also higher than wt PAK4 under normal
culture conditions (P 0.05 [*]).
1292 The Journal of Cell Biology | Volume 158, Number 7, 2002
we constructed Flag-tagged PAK4 mutants that lack the
binding capacity for Cdc42/Rac (PAK4-L19, 22), the IBD
(PAK4-IBD), or PAK4 kinase activity (PAK4-M350) as il-
lustrated in Fig. 5 A. Human M21 melanoma cells were
transfected with these PAK4 mutants and compared with
cells transfected with wild-type (wt) Flag–PAK4 and a vector
containing a nonrelated Flag-tagged BAP protein. Under
normal culture conditions, wt PAK4 mainly localized in the
cytosol (Fig. 5 B). However, upon cell replating onto VN,
the majority of cells transfected with PAK4 displayed a relo-
calization to lamellipodia (Fig. 5 C). A similar relocalization
to lamellipodia upon replating was also observed for the ki-
nase-dead and IBD PAK4 mutants, both of them lacking
kinase activity (unpublished data) and PAK4-IBD also
lacking integrin-binding capacity (Fig. 3 B). However, the
PAK4-L19, 22 that lacks GTPase-binding capacity was
found in lamellipodia in almost half of the cells in regular
culture and was then redistributed to the membrane in the
remaining cells upon replating onto VN. A quantification of
the PAK4 relocalization by counting the number of cells
with membrane-localized PAK4 is displayed in Fig. 5 D.
Taken together, these results suggest that PAK4 relocaliza-
tion to lamellipodia does not require its kinase activity or in-
tegrin or Cdc42/Rac binding. However, the Cdc42/Rac
binding capacity of PAK4 might be inhibitory for PAK4 lo-
calization in lamellipodia.
Dynamic distribution of PAK4
in actively reshaping lamellipodia
To study the temporal and spatial localization of PAK4 in
living cells, we established MCF-7 human breast carci-
noma cells stably expressing a fluorescent EGFP–PAK4
fusion protein. These cells were plated onto VN-coated
glass slides and analyzed by time-lapse fluorescent micros-
copy. Consistent with our immunofluorescent staining of
endogenous PAK4 lamellipodial localization in MCF-7
cells (Fig. 4 A) and of Flag-tagged PAK4 (Fig. 5), EGFP–
PAK4 also localized in lamellipodial protrusions after re-
plating onto VN (Fig. 6 A). Interestingly, the PAK4 dis-
tribution changed in a highly dynamic fashion, whereas
EGFP control cells exhibited only cytoplasmic and nu-
clear or perinuclear distribution (Fig. 6 B). Furthermore,
like endogenous PAK4 (Fig. 4 B), EGFP–PAK4 was also
found to partially colocalize with integrin v5 in lamelli-
podia (unpublished data). The transient localization of
PAK4 in lamellipodia coinciding with lamellipodia of ac-
tively forming and retracting extensions indicates that
PAK4 may modulate these processes. Given that PAK4 as-
sociated with integrin v5 and colocalized with integrin
v5 in lamellipodia, the dynamic distribution of PAK4
in lamellipodia may reflect a transient and periodic inter-
action between PAK4 and integrin v5. This led us to
hypothesize that PAK4 may not only interact with v5,
but that PAK4 may also influence integrin-mediated mo-
PAK4 stimulates integrin v5–specific cell migration
in human breast carcinoma cells
Based on the above hypothesis, we examined the potential
effect of PAK4 on v5-mediated cell motility. Integrins
v3 and v5 both participate in cell attachment and cell
migration toward VN (Wayner et al., 1991). To assess the
integrin v5–mediated cell motility, it is ideal to use a cell
line expressing v5, but not v3, because v3 usually
dominates as VN receptor for cell migration if present,
which is the case in most cultured cell lines. Therefore, we
chose MCF-7 human breast carcinoma cells, which express
v5 but not v3 (unpublished data; Brooks et al., 1997;
Wong et al., 1998). In addition, we found that both PAK4
mRNA and protein are highly expressed in MCF-7 cells
compared with a number of other tumor cell lines tested
(unpublished data), consistent with the recent study by Cal-
low et al. (2002).
In a haptotactic cell migration assay, we found that tran-
sient expression of EGFP–PAK4 in MCF-7 cells specifically
induced MCF-7 cell migration on VN, but not integrin 1–
mediated cell migration on collagen type I (Fig. 7 A). Fur-
thermore, EGFP–PAK4-induced cell migration was blocked
by a functional blocking anti-v5 mAb, but not by an
anti-v3 mAb (Fig. 7 A, left). Taken together, this demon-
strates that PAK4 specifically induces integrin v5–medi-
ated cell motility. Moreover, stable expression of EGFP–
PAK4 in MCF-7 cells yielded numerically almost identical
results on induction of v5-mediated cell migration as
transient PAK4 expression, but did not influence cell motil-
ity on collagen (Fig. 7 B). Similarly, stable overexpression of
Figure 6. Dynamic localization of PAK4 in lamellipodia. MCF-7
human breast carcinoma cells stably transfected with EGFP–PAK4 or
EGFP were plated onto VN and visualized by immunofluorescent
microscopy after being allowed to attach for 30 min. Images were
taken by a CCD camera every minute and the displayed cells are
representative for the respective transfection. (A) EGFP–PAK4 is
transiently localized in actively reshaping lamellipodia. (B) EGFP
control exhibits only nuclear or perinuclear localization. Bars, 20 m.
PAK4 interacts with and modulates integrin v5 function | Zhang et al. 1293
a Flag-tagged constitutively active PAK4 mutant (S474E;
Callow et al., 2002) induced MCF-7 cell migration to VN
to the same degree as EGFP–PAK4 (unpublished data). This
indicates that overexpression of EGFP–PAK4 may saturate
PAK4-inducible motility in this cell type, which might be
explained by the observation that a large GST fusion partner
at the NH
terminus of PAK1 causes constitutive PAK1 ac-
tivation, suggesting that the EGFP fusion to PAK4 may
cause PAK4 activation.
Given that cell adhesion is the basis for cell migration, it
was interesting to examine whether PAK4 may also im-
pact cell adhesion on VN. To this end, MCF-7 cells were
transfected with EGFP–PAK4 or control EGFP. As shown
in Fig. 7 C, overexpression of EGFP–PAK4 markedly in-
hibited cell adhesion on VN compared with EGFP-trans-
fected cells. One possibility that may explain the inhibi-
tion of cell adhesion on VN could be a down-regulation of
integrin v5 cell surface expression by PAK4. To exam-
ine this, we performed a flow cytometry analysis to deter-
mine the cell membrane distribution of integrin v5.
However, PAK4 overexpression did not change the abun-
dance of integrin v5 expressed on the cell membrane
(Fig. 7 D), or that of integrin 1 (unpublished data). This
suggests that PAK4 inhibition of cell adhesion might be
caused by an alteration of integrin v5 binding capacity
for its ligand VN.
PAK interaction with integrins
Previously, only one intracellular molecule, Rack1 (Liliental
and Chang, 1998), had been identified to directly interact
with the integrin 5 cytoplasmic domain. By yeast two-
hybrid screening, we now add six novel intracellular interac-
tors of integrin 5, including PAK4. The PAK4 binding sites
within the integrin 5 cytoplasmic domain was mapped to a
conserved, membrane-proximal region (aa 759–767). Inter-
estingly, the Rack1-binding site within 5 cytoplasmic do-
main was also mapped to the membrane-proximal region
partially overlapping with the PAK4-binding region, but
more extended to the NH
terminus (Fig. 2 C). Rack1 is a re-
ceptor for PKC and may play a role in linking PKC to inte-
grins. Slightly upstream of the PAK4-binding region, a con-
served integrin membrane-proximal region of 3 has been
shown to form a hinge with the integrin
controlling extracellular ligand-binding affinity of integrin
3 (Hughes et al., 1995). In addition, a conserved region
in the 2 integrin membrane-proximal region (733–742),
which almost corresponds to the PAK4-binding region in 5,
has been suggested to be critical for endoplasmic reticulum
retention, – dimerization, and cytoskeletal association of
leukocyte integrin L2 (Pardi et al., 1995). The conserved
-integrin membrane-proximal region has also been shown
Figure 7. PAK4 stimulates integrin
v5–mediated cell migration. (A) MCF-7
cells transiently transfected with EGFP–
PAK4 or EGFP control were analyzed for
haptotactic cell migration toward VN in
the presence or absence of normal mouse
IgG and functional blocking mAbs
LM609 (anti-v3) or P1F6 (anti-v5)
(left panel) and toward collagen type I
(right panel). Data represent the average
of three independent experiments and
were normalized to the transfection
efficiency of individual vectors, as
determined by flow cytometry. Statistical
evaluation comparing EGFP to EGFP–
PAK4 on VN gave P 0.05 by t test.
(B) Cell migration analyzed as in A of
MCF-7 cells stably expressing EGFP–PAK4 or EGFP. All results are expressed as mean values SEM of three independent experiments using
triplicate analysis in each experiment. Statistical evaluation by t test gave P 0.05 for EGFP–PAK4 compared with EGFP on VN. (C) Overexpression
of PAK4 decreases cell adhesion on VN. Cell attachment of MCF-7 cells stably expressing EGFP–PAK4 or EGFP at different coating concentrations
of VN was determined. (D) Transient EGFP–PAK4 expression does not change the membrane expression levels of integrin v5 in MCF-7
cells as measured by flow cytometry. v5 expression was plotted versus EGFP content and the v5 cell surface levels in EGFP-transfected cells
(bottom left) was compared with that in EGFP–PAK4 cells (bottom right). Values indicate the mean fluorescence of v5 staining in EGFP-positive
cells. Unstained MCF-7 cells (top left) and staining without primary mAb (top right) were used to determine specificity and background.
1294 The Journal of Cell Biology | Volume 158, Number 7, 2002
to mediate integrin oligomerization, inhibition of integrin
conformation, and constraining of an integrin in the inactive
state (Zage and Marcantonio, 1998; Bodeau et al., 2001; Lu
et al., 2001). However, although it is possible that PAK4
binding to integrin 5 membrane-proximal region may affect
the association between the integrin v and 5 subunits or
binding between v5 and VN, further studies are required
to elucidate if modulation of the PAK4-binding region of 5
can affect integrin hinge formation or the integrin extracellu-
lar binding affinity to the ECM.
Intriguingly, interactions between PAK family members
and integrins may be conserved, because the IBD within
PAK family members is highly conserved (Fig. 3 C) and be-
cause we found that in addition to PAK4, both hPAK1 and
the Drosophila PAK4 homologue MBT are able to interact
with various integrin subunits (unpublished data). The
wide interaction spectra between integrins and PAK family
members of man and Drosophila suggest that the capacity for
these interactions might be highly conserved during evolu-
tion and may thus fulfill vital functions in various species.
Relocalization of PAK4 to lamellipodia
PAK4 has been found to be localized in the cytosol and in
the Golgi apparatus (Abo et al., 1998; Callow et al., 2002).
In the current study, we found that PAK4 relocalized to mo-
tile structures in the cell membrane upon replating onto
VN, including relocalization of PAK4 to lamellipodia and
ruffles. In comparison, PAK1 can localize to focal adhesions
upon replating (unpublished data; Manser et al., 1997). The
-terminal regulatory domain of PAK1:1–329, including
its Cdc42/Rac-binding motif, is sufficient to localize PAK1
to focal adhesions, and PAK1 has been indicated to be re-
cruited to focal adhesions dependent on its binding to
Cdc42/Rac (Manser et al., 1997; Brown et al., 2002). How-
ever, in a substantial part of the cells (40%), a PAK4 mu-
tant deficient in binding to Cdc42/Rac localized to the
membrane before cell replating onto VN, whereas wt PAK4
only localized to the membrane in a few cells before replat-
ing. This suggests that binding to Cdc42/Rac might nega-
tively regulate PAK4 membrane relocalization, which is
consistent with the finding by Abo et al. (1998) that over-
expression of activated Cdc42 results in localization of
PAK4 in the Golgi apparatus. In addition, binding to Nck
has been shown to mediate PAK1 membrane localization
(Lu et al., 1997), whereas Pix, but not Nck, binding is re-
quired for PAK1:1–329 localization to focal adhesions
(Brown et al., 2002). However, the Nck- and Pix-binding
regions of PAK1 are not conserved in PAK4, which might
explain why PAK1 can be readily detected in focal adhesions
(unpublished data; Manser et al., 1997), whereas PAK4 in-
stead mainly relocalizes to lamellipodia upon replating and is
rarely found in focal adhesions (unpublished data). It is un-
clear how PAK4 relocalization is mediated, but it appears to
be independent of its catalytic activity, Cdc42/Rac binding,
and integrin binding capacity, because PAK4 mutants defi-
cient in these capacities all still relocalized to lamellipodia
upon replating. However, it will be highly interesting to elu-
cidate how relocalization of PAK4 is regulated, because the
PAK4 localization at motile cellular structures may be im-
portant for its function in motility.
Role of PAK4 in the regulation of cell motility
Cell migration is important in many physiological and
pathological processes and the regulation of cell motility is
complicated, including both extracellular and intracellular
events. Among these, PAK1 has been found to regulate cell
motility in mouse fibroblasts (Sells et al., 1999), endothelial
cells (Kiosses et al., 1999; Master et al., 2001), and tracheal
smooth muscle cells (Dechert et al., 2001). Previous studies
have suggested that the regulation of phosphorylation of
myosin light chain kinase and LIM kinase (LIMK) by PAK1
and PAK2 might account for the role of these PAK family
members in regulation of cell motility on various ECM sub-
strates, but without displaying any apparent integrin speci-
ficity (Edwards et al., 1999; Sanders et al., 1999; Goeckeler
et al., 2000). However, in this report, we demonstrate that
PAK4 specifically induces v5-mediated cell migration on
VN, whereas 1 integrin–mediated cell migration on col-
lagen type I is not influenced.
A recent study indicated that unlike PAK1, PAK4 is un-
able to phosphorylate myosin light chain kinase (Qu et al.,
2001), which may rule out one possible route by which
PAK4 could stimulate cell migration. Therefore, the mecha-
nisms for induction of cell motility by PAK1 and PAK4 may
be distinct, which is also supported by their distinct localiza-
tion in cell adhesive structures of focal adhesions and lamelli-
podia, respectively. PAK4 was recently found to interact with
LIMK1, to phosphorylate LIMK1 and stimulate the ability
of LIMK1 to phosphorylate cofilin (Dan et al., 2001a). As a
consequence, PAK4 and LIMK1 may cooperatively regulate
cytoskeletal changes that impact cell motility. However, the
PAK4 interaction with integrin 5 cytoplasmic domain may
also directly modulate the extracellular motility machinery,
including cell adhesion to ECM for which PAK4 has been
indicated to play a functional role (Qu et al., 2001). PAK4
binding to integrin might directly effect the integrin func-
tion, and thereby cell motility, and/or localize PAK4 effects
to integrin-proximal sites of migratory regulation. For exam-
ple, we found that PAK4 can phosphorylate the integrin 5
cytoplasmic domain and this way might affect the integrin
v5 extracellular binding capacity (unpublished data).
Taken together, our study suggests a model where PAK4
binds to integrin 5 cytoplasmic domain in motile cellular
structures and modulates integrin v5–mediated cell mi-
gration. This may be brought about by PAK4 regulation of
cytoskeletal components and/or by directly influencing inte-
grin v5 function, thereby facilitating cell migration.
Possible role of PAK4 in tumor progression
Intriguingly, PAK4 was recently found to be overexpressed
in 78% of an array of human cancer cell lines where its func-
tion may be to promote cell transformation (Callow et al.,
2002). In addition to this potential function, our study indi-
cates a role for overexpressed PAK4 in breast carcinoma cell
migration, suggesting a potential role also in metastasis. The
predominant VN receptor in human carcinomas in vivo is
integrin v5 (Lehmann et al., 1994; Jones et al., 1997), an
integrin that can be activated by growth factors for cell mi-
gration (Klemke et al., 1994; Yebra et al., 1996). In fact,
growth factor stimulation of breast and pancreatic carci-
PAK4 interacts with and modulates integrin v5 function | Zhang et al. 1295
noma cells has been shown to cause tumor dissemination
and metastasis in vivo, which was functionally linked to acti-
vation of integrin v5–mediated cell migration (Brooks et
al., 1997). Given that PAK4 stimulated v5-mediated cell
migration in breast carcinoma cells, elucidation of its poten-
tial role in growth factor signaling pathways governing inte-
grin v5 activation will be very interesting; for example if
PAK4 might effect src and/or FAK pathways recently impli-
cated in integrin v5 activation (Eliceiri et al., 2002). In
addition, stimulation of angiogenesis by VEGF or TGF-
depends on integrin v5 activation (Friedlander et al.,
1995). Therefore, it will also be interesting to assess the po-
tential role of PAK4 in in vivo progression of carcinoma me-
tastasis as well as angiogenesis.
In conclusion, we report a novel cell motility pathway me-
diated by the serine/threonine kinase PAK4 that directly in-
teracts with integrin v5 and selectively induces v5-
mediated cell motility, a mechanism previously demonstrated
to mediate carcinoma dissemination.
Materials and methods
Cell culture, cDNA expression vectors, and antibody production
African green monkey kidney COS-7 cells, human breast carcinoma
MCF-7 cells, and human melanoma M21 cells were grown in DME suppl-
emented with 10% FCS, 10 g/ml Gentamycin (Life Technologies). Clones
of MCF-7 cells stably expressing EGFP or EGFP–PAK4 were selected in the
presence of 0.5 mg/ml G418 (Life Technologies). Pools of G418-resistant
EGFP and EGFP–PAK4-expressing clones were used for cell migration
studies. hPAK4 expression vector HA–PAK4-SR3 was provided by Dr.
Audrey Minden (Columbia University, New York, NY). Flag-tagged hPAK4
was constructed by cloning the full-length hPAK4 cDNA into the vector
p3FLAG-CMV-10 (Sigma-Aldrich). EGFP–PAK4 expression vector was
generated by cloning the full-length hPAK4 cDNA into pEGFP-C2 (CLON-
TECH Laboratories, Inc.) and confirmed by sequencing. The PAK4 mutants
PAK4-M350, PAK4-L19, 22, and PAK4-IBD (deletion of aa 505–530)
were created by site-directed mutagenesis using the QuickChange kit
(Stratagene), followed by sequence confirmation of the mutated regions.
For anti-PAK4 antibody production, a PAK4 NH
-terminal sequence (aa
116–323) was amplified by PCR and cloned into the GST fusion protein
expression vector pGEM-1T (Amersham Biosciences). GST fusion pro-
teins were purified using glutathione-Sepharose beads (Amersham Bio-
sciences), according to the manufacturer’s protocol, followed by separa-
tion in PAGE and extraction of the GST–PAK4 fusion protein. Two rabbits
were immunized with the GST–PAK4 fusion protein and the sera collected
by Dr. Arturo Galvani at Pharmacia Corp. (Nerviano, Italy). The anti-PAK4
antibody was purified by protein G affinity column (Pierce Chemical Co.)
after absorption by bacteria powder containing expressed GST protein.
The purified anti-PAK4 antibody was tested for reactivity with other PAK
family members and found to be specific for PAK4 (unpublished data).
Yeast two-hybrid screening and yeast mating tests
Integrin 5 cytoplasmic domain (aa 753–799; Ramaswamy and Hemler,
1990) was fused to Lex A-DBD in the pEG202 vector (OriGene). The insert
sequence and reading frame were confirmed by sequencing. Approxi-
mately 2 10
yeast transformants of a 19-d mouse whole embryo expres-
sion library (OriGene) were pooled and screened by the activation of leu-
cine and -galactosidase marker genes. 98 clones were His
and 25 of them specifically interacted with 5 cytoplasmic
domain in yeast mating tests. Sequence analysis and bioinformatics studies
indicated that 6 out of 25 5 cytoplasmic domain–interacting sequences
represented mPAK4. For yeast mating tests, different regions of the integrin
5 cytoplasmic domain were cloned into the pEG202 bait vector using
yeast strain RFY206 as host, and different regions of the KD of hPAK4 were
cloned into pJG4-5 prey vector using yeast strain EGY48 as host.
Protein–protein interaction assays
COS-7 cells were transfected with 4 g expression vector HA–PAK4-SR3
by LipofectAmine plus (Life Technologies). Cells were harvested 48 h after
transfection and lysed in RIPA buffer (1 PBS, pH 7.4, 0.5% sodium de-
oxycholate, 1% Triton X-100, 0.1% SDS) with protease inhibitors (0.5 g/
ml leupeptin, 1 mM EDTA, 1 g/ml pepstatin A, 0.2 mM PMSF). Approxi-
mately 500 g of precleared lysates were immunoprecipitated by 4 g
rabbit anti-HA pAb Y11 (Santa Cruz Biotechnology, Inc.), anti-v5 mAb
P1F6 (Life Technologies), or anti-3 mAb AP3 (GTI) and were separated
and probed with rabbit anti–human integrin 5 cytoplasmic pAb, anti–
integrin v pAb (Chemicon), or anti–HA tag mAb F-7 (Santa Cruz Biotech-
nology, Inc.) and then with an HRP-conjugated secondary pAb (Jackson
ImmunoResearch Laboratories) and visualized by ECL technique as previ-
ously described (Bao et al., 2002). Alternatively, IP of endogenous PAK4
was performed using 2 g anti-PAK4 pAb. For testing the in vivo associa-
tion of PAK4-IBD mutant with integrin v5, COS-7 cells were trans-
fected with Flag–PAK4 and Flag–PAK4-IBD. Precleared lysates (100 g)
were immunoprecipitated by 6 l ascites fluid of anti–integrin v5 mAb
P1F6 (Life Technology) or rabbit IgG or -Rab pAb (C-19; Santa Cruz Bio-
technology, Inc.) followed by Western blot analysis using anti-Flag tag
mAb M2 (Sigma). As positive controls for the Western blot analyses, 10–15
g lysates were applied without IP. To make GST fusion proteins, PAK4
KD (aa 324–591) and 5 (aa 753–799) cytoplasmic domains were cloned
separately into the GST fusion protein expression vector pGEM-1T (Amer-
sham Biosciences). GST fusion proteins were purified using glutathione-
Sepharose beads (Amersham Biosciences) according to the manufacturer’s
protocol. To pull down hPAK4, 2 g of GST 5 tail fusion protein was
mixed with 500 g cell lysate containing HA–hPAK4 in RIPA buffer. Re-
ciprocally, to pull down the endogenous integrin 5 subunit, 200 g COS-7
cell lysate was mixed with 5 g purified GST–hPAK4 KD fusion protein in
RIPA buffer and incubated overnight at 4C. Glutathione-Sepharose beads
(Amersham Biosciences) were used to capture the GST fusion proteins and
the interacting proteins. The bound proteins were visualized by Western
blotting with mAb F-7 or rabbit anti–human integrin 5 cytoplasmic do-
main pAb (Chemicon), respectively.
Fluorescent microscopy and time-lapse video microscopy
MCF-7 cells stably expressing EGFP–PAK4 or EGFP were established under
the selection of G418 (0.5 mg/ml). Cells were fixed by 4% paraformalde-
hyde after attachment on VN. For staining of endogenous PAK4 in MCF-7
cells, rabbit anti-PAK4 pAb was used. For integrin v5 staining, cells
were replated in the absence of FCS and Mn
(RPMI 1640, 2 mM CaCl
, and 0.5% BSA), and anti–integrin v5 mAb clone 15F11
(Chemicon) was used for staining. Anti-Flag mAb M2 (Sigma-Aldrich) was
used for Flag tag staining. For quantification of cells with lamellipodial
PAK4 localization, six microscopic fields were chosen randomly and
counted directly through 20 objective. Statistical analysis was performed
using Origin version 6.0 (Microcal Software, Inc.). Stained cells were pho-
tographed by fluorescent microscopy using a digital camera. Time-lapse
studies were performed 30 min after plating EGFP–PAK4 stably trans-
fected MCF-7 cells onto VN-coated chamber slides in the absence of FCS
using a fluorescent microscope (Leica). Pictures were captured every
minute using Slidebook software version 2.06 (Intelligent Imaging Innova-
tions, Inc.). The acquired pictures were further processed and assembled
using Adobe Photoshop
5.0 and Adobe Illustrator
Cell migration and cell adhesion assays
Haptotactic cell migration assays were performed using Transwell cham-
bers (Costar Inc.) with 8.0 m pore size. The Transwell membranes were
coated with VN (10 g/ml), collagen type I (10 g/ml), or 1% BSA at the
bottom surfaces for 2 h at 37C. MCF-7 cells were transfected with EGFP or
EGFP–PAK4 for 48 h, and then cells were trypsinized, washed, and
counted in the presence of soybean trypsin inhibitor (0.25 mg/ml). Cells
) were then added on the top of Transwell membranes and al-
lowed to migrate toward VN or collagen type I in the presence or absence
of anti–integrin v3 mAb LM609 (Chemicon) or anti-v5 mAb P1F6 (25
g/ml) for 6 h at 37C in migration buffer (RPMI 1640, 2 mM CaCl
, 1 mM
, 0.2 mM MnCl
, and 0.5% BSA). After thoroughly cleaning the up-
per chambers of the Transwells, the migrated cells expressing EGFP or
EGFP–PAK4 were counted using a fluorescent microscope; typically 12
microscopic fields were randomly chosen and counted. For comparison,
the number of migrating cells was calibrated to the transfection efficiency
within the cell population as determined by flow cytometry. Quantifica-
tion of stably transfected cells was performed by staining using crystal vio-
let followed by counting of random microscopy fields. For the cell adhe-
sion assay, nontreated 48-well plates (Corning Costar Corp.) were used.
Wells were coated with 0.5–10 g/ml VN overnight at 4C. 1% heat-dena-
tured BSA was applied to block nonspecific adhesion. MCF-7 cells stably
transfected with EGFP–PAK4 or EGFP control were plated into the wells in
triplicate at 5 10
cells/well in cell adhesion buffer (RPMI 1640, 2 mM
1296 The Journal of Cell Biology | Volume 158, Number 7, 2002
, 1 mM MgCl
, 0.2 mM MnCl
, and 0.5% BSA) and allowed to attach
for 60 min. After careful washing of nonbound cells using adhesion buffer,
MTT was used to quantify the number of stably transfected cells attached.
Flow cytometry analyses
The efficiency for cell transfections and the cell surface expression levels
of integrins were analyzed by measurement of EGFP content and phyco-
erythrin staining intensity, respectively, by FACScan
flow cytometer using
CellQuest software (Becton Dickinson) after staining with anti–integrin
v5 mAb P1F6 and a phycoerythrin-conjugated secondary goat anti–
mouse pAb (Jackson ImmunoResearch Laboratories).
We thank Drs. Audrey Minden and Errki Ruoslahti (Burnham Institute, La
Jolla, CA) for providing the hPAK4 and the human integrin 5 cDNA, re-
spectively. We thank Dr. Arturo Galvani for help with the anti-PAK4 serum
production and Dr. Pontus Aspenström (Ludwig Institute for Cancer Re-
search, Uppsala, Sweden) for critical reading of the manuscript.
This study was supported by grants to S. Strömblad from the Swedish
Cancer Society, the Swedish Science Research Council, the Swedish Stra-
tegic Research Foundation, and the Magnus Bergvall Foundation, and to
H. Zhang from the Swedish Society of Medicine. H. Zhang was also sup-
ported by the Wenner-Gren Foundation.
Submitted: 1 July 2002
Revised: 19 August 2002
Accepted: 20 August 2002
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