Báo cáo Y học: Interaction between p21-activated protein kinase and Rac during differentiation of HL-60 human promyelocytic leukemia cell induced by all-trans-retinoic acid pdf

Interaction between p21-activated protein kinase and Rac
during differentiation of HL-60 human promyelocytic leukemia cell
induced by all-
trans
-retinoic acid
Yukio Nisimoto
1
and Hisamitsu Ogawa
2
1
Department of Biochemistry, Aichi Medical University, School of Medicine, Nagakute, Aichi, Japan;
2
Department of Biology,
Fujita Health University School of Medicine, Toyoake, Aichi, Japan
Undifferentiated human promyelocytic leukemia HL-60
cells show little or no superoxide production, but generate a
very low O
2
–
concentration upon incubation with all-trans-
retinoic acid (ATRA). Its production reaches a maximum
within 20 h, and thereafter is maintained at an almost con-
stant level. The differentiated cells show phorbol 12-myri-
state 13-acetate (PMA)-stimulated NADPH oxidase activity
consistent with the amount of gp91phox (phagocytic oxid-
ase) expressed in the plasma membrane. Three isoforms of
p21-activated serine/threonine kinases, PAK68, PAK65 and
PAK62, were found in both cytosolic and membrane frac-
tions, and their contents were significantly increased during
induced differentiation. The amount of Rac identified in the
two fractions was also markedly enhanced by ATRA-

induced differentiation. In contrast, neither PAK nor Rac
was seen in the plasma membrane of undifferentiated HL-60
or human neutrophil, but they were abundant in the cyto-
plasmic fraction. Binding of Rac with PAK isoforms was
shown in the membrane upon induced differentiation of
HL-60 cells. Direct binding of purified Rac1 to PAK68 was
quantified using a fluorescent analog of GTP (methylanth-
raniloyl guanosine-5¢-[b,c-imido]triphosphate) bound to
Rac as a reporter group. Rac1 bound to PAK68 with a 1 : 1
stoichiometry and with a K
d
value of 6.7 n
M
.
Keywords:Rac;PAK;HL-60;GTPase;MAPkinase.
A phagocytic superoxide-generating system is expressed
upon induced differentiation of HL-60 human promyelo-
cytic leukemia cells with dimethylsulfoxide or retinoic acid
[1–4]. Following initiation of differentiation, the synthesis of
flavocytochrome b
558
, which utilizes reducing equivalents
from NADPH to reduce oxygen to superoxide, has been
observed in the membrane spectrophotometrically [1] and
by our present immunoblot analysis. Rac protein shows
little or no interaction with the NADPH oxidase compo-
nents in the process of differentiation of HL-60 cells.
However, Rac translocates to the plasma membrane and
binds specifically with p67phox (phagocytic oxidase) to
produce O

2
–
when HL-60 cells are differentiated into
granulocytes and exposed to bacteria or to a variety of
soluble stimuli. In fact, earlier studies reported that Rac was
found to interact specifically with p67phox translocated to
the plasma membrane of stimulated neutrophils [5,6].
There are few reports on the action of Rac-activated
protein kinase (PAK) during the differentiation of HL-60
induced by all-trans-retinoic acid (ATRA). Manser et al.[7]
have isolated a brain protein kinase, PAK68, by purifying a
protein with Rac1/Cdc42-GTP binding ability. Because
the kinase binds tightly to an affinity column loaded with
Rac/Cdc42-GTP or guanosine 5¢-O-(3-thiotriphosphate)
(GTPcS) but not the GDP-bound form, affinity chroma-
tography was then used to purify PAK68. The autophos-
phorylation and kinase activity of PAK were stimulated by
binding to activated Rac/Cdc42, which thereby directly
modulates the enzyme activity [7–11]. However, Rho did
not show binding activity to PAK [8]. In apparent
agreement with this, several groups of investigators have
reported that Rac and Cdc42, but not Rho, regulate the
c-jun N-terminal or stress-activated MAP kinase and p38/
HOG MAP kinase cascade [12–17].
In the present study, in order to investigate the PAK
expression and its binding to Rac, we quantitated PAK,
Rac and their complex in both cytosol and membrane
fractions in the process of HL-60 differentiation. We show
that HL-60 cells produce low levels of O
2

–
at an early stage
of incubation with ATRA, and also that the PAK–Rac
association observed in the plasma membrane appears to be
involved in the differentiation of HL-60 to granulocytes.
MATERIALS AND METHODS
Materials
Diisopropyl fluorophosphate, protease inhibitor cocktail,
cytochrome c, NADPH and superoxide dismutase
and Ponceau S solution were from Sigma. Hessol (6%
hetastarch in 0.9% NaCl) was from Green Cross Corp., and
lymphocyte separation medium (6.2% Ficoll, 9.4% sodium
Correspondence to Y. Nisimoto, Department of Biochemistry,
Aichi Medical University, School of Medicine, Nagakute,
Aichi 480-1195, Japan.
Fax: + 81 0561 62 4056, Tel.: + 81 0561 62 3311,
E-mail:
Abbreviations: ATRA, all-trans-retinoic acid; mant-GppNHp,
methylanthraniloyl guanosine-5¢-[b,c-imido]triphosphate; PMA,
phorbol 12-myristate 13-acetate; PAK, p21-activated protein kinase;
phox, phagocytic oxidase; GST, glutathione S-transferase.
Enzymes: p21-activated protein kinases PAK68, PAK65,
PAK62 (EC 2.7.1 ).
(Received 15 January 2002, revised 15 April 2002,
accepted 18 April 2002)
Eur. J. Biochem. 269, 2622–2629 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.02939.x
diatrizoate) was obtained from Flow Laboratories. GTPcS
was purchased from Boehringer Mannheim, and mant-
GppNHp was synthesized as previously described [18].
Polyclonal antibodies against PAK68 (C-terminal residues

525–544) which are partially cross-reactive with PAK65 and
PAK62, agarose-conjugated PAK68 antibodies, and poly-
clonal antibodies to PAK65 and to PAK62 were obtained
from Santa Cruz Biotech, Inc. Polyclonal antibodies to
human gp91phox and Rac1 were kindly provided by D. J.
Lambeth (School of Medicine, Emory University, Atlanta,
GA). The polyclonal antibodies to Rac1 gave a positive
cross-reactivity to Rac2, which exists dominantly in gra-
nulocytes. The anti-(rabbit IgG) and anti-(goat IgG)
secondary antibodies linked to horseradish peroxidase were
purchased from Bio-Rad. DEAE-Sepharose, 2¢,5¢-ADP–
Sepharose, glutathione–Sepharose and ECL reagent were
from Pharmacia Biotech. All other reagents were of the
highest grade available commercially.
Isolation of ATRA-induced differentiated HL-60 cells
Human promyelocytic leukemia HL-60 cells were grown in
suspension in 55-cm
2
Falcon tissue culture dishes containing
20 mL of RPMI 1640 (Gibco BRL) supplemented with
10 m
M
Hepes, pH 7.4, 10% heat-inactivated fetal bovine
serum and kanamycin (50 lgÆmL
)1
)at37°C in a humid-
ified incubator with 5% CO
2
. Differentiation was induced
by the addition of 1 l

M
ATRA for 1, 3, 5 and 7 days.
Undifferentiated and differentiated HL-60 cells were har-
vested by centrifugation and washed three times with
100 mL of NaCl/P
i
. After centrifugation, the number of
packed cells was 2–5 · 10
7
.
Separation of human neutrophil
Human neutrophils were obtained from the peripheral
blood of normal healthy donors after obtaining informed
consent. Erythrocytes were sedimented with Hessol, and the
mononuclear cells were removed from the resulting super-
natant by centrifugation through lymphocyte separation
medium [19]. The resulting cells were more than 95%
neutrophil granulocytes.
Measurements of reactive oxygen species
Superoxide generating activity was spectrophotometrically
assayed by monitoring SOD-inhibitable ferricytochrome c
reduction at 550 nm. 2¢,5¢-Dichlorofluorescin fluorescence
was measured to determine intracellular H
2
O
2
using
5.95 · 10
5
cellsÆmL

)1
of NaCl/P
i
solution. Fluoroskan
Ascent FL (Labsystems) was used for the emission meas-
urements at 525 nm when excited at 488 nm.
Preparation of cytosolic and membrane fractions
Cytosol and plasma membrane from neutrophils or HL-60
cells were prepared as described previously [20]. Cells were
suspended in buffer A (0.1
M
Tris/HCl buffer, pH 7.4,
containing 0.1
M
KCl, 5.5 m
M
NaCl, 10% glycerol, 1 m
M
EDTA, 50 l
M
diisopropylfluorophosphate and 1 lgÆmL
)1
protease inhibitor cocktail), and then disrupted by nitrogen
cavitation after being pressurized at 500 p.s.i. for 30 min at
3 °C [21]. The cavitate was centrifuged (800 g,5min)to
remove nuclei and unbroken cells. The supernatant was
further centrifuged at 150 000 g for 1 h. The precipitates
were washed with 25 m
M
phosphate buffer, pH 7.3, contain-

ing 10% glycerol, and then stored as a membrane fraction at
)70 °C. The supernatant was used as a cytosol fraction.
Purifications of glutathione
S
-transferase (GST)-Rac1
expressed in
E. coli
and PAK 68 from human neutrophil
cytosol
Using previously reported methods [22–24], the Rac1 gene
was engineered with flanking BamH1 and EcoR1 restriction
enzyme sites, and the sequence was mutated to replace
Cys189 with Ser, and thus increasing the stability of the
protein and eliminating the possibility of isoprenylation.
Recombinant Rac1 protein was expressed in E. coli as a
fusion protein with an N-terminal GST using the pGEX-2T
fusion vector and was purified to about 95% homogeneity
using thrombin cleavage from a glutathione affinity matrix.
Approximately 1.18 g of cytosol was mixed and incubated
with 10 mL of 2¢,5¢-ADP-Sepharose beads for 12 h at 3 °C.
The beads were transferred into a column (10 · 100 mm)
and washed well with buffer A containing 2 m
M
NADPH.
The fractions released from the column were incubated
while gently stirring with agarose-conjugated PAK68 anti-
bodies for 12 h at 3 °C. The agarose beads were transferred
into a column (10 · 50 mm) and washed extensively with
50 m
M

buffer A containing 0.1% Triton X-100. The PAK
was eluted from the column with 25 m
M
glycine/HCl buffer,
pH 3.0, and the eluted fractions were quickly neutralized at
pH 7.0 by adding 0.2
M
Tris/HCl buffer, pH 9.0, containing
20% glycerol and 1 m
M
dithiothreitol. Samples with high
PAK activity were pooled, then concentrated using a
Centricon-10 microconcentrator, and employed in subse-
quent studies.
Binding assay between Rac and PAK
Approximately 7.55 mg of cytosol or 6.90 mg of plasma
membrane prepared from either neutrophils or differenti-
ated HL-60 cells was mixed and incubated with 3.0 mL of
2¢,5¢-ADP–Sepharose beads for 3 h at 3 °C. The beads were
transferred into a column (10 · 20 mm) and washed well
with buffer A containing 0.1% Triton X-100. The column
was then eluted with buffer A containing 2 m
M
NADPH.
The fractions released from the column were pooled and
employed to detect PAK and Rac by Western blot. Protein
was quantitated by the method of Bradford [25], using BSA.
Immunoprecipitation
Protein samples of cytosol and plasma membranes obtained
from either neutrophils or HL-60 were mixed with anti-

Rac1 IgG or preimmune rabbit IgG (negative control) for
3 h. Then protein A–agarose beads were added and the
mixtures were incubated for 1 h. After washing the beads
with buffer A containing 0.1% Triton X-100, the immuno-
precipitates were analyzed using Western blotting.
SDS/PAGE and Western blot analysis
SDS/PAGE (0.1% SDS and 10% gel) was carried out at
25 °C for 3 h, and the gels were subjected to silver
Ó FEBS 2002 Rac–PAK interaction in HL-60 (Eur. J. Biochem. 269) 2623
staining. Proteins separated by SDS/PAGE were also
transferred to an Immobilon-P membrane (Millipore
Corp.) [26]. The membrane was incubated at 25 °Cfor
2 h in 5% skim milk in 20 m
M
phosphate buffer, pH 7.3,
containing 0.14
M
NaCl and 2.7 m
M
KCl. Polyclonal
antibodies used were those to PAK68 and Rac1. After
washing, the membrane was reacted with antibodies
(1.5 lgÆmL
)1
) and then with a horseradish peroxidase-
linked secondary antibody (IgG, 1: 5000 dilution) raised in
goat. The membrane was washed extensively three times
with 20 m
M
NaCl/P

i
, pH 7.3, containing 0.1% Tween 20
(20 min each), and immune complexes were detected with
ECL reagents.
Emission titration and calculation of dissociation
constants
Rac1 and mant-GppNHp were incubated at 20 °Cin
0.3mL of 50m
M
Tris/HCl buffer, pH 7.5, containing
3m
M
NaCl, 50 m
M
KCl and 0.1 l
M
MgCl
2
. Preloading of
Rac with mant-GppNHp was carried out for 15 min, by
which point the fluorescence change due to guanine
nucleotide binding was stable. Very low MgCl
2
concentra-
tion was essential to facilitate a complete guanine nucleotide
exchange. Titration was carried out by adding PAK68 to
Rac1 preloaded with mant-GppNHp and recording
fluorescence changes until stable readings were obtained.
Fluorescence changes induced by PAK68 occurred within
3–4 min and did not change further even with prolonged

incubation. Spectral resolution was 5 nm for both the
excitation and emission paths. Fluorescence titrations were
fit to a single site-binding equation to calculate K
d
values as
described previously [27].
RESULTS
Induction of gp91
phox
and superoxide generating
activity of HL-60 Cells
Utilizing the induced differentiation of HL-60 promyelo-
cytic leukemia cells as a model of myeloid maturation, we
examined the expression and location of Rac and PAK.
The interaction between the two proteins during HL-60
myeloid differentiation has received little attention. On the
other hand, it is well known that, in the process of
myeloid maturation, differentiated HL-60 cells are capable
of most neutrophil functions: chemotaxis, ingestion, res-
piratory burst oxidase activity and bacterial killing [28–30].
Our present study showed that HL-60 cells cultured with
ATRA increased the rate of O
2
–
production in responce to
phorbol 12-myristate 13-acetate (PMA) from 6.5 ±
5 nmol per 10 min per 10
7
cells on day 0 of incubation
to 495 ± 110 on day 5. The corresponding reference

value of neutrophils was 980 nmol of O
2
–
per 10 min per
10
7
cells. Concomitantly, plasma membrane-associated
gp91phox content, which is a large subunit of flavocyto-
chrome b
558
and responsible for superoxide generation,
increased with the induction of differentiation in propor-
tion to the change in NADPH oxidase activity (Fig. 1,
inset). The NADPH oxidase was inactive in resting HL-60
cells on day 5, although they showed very low superoxide
generating activity (15 ± 5 nmol O
2
–
per 10 min per
10
7
cells). The dormant cells exhibited a very low level of
O
2
–
production both with and without ATRA induction.
The increase in hydrogen peroxide, which is formed via
dismutation of O
2
–

, is also measured by using 2¢,5¢-
dichlorofluorescin. As shown in Fig. 2, HL-60 cells gave
intracellular 2¢,5¢-dichlorofluorescin fluorescence that was a
little higher in the cells treated with ATRA. The emission
difference reached a peak around 10 h after the start of
the incubation. Induced and uninduced HL-60 cell pop-
ulations are heterogeneous in each stage of differentiation.
Although induction causes a shift to a much higher
proportion of mature cell types, all stages from promyel-
ocytes to polymorphonuclear leukocytes are present in
both. HL-60 cells induced with ATRA for 5 days showed
40–60% of the NADPH oxidase activity observed in
human neutrophils. The presence of these active phago-
cytic cells was negligible before the induced differentiation
of HL-60. Thus, in the present study the O
2
–
generating
activity was measured to estimate the rate of differenti-
ation of HL-60 cells induced with ATRA.
Induction of Rac and PAK
HL-60 cells were treated with 1 l
M
ATRA for a week,
andRacandPAKwereassayedinbothcytosoland
membrane fractions. Rac occurs as two isoforms (Rac1
and Rac2) that are 92% identical in amino-acid sequence
and Rac2 is more abundantly expressed in HL-60 [31]. In
the two fractions of undifferentiated HL-60 cells, the
expression of Rac was weak and was hardly detected in

the membrane. However, its content was significantly
Fig. 1. ATRA-induced changes in gp91phox production and superoxide
generating activity in HL-60 cells. After stimulation of the cells with
10 l
M
PMA, SOD-inhibitable superoxide production was measured in
thepresenceof0.1m
M
cytochrome c with or without added 50 lg
superoxide dismutase (black bars). Superoxide generation of dormant
cells was assayed before stimulation with PMA (hatched bars). Each
value represents the mean ± SD of three independent experiments.
The inset shows immunoblot analysis of gp91phox in the plasma
membrane fraction. A major band corresponding to an apparent
molecular mass of 91 kDa was indicated by an arrow. Induced dif-
ferentiation times (days) are numbered on the top of each lane.
Neutrophil membrane proteins were loaded onto lane N.
2624 Y. Nisimoto and H. Ogawa (Eur. J. Biochem. 269) Ó FEBS 2002
increased concomitant with induced differentiation
(Fig. 3). Using the antibodies that show cross-reactivity
to the three isoforms of p21-activated protein kinase,
PAK68, PAK65 and PAK62 were found in the cytosol of
undifferentiated HL-60, and they increased upon induced
differentiation (Fig. 4A). In addition, each antibody
specific to PAK68, PAK65 or PAK62 demonstrated that
their relative abundance in the cytosol was about 45, 15
and 40% of total protein, respectively. The molar ratio of
these PAK proteins was almost constant before and after
differentiation. They were also detected and increased in
the membrane fraction during the ATRA-induced differ-

entiation to granulocytes (Fig. 4B). However, the PAK
proteins were not clear in the plasma membrane fraction
from either the undifferentiated cells or mature neutrophils
(Fig. 4B). These data suggest that Rac and PAK located
in the membrane may interact to play a role in the
differentiation of HL-60 cells.
Nonimmune serum did not show any positive bands in
either cytosol or membrane fractions (data not shown).
Binding of PAK to Rac1 and Rac2 isoforms
in the membrane
The plasma membrane fraction was separated from
neutrophils and HL-60 cells were harvested at 0, 1, 3, 5
and 7 days after ATRA treatment. Rac-PAK binding
assay was carried out by immunoprecipitation using
antibodies to Rac1. The Rac protein from HL-60 mem-
brane was efficiently coimmunoprecipitated with PAK68,
PAK65 and PAK62 proteins. Interactions between Rac
and the three isoforms of PAK were observed in the
plasma membrane at each stage of the induced differen-
tiation of HL-60 (Fig. 5). In agreement with Figs 3 and 4,
little or no binding complex between Rac and PAK was
found in the membrane from undifferentiated cells and
fully mature neutrophils. In addition, proteins solubilized
Fig. 2. Time-dependent intracellular superoxide generation in dormant
HL-60 after starting the incubation with and without ATRA. HL-60 cells
were incubated with 2¢,5¢-dichlorofluorescin for 30 min and then the
reagent was removed by washing the cells twice with 10 mL each of
phosphate buffered saline. The 2¢,5¢-dichlorofluorescin-treated cells
wereincubatedintheabsence(s) and presence of 1 l
M

ATRA (d).
Fluorescence assay for reactive oxygen species was performed by
monitoring the emission at 525 nm. Fluorescence differences between
ATRA-treated and nontreated cells were indicated by close triangles.
Data are means from three independent experiments.
Fig. 3. Immunoblot analysis of Rac in the cytosolic and membrane
fractions of HL-60 cells. The differentiation of the cells were induced
with 1 l
M
ATRAfor0,1,3,5and7days(lane0–7)andlaneN
indicates human neutrophil. Cells were disrupted in the presence of
protease inhibitor cocktail, and fractionated into cytosol (A) and
plasma membrane (B). Each fraction (20 lg as protein) was loaded
onto SDS/PAGE, followed by transferring to Immobilon PVDF
membrane and then the membrane was incubated with antibodies
raised against Rac1. An immuno-reactive protein corresponding to
Rac1andRac2wasindicatedbyanarrow.
Fig. 4. Increase of PAK proteins in subcellular fraction during ATRA-
induced granulocytic differentiation of HL-60 cells. The differentiation
of the cells were induced with 1 l
M
ATRAfor0,1,3,5and7days
(lane 0–7). Lane N contained proteins of human neutrophil cytosol (A)
and plasma membrane (B). The cytosol (20 lg protein) and solubilized
membrane (15 lg protein) were subjected to SDS/PAGE (10% gel),
andthenelectricallyblottedtoImmobilonPVDFmembrane.The
PVDF membrane was treated with polyclonal antibodies to C-ter-
minal peptide (C-19) of PAK68. The arrows on the right side denote
immuno-positive PAK68, PAK65 and PAK62 bands. Lane MW
contained molecular weight standard proteins.

Ó FEBS 2002 Rac–PAK interaction in HL-60 (Eur. J. Biochem. 269) 2625
from the plasma membrane of HL-60 cultured for 5 days
were immobilized with 2¢,5¢-ADP–Sepharose and eluted by
NADPH as shown in Fig. 6A. The separately pooled
fractions 4–8 indicated major protein bands with their
molecular masses of about 68, 35 and 21 kDa, respectively
(Fig.6B).IneachfractionthethreetypesofPAKwere
effectively immunoprecipitated by antibodies to PAK68,
and Rac appeared to be coimmunoprecipitated with PAK
in a concentration-dependent manner. The 35-kDa protein
did not precipitate with anti-PAK68 IgG and its identity
was not clear, demonstrating that activated Rac binds to
PAK in the membrane of HL-60 during induced differ-
entiation. As neither PAK nor Rac immunoprecipitated
with nonimmune rabbit IgG, the results of coimmunopre-
cipitation suggested that their binding was specific. The
investigation to understand more about the expression and
functional diversity on each PAK isoform in the process of
differentiation is in progress.
From the fluorescence titration of the mant-GppNHp
complex of Rac1 with purified PAK68, the binding strength
of Rac1 to PAK68 was determined. As shown in Fig. 7, the
result indicates an approximate 1 : 1 binding of PAK to Rac
(dotted line in the inset) with a K
d
value of about 6.7 n
M
,
which is 10-fold or more stronger than the binding of
p67phox to Rac1 [32]. In the present study p67phox was also

observed in the cytosol but not in the membrane during the
induced differentiation of HL-60 into granulocytes (data
not shown).
DISCUSSION
PAK is a member of the serine/threonine kinase family,
which includes three types of isoform, PAK68, PAK65 and
PAK62. They have been shown to have a high degree of
sequence homology with the Saccharomyces cerevisiae
kinase STE20, involved in pheromone signaling [7, 33].
The three types of PAK are widely expressed in many
human tissues, and they are also found in undifferentiated
and differentiated HL-60 cells. These PAK proteins are
highly homologous to each other and bind specifically with
Rac or Cdc42 in its active, GTP-bound state through the
small GTPase binding (CRIB) domains.
Rac (or Cdc42)–PAK interactions lead to PAK auto-
phosphorylation and, once phosphorylated, its binding
affinity for Rac (or Cdc42) is reduced, and PAK dissociates
Fig. 5. Immunoprecipitated Rac exhibits PAK binding activity in the
membrane fraction of differentiated HL-60 cells. Plasma membrane
(1.25 mg protein) was incubated with antibodies to Rac1 for 3 h at
3 °C, and then protein A–agarose beads were added and gently
stirred for 1 h. After washing the beads, the immunoprecipitates
(150 lg protein) were loaded onto SDS/PAGE and then PAK (A)
and Rac (B) were detected by their antibodies. The arrows indicate
immunoreactive protein bands. Lane MW shows molecular mass
standard proteins.
Fig. 6. Binding of Rac and PAK in the plasma membrane of HL-60 cells. Solubilized membrane (6.90 mg protein) from HL-60 cells differentiated by
1 l
M

ATRA for 5 days was stirred gently with 3.0 mL of 2¢,5¢-ADP-Sepharose beads in the presence of 25 m
M
Tris/HCl buffer, pH 7.5, containing
10 m
M
NaCl, 0.12
M
KCl and 0.1% Triton X-100 for 3 h at 3 °C. The mixture was transferred to the column (10 · 20 mm) and Sepharose beads
were washed three times with the same buffer as above. Proteins were eluted by 50 m
M
Tris/HCl, pH 7.5, containing 2 m
M
NADPH and protease
inhibitors. Elution profile was indicated in (A) and fraction number 4, 5, 6, 7 and 8 were pooled (black bar), respectively. Proteins in each fraction
were separated by SDS/PAGE and subjected to silver stain (B). The numbers on the left side exhibit molecular weight standards. PAK68, PAK65
and PAK62 (C, top) and Rac (C, bottom) were visualized, respectively, by Western blot. Their positions were indicated by arrows on the right side.
Top numbers on each panel correspond to those of fraction eluted from the ADP–Sepharose column shown in panel A.
2626 Y. Nisimoto and H. Ogawa (Eur. J. Biochem. 269) Ó FEBS 2002
from the complex to phosphorylate downstream target
proteins in MAP kinase cascades. However, very little
information is available concerning the signaling pathways
beyond this point.
It has been demonstrated that in resting phagocytes Rac
protein is located in a cytosolic complex with an inhibitor
protein, RhoGDI [34–36]. Upon the stimulation of cells
exposed to bacteria or to a variety of soluble stimuli, Rac1
and Rac2 (the more abundant isoform in neutrophils)
become associated with the plasma membrane [37]. The
binding of activated Rac with p67phox in the membrane
facilitates the formation of assembled NADPH oxidase

complex, producing superoxide anion; however, Cdc42 is
inactive in this process [5]. Thus, Rac, PAK and p67phox
proteins were not detected in the plasma membrane of
dormant granulocytes in spite of the fact that they were
observed abundantly in cytoplasm. Whereas reactive
oxygen species are classically thought of as cytotoxic and
mutagenic or as inducers of oxidative stress, recent
evidence suggests that O
2
–
plays a role in signal transduc-
tion. The production of low levels of intracellular reactive
oxygen in growth factor-stimulated nonphagocytic cells
was reported [38,39], but its function is unclear. Immedi-
ately following induction of the differentiation with
ATRA, HL-60 cells show higher levels of O
2
–
and H
2
O
2
than those produced in cells cultured without added
ATRA (Fig. 2). These results suggest that a slightly higher
level of reactive oxygen species generated by signaling
responses to ATRA may trigger the activation of MAP
kinase cascades related to cell differentiation. Thus, during
the differentiation, the interactions between Rac and PAK
proteins located upstream of the signal pathways were
examined.

The present study revealed that Rac and PAK isoforms
increased in both cytosol and membrane fractions upon
the induced differentiation of HL-60 cells. No remarkable
Racwasseenintheplasmamembranefractionof
undifferentiated HL-60 cells. In addition, Rac and PAK
were distributed in the cytosol of neutrophils but were not
found in the plasma membrane. However, upon ATRA-
induced differentiation, Rac appeared in the membrane
and specifically bound to PAK protein to activate its
autophosphorylation, suggesting that Rac–PAK interac-
tions in the membranes possibly work as an ATRA-
responsive signaling mechanism to activate MAP kinase
linked to cell differentiation. Although the Rac–PAK
complex was also observed in the cytosol of HL-60 (data
not shown), it is not clear yet if the membrane-associated
Rac–PAK complex has a distinctive function from that of
the cytosolic one, or whether both complexes synergize
upon the cell differentiation. Further studies are required
to investigate the functional roles of Rac-PAK binding
seen in cytosol. The mammalian Rho subfamily of GTP
binding proteins, including Rac, Cdc42 and Rho, are
reported to participate in the regulation of diverse cellular
functions such as actin cytoskeletal dynamics, superoxide
generation, membrane trafficking, apoptosis, cell cycle
control, activation of phospholipases C and D, and cell
chemotaxis [40–46]. Besides these functions, our present
data suggest the possibility that membrane-bound Rac
is involved in the differentiation of HL-60 cells through
its binding to PAK protein in downstream signaling
pathways.

ACKNOWLEDGEMENT
We thank Dr Ryouko Tsubouchi for preparing HL-60 cells differen-
tiated with ATRA, and this study was supported by the fund from
Aichi Medical University, Medical School.
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Fig. 7. Binding affinity of purified PAK68 to Rac1 quantitated by
fluorescence titration. Before addition of Rac, the fluorescence emission
spectrum (excitation, 355 nm) of free mant-GppNHp (0.025 l
M

)was
measured (spectrum 1). After adding 0.035 l
M
Rac1 and incubating
for 15 min at 20 °C, the fluorescence spectrum was recorded (spectrum
2). Ten minutes after the addition of 8, 16, 24, 32, 40, 45, 55 and 70 n
M
PAK68 to the incubation mixture, the emission spectra (spectra 3–12)
were recorded. The increase in fluorescence intensity (DF
440
)isshown
as a function of the concentration (8–70 n
M
)oftheaddedPAK68
(inset). The observed fluorescence was corrected for volume changes.
The stoichiometry and K
d
for the binding of PAK68 with mant-
GppNHp-Rac1 complex were determined.
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