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-

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

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

)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-

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-

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

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
–

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-

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.

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

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.

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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Ó FEBS 2002 Rac–PAK interaction in HL-60 (Eur. J. Biochem. 269) 2629