Interaction between p21-activated protein kinase and Racduring differentiation of HL-60 human promyelocytic leukemia cell induced by all- trans -retinoic acid Yukio Nisimoto1and Hisamits
Trang 1Interaction between p21-activated protein kinase and Rac
during differentiation of HL-60 human promyelocytic leukemia cell induced by all- trans -retinoic acid
Yukio Nisimoto1and Hisamitsu Ogawa2
1
Department of Biochemistry, Aichi Medical University, School of Medicine, Nagakute, Aichi, Japan;2Department 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 O2 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 Kdvalue of 6.7 nM
Keywords: Rac; PAK; HL-60; GTPase; MAP kinase
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 b558, 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 O2 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 O2 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
M A T E R I A L S A N D M E T H O D S
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: nisiio@amugw.aichi-med-u.ac.jp
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)
Trang 2diatrizoate) 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-cm2Falcon tissue culture dishes containing
20 mL of RPMI 1640 (Gibco BRL) supplemented with
10 mM Hepes, pH 7.4, 10% heat-inactivated fetal bovine
serum and kanamycin (50 lgÆmL)1) at 37C in a
humid-ified incubator with 5% CO2 Differentiation was induced
by the addition of 1 lM 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/Pi After centrifugation, the number of
packed cells was 2–5· 107
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 H2O2 using
5.95· 105cellsÆmL)1 of NaCl/Pi 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 (0.1M Tris/HCl buffer, pH 7.4,
containing 0.1MKCl, 5.5 mMNaCl, 10% glycerol, 1 mM
EDTA, 50 lMdiisopropylfluorophosphate 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
3C [21] The cavitate was centrifuged (800 g, 5 min) to
remove nuclei and unbroken cells The supernatant was further centrifuged at 150 000 g for 1 h The precipitates were washed with 25 mMphosphate 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 glutathioneS-transferase (GST)-Rac1 expressed inE 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 mMNADPH The fractions released from the column were incubated while gently stirring with agarose-conjugated PAK68 anti-bodies for 12 h at 3C The agarose beads were transferred into a column (10· 50 mm) and washed extensively with
50 mMbuffer A containing 0.1% Triton X-100 The PAK was eluted from the column with 25 mMglycine/HCl buffer,
pH 3.0, and the eluted fractions were quickly neutralized at
pH 7.0 by adding 0.2MTris/HCl buffer, pH 9.0, containing 20% glycerol and 1 mMdithiothreitol 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 incubdifferenti-ated 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 mMNADPH 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
25C for 3 h, and the gels were subjected to silver
Trang 3staining Proteins separated by SDS/PAGE were also
transferred to an Immobilon-P membrane (Millipore
Corp.) [26] The membrane was incubated at 25C for
2 h in 5% skim milk in 20 mMphosphate buffer, pH 7.3,
containing 0.14M NaCl and 2.7 mM 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 mM NaCl/Pi, 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 20C in
0.3 mL of 50 mM Tris/HCl buffer, pH 7.5, containing
3 mMNaCl, 50 mMKCl and 0.1 lMMgCl2 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 MgCl2
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 Kdvalues as
described previously [27]
R E S U L T S
Induction of gp91phox 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 O2 production in responce to
phorbol 12-myristate 13-acetate (PMA) from 6.5 ±
5 nmol per 10 min per 107 cells on day 0 of incubation
to 495 ± 110 on day 5 The corresponding reference
value of neutrophils was 980 nmol of O2 per 10 min per
107 cells Concomitantly, plasma membrane-associated
gp91phox content, which is a large subunit of
flavocyto-chrome b558 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 O2 per 10 min per
107 cells) The dormant cells exhibited a very low level of
O2 production both with and without ATRA induction The increase in hydrogen peroxide, which is formed via dismutation of O2, 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 leukpromyel-ocytes 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 O2 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 lM ATRA for a week, and Rac and PAK were assayed in both cytosol and 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 the presence of 0.1 m 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.
Trang 4increased 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
were incubated in the absence (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 ATRA for 0, 1, 3, 5 and 7 days (lane 0–7) and lane N
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
Rac1 and Rac2 was indicated by an arrow.
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 ATRA for 0, 1, 3, 5 and 7 days (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), and then electrically blotted to Immobilon PVDF membrane 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.
Trang 5from 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) In each fraction the three types of PAK were
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 Kdvalue of about 6.7 nM, 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)
D I S C U S S I O N
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.
Trang 6from 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 O2 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 O2 and H2O2
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 Rac was seen in the plasma membrane fraction of 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
A C K N O W L E D G E M E N T
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
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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 ) is shown
as a function of the concentration (8–70 n M ) of the added PAK68
(inset) The observed fluorescence was corrected for volume changes.
The stoichiometry and K d for the binding of PAK68 with
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