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

doi:10.1046/j.1432-1033.2002.02939.x

Eur. J. Biochem. 269, 2622–2629 (2002) (cid:2) FEBS 2002

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

Yukio Nisimoto1 and Hisamitsu Ogawa2 1Department of Biochemistry, Aichi Medical University, School of Medicine, Nagakute, Aichi, Japan; 2Department of Biology, Fujita Health University School of Medicine, Toyoake, Aichi, Japan

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 Kd value of 6.7 nM.

Keywords: Rac; PAK; HL-60; GTPase; MAP kinase.

Undifferentiated human promyelocytic leukemia HL-60 cells show little or no superoxide production, but generate a – concentration upon incubation with all-trans- very low O2 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

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].

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 – when HL-60 cells are differentiated into produce O2 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

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 – at an early stage that HL-60 cells produce low levels of O2 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

superoxide

Diisopropyl fluorophosphate, protease inhibitor cocktail, cytochrome c, NADPH and 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)

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remove nuclei and unbroken cells. The supernatant was further centrifuged at 150 000 g for 1 h. The precipitates were washed with 25 mM phosphate buffer, pH 7.3, contain- ing 10% glycerol, and then stored as a membrane fraction at )70 (cid:3)C. The supernatant was used as a cytosol fraction.

Purifications of glutathione S-transferase (GST)-Rac1 expressed in E.coliand PAK 68 from human neutrophil cytosol

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-cm2 Falcon 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 37 (cid:3)C 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

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 (cid:3)C. The beads were transferred into a column (10 · 100 mm) and washed well with buffer A containing 2 mM NADPH. The fractions released from the column were incubated while gently stirring with agarose-conjugated PAK68 anti- bodies for 12 h at 3 (cid:3)C. The agarose beads were transferred into a column (10 · 50 mm) and washed extensively with 50 mM buffer A containing 0.1% Triton X-100. The PAK was eluted from the column with 25 mM 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 mM 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

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

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 (cid: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 mM 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

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 · 105 cellsÆmL)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

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

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 mM NaCl, 10% glycerol, 1 mM EDTA, 50 lM 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 (cid:3)C [21]. The cavitate was centrifuged (800 g, 5 min) to

SDS/PAGE (0.1% SDS and 10% gel) was carried out at 25 (cid:3)C for 3 h, and the gels were subjected to silver

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staining. Proteins separated by SDS/PAGE were also transferred to an Immobilon-P membrane (Millipore Corp.) [26]. The membrane was incubated at 25 (cid:3)C for 2 h in 5% skim milk in 20 mM phosphate buffer, pH 7.3, containing 0.14 M NaCl and 2.7 mM KCl. Polyclonal antibodies used were those to PAK68 and Rac1. After the membrane was reacted with antibodies washing, (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 20 (cid:3)C in 0.3 mL of 50 mM Tris/HCl buffer, pH 7.5, containing 3 mM NaCl, 50 mM KCl and 0.1 lM MgCl2. 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 Kd values as described previously [27].

Fig. 1. ATRA-induced changes in gp91phox production and superoxide generating activity in HL-60 cells. After stimulation of the cells with 10 lM PMA, SOD-inhibitable superoxide production was measured in the presence of 0.1 mM 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.

R E S U L T S

–,

Induction of gp91phoxand superoxide generating activity of HL-60 Cells

it is well known that,

– production both with and without ATRA induction. O2 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 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 – generating of HL-60. Thus, in the present study the O2 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 its content was significantly the membrane. However,

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, 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 – production in responce to ATRA increased the rate of O2 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 – per 10 min per value of neutrophils was 980 nmol of O2 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 – per 10 min per generating activity (15 ± 5 nmol O2 107 cells). The dormant cells exhibited a very low level of

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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 lM 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.

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

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 lM 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.

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

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. 3. Immunoblot analysis of Rac in the cytosolic and membrane fractions of HL-60 cells. The differentiation of the cells were induced with 1 lM 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.

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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 Kd value 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

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). 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

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 (cid: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 lM ATRA for 5 days was stirred gently with 3.0 mL of 2¢,5¢-ADP-Sepharose beads in the presence of 25 mM Tris/HCl buffer, pH 7.5, containing 10 mM NaCl, 0.12 M KCl and 0.1% Triton X-100 for 3 h at 3 (cid: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 mM Tris/HCl, pH 7.5, containing 2 mM 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.

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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 including Rac, Cdc42 and Rho, are binding proteins, 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.

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 lM) was measured (spectrum 1). After adding 0.035 lM Rac1 and incubating for 15 min at 20 (cid:3)C, the fluorescence spectrum was recorded (spectrum 2). Ten minutes after the addition of 8, 16, 24, 32, 40, 45, 55 and 70 nM PAK68 to the incubation mixture, the emission spectra (spectra 3–12) were recorded. The increase in fluorescence intensity (DF440) is shown as a function of the concentration (8–70 nM) of the added PAK68 (inset). The observed fluorescence was corrected for volume changes. The stoichiometry and Kd for the binding of PAK68 with mant- GppNHp-Rac1 complex were determined.

A C K N O W L E D G E M E N T

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.

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.

R E F E R E N C E S

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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 – plays a role in signal transduc- evidence suggests that O2 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 – and H2O2 ATRA, HL-60 cells show higher levels of O2 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

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