©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Activation Induces Dephosphorylation of Cofilin and Its Translocation to Plasma Membranes in Neutrophil-like Differentiated HL-60 Cells (*)

(Received for publication, April 18, 1995)

Kazuhiro Suzuki (1)(§) Teruhide Yamaguchi (1) Toshikazu Tanaka (1) Toru Kawanishi (1) Tomoko Nishimaki-Mogami (1) Kazuo Yamamoto (2) Tsutomu Tsuji (2) Tatsuro Irimura (2) Takao Hayakawa (1) Atsushi Takahashi (1)

From the  (1)National Institute of Health Sciences, 18-1, Kamiyoga 1-chome, Setagaya-ku, Tokyo 158, Japan and (2)Faculty of Pharmaceutical Sciences, University of Tokyo, 3-1, Hongo 7-chome, Bunkyo-ku, Tokyo 113, Japan

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

We suggested that a cytosolic 21-kDa phosphoprotein played an important role in opsonized zymosan-triggered activation of superoxide-generating enzyme in neutrophil-like HL-60 cells through dephosphorylation (Suzuki, K., Yamaguchi, T., Oshizawa, T., Yamamoto, Y., Nishimaki-Mogami, T., Hayakawa, T., and Takahashi, A (1995) Biochim. Biophys. Acta 1266, 261-267). In the present study, we characterized the phosphoprotein and studied changes in its localization upon activation of phagocytes. The 21-kDa phosphoprotein was rapidly dephosphorylated upon activation not only with opsonized zymosan but also with formyl-Met-Leu-Phe and arachidonic acid. The peptide fragments derived from the 21-kDa phosphoprotein were found to have the same amino acid sequences as those of cofilin, an actin-binding protein. The phosphoprotein reacted exclusively with anti-cofilin antibody on two-dimensional immunoblots. Accordingly, together with its apparent molecular weight, isoelectric point, and detection of phosphoserine as a phosphoamino acid, we concluded that the 21-kDa phosphoprotein was a phosphorylated form of cofilin. The amount of cofilin in membranous fractions was increased upon activation. Furthermore, confocal laser scanning microscopy showed that cofilin existed diffusely in the cytosol and nuclear region of the resting cells, while in the activated cells, it was accumulated at the plasma membrane area, forming ruffles or endocytic vesicles on which O should be produced. These results suggested that in resting cells cofilin exists as a soluble phosphoprotein in the cytosol and nuclei, while upon stimulation a large portion of cofilin is dephosphorylated and translocated to the plasma membrane regions.


INTRODUCTION

Phagocytes including neutrophils and macrophages play a crucial role in host defense systems against microorganisms and harmful substances. They are activated by a wide variety of stimulants such as opsonized zymosan (OZ), (^1)formyl peptides, and arachidonic acid (AA). Activated cells show complicated responses including superoxide production, endocytosis, degranulation, and release of cytokines or lipid mediators(1) . On the other hand, protein phosphorylation has been extensively studied in various functional proteins of eukaryotes such as enzymes, receptors, and cytoskeletal proteins. Recently, it has been recognized that not only phosphorylation but also dephosphorylation of the proteins is important for the regulation of numerous cellular processes, including signal transduction(2) . In the case of neutrophils, many reports have shown the importance of the increased phosphorylation of certain proteins (e.g. p47, p67, and substrates of tyrosine kinases) in the activation of respiratory burst oxidase(3, 4) . It has been hypothesized that increased phosphorylation of p47 may be a triggering event for the translocation of the cytosolic components to membranes to form an active complex of the O-generating enzyme(5, 6) . Recently, the seven phosphorylated sites of p47 have been investigated in detail(7) . However, the physiological roles of protein phosphorylation in the activation of O-generating enzyme remain to be established, partly because the events of cellular phosphorylation could not be reconstituted in an O-producing cell-free system (8, 9, 10) . Although stimuli-induced dephosphorylation of a low molecular mass protein (18-21 kDa) in neutrophils has been described by Andrews and Babior (11) and others(12, 13, 14) , its characterization has not been performed. Very recently, we suggested that a similar cytosolic 21-kDa phosphoprotein may be involved in O production through dephosphorylation in neutrophil-like HL-60 cells because 1) OZ-triggered O production was accompanied with the dephosphorylation of 21-kDa protein; 2) when the dephosphorylation of 21-kDa protein was inhibited by okadaic acid, the respiratory burst could not be triggered even though p47was heavily phosphorylated; and 3) the 21-kDa protein was, at least in part, coimmunoprecipitated with the components of respiratory burst oxidase(15) .

In this work, we identified the 21-kDa phosphoprotein as cofilin, a widely distributed actin-binding protein discovered by Nishida et al.(16) . Moreover, we present data on the activation-dependent translocation of cofilin to the membranous region and discuss the roles of this protein in the activation of phagocytes.


EXPERIMENTAL PROCEDURES

Cells

HL-60 cells obtained from the Japanese Cancer Research Resources Bank were cultured in RPMI 1640 medium supplemented with 10% fetal calf serum. The details of culture conditions were previously described(15) . The cells were induced to neutrophil-like cells with 1.25% dimethyl sulfoxide for 6 days according to Collins et al.(17) .

Antibodies

The affinity-purified rabbit anti-cofilin and anti-destrin antibodies were generously provided by Drs. I. Yahara and K. Iida (Tokyo Metropolitan Institute of Medical Science)(18) . Monoclonal anti-cofilin antibody (MAB22) was kindly donated by Drs. T. Obinata and H. Abe (Chiba University, Japan)(19) . Affinity-purified rabbit anti-nucleoside diphosphate kinase was a kind gift from Dr. N. Kimura (Tokyo Metropolitan Institute of Gerontology). Preparation and purification of anti-p47 peptide(340-355) antibody were described before(15) . Dichlorotriazinyl amino fluorescein-conjugated F(ab`)(2) fragment goat anti-mouse IgG (H+L) was obtained from Immunotech (Marseille, France).

Reagents

Lysyl endopeptidase was obtained from Wako Chemicals (Osaka, Japan). P(i) and immunostaining chemiluminescence reagents were obtained from DuPont NEN. OZ was prepared according to the method of Markert et al.(20) . Briefly, 700 mg of zymosan A (Sigma) was incubated with 70 ml of human serum at 37 °C for 30 min and washed with HBSS. The OZ suspended in HBSS (10 mg/ml) was divided and stored at -80 °C until use.

All other chemicals used were commercial preparations of the highest purity.

Two-dimensional Electrophoresis of Phosphoproteins

The differentiated HL-60 cells, which had been preincubated with P(i) at 37 °C for 60 min, were stimulated with either 0.5 µM FMLP, 20 µM AA, or OZ (1 mg/ml) for 0.5-2.0 min. After subcellular fractionation, the cytosolic fractions were subjected to two-dimensional gel electrophoresis followed by autoradiography. The details of the experimental conditions were the same as described before(15) . For immunoblotting, the first dimensional isoelectric focusing with a different pH gradient was also adopted by using 2.0% ampholine, pH 3.5-10 (Pharmacia, Japan), only. The P incorporated in a protein was determined by Bioimage Analyzer BAS2000 (Fuji Film Co., Tokyo). To calculate the means of data, each value was normalized and expressed as a percent of the control (not activated).

Protein Sequencing

The 21-kDa protein was excised from two-dimensional gels (about 30 gels), which were derived from about 1 10^8 cells. Then, in-gel proteinase digestion using lysyl-endopeptidase was performed essentially according to Kawasaki et al.(21) . The proteolysis was carried out in the absence of SDS at enzyme/substrate = 1/500. The resulting peptides were purified by reverse phase high performance liquid chromatography on a Waters Puresil C18 column (4.6 150 mm) heated at 40 °C using a 1-h linear gradient of 0-18% acetonitrile, 0-42% isopropyl alcohol, and 0.05-0.04% trifluoroacetic acid at a flow rate of 1.0 ml/min(22) . The absorbance of the eluate was monitored at 220 nm, and each peak was subjected to a gas phase peptide sequencer (PPSQ-10, Shimadzu Co., Kyoto, Japan). GENETYX-CD biodata base software (version 25.0) was used to search the SWISS-PROT protein data base.

Immunoblotting

The detailed conditions for electroblotting were previously described(15) . Briefly, the proteins separated on the slab gels (1-mm thick) were electrophoretically transferred to polyvinylidene difluoride membranes (Bio-Rad) at pH 11.0. After being treated with Block Ace (Dainippon Pharmaceutical Co., Osaka, Japan) at 4 °C for 17 h, the polyvinylidene difluoride membranes were probed with affinity-purified rabbit anti-cofilin, anti-destrin, anti-nucleoside diphosphate kinase, or anti-p47 peptide antibody. Immunochemical detection by chemiluminescence was performed essentially according to the DuPont NEN technical brochure.

Nitrogen Cavitation and Subcellular Fractionation

The cells (3 10^7), which had been preincubated at 37 °C for 5 min, were activated with 0.5 µM FMLP, 20 µM AA, or OZ (final 6 mg/ml) in HBSS (l ml) at 37 °C for 2 min. The activation was stopped by the addition of 5 ml of ice-cold inhibitor solution, which contained 5 mM diisopropyl fluorophosphate, 20 µM leupeptin, 20 µM pepstatin, 0.1 M NaF, 10 mM EDTA, 2 mMN-ethylmaleimide, 1 mM ammonium molybdate, 1 mM iodoacetic acid, 1 mM benzamidine, 75 mM NaCl, 2.5 mM KCl, 3 mM Tris, and 3 mM Hepes (pH 7.3), and the cells were chilled by immersing them in melting ice for 20 min. The cells were then packed by centrifugation (800 rpm, 15 min) and resuspended in 0.7 ml of 0.125 M sucrose solution containing the above inhibitors except diisopropyl fluorophosphate. The cells were pressurized with N(2) for 20 min at 350 p.s.i. in a nitrogen bomb (model 4639, Parr Instrument Co., Moline, IL) according to Borregaard et al.(23) , and the cavitates were separated into three fractions by successive centrifugation as described before(12) . A fraction including zymosan-containing phagosomes and nuclei was obtained by the first centrifugation (500 g, 15 min). Membranous and cytosolic fractions were obtained by the following centrifugation (100,000 g, 30 min). All the fractionation procedures were performed at 0-4 °C. The phagosome and nucleus fractions were suspended in 100 µl of 125 mM Tris-HCl buffer (pH 6.8) containing 0.1% SDS and 2 mM MgCl(2) by sonication and treated with 25 units of benzone nuclease (Merck, Darmstadt, Germany) at 37 °C for 30 min to break the DNA. Then, each phagosome and nucleus fraction was mixed with 15 µl of 3% SDS solution containing 15 mM dithiothreitol, sonicated for 5 min, and incubated at 70 °C for 2 min. Finally, the mixtures were centrifuged at 1,400 g for 10 min to remove the unsolubilized materials including zymosan. The membranous fraction was solubilized by sonication in 100 µl of 125 mM Tris-HCl buffer (pH 6.8) containing 0.4% SDS and 1 mM dithiothreitol, and heat treatment was performed at 75 °C for 2 min. 100 µl of the cytosolic fraction was mixed with 5 µl of 5% SDS solution containing 20 mM dithiothreitol and heated as above. 10 µl of the phagosome and nucleus fractions and 15 µl of the membranous and cytosolic fractions were subjected to SDS-polyacrylamide gel electrophoresis according to Laemmli (24) using 12.5% gel.

Confocal Laser Scanning Microscopy

The cells were activated as above, and the activation was stopped by the addition of formaldehyde (final 4%) and immediate chilling in melting ice for 5 min. Incubation for formaldehyde fixation was continued at room temperature for 30 min. Then, the fixed cells were washed three times with phosphate-buffered saline (PBS) for three times and suspended and diluted in PBS (about 5 10^4 cells/ml). 700 µl of each cell suspension was plated on a slide glass, which had one poly-L-lysine-coated well surrounded by a silicon-coated surface (Matsunami Co., Japan) by centrifugation at 700 rpm and 4 °C for 4 min using a cytospin apparatus (inner diameter, 8 mm) (model SC-2, Tomy Seiko Co., Tokyo). The cells were rinsed with PBS and treated with methanol for 5 min. After three rinses with PBS, they were blocked with 2% bovine serum albumin in PBS at room temperature for 1 h. Then, the slides were incubated with 50 µl of monoclonal anti-cofilin antibody (MAB22) in RPMI 1640 medium containing 10% fetal bovine serum or with the same medium (control for background) for 1 h at 37 °C in a humidified incubator. After three washes with PBS, they were incubated at 37 °C for 1 h with 5-(4,6-dichlorotriazinyl)aminofluorescein-conjugated F(ab`)(2) fragment goat anti-mouse IgG (H+L) diluted 1:130. After three rinses with PBS, the slides were covered with PermaFluor (Immunon, Pittsburgh, PA). The nuclear region of the cells was identified by staining with Hoechst 33258 (Sigma). Digitized images were generated by using a confocal laser scanning microscope (RCM8000, Nikon, Japan) with an excitation wavelength of 488 nm and emission of at least 520 nm. The objective was a Nikon 40X/NA1.15 water immersion.


RESULTS

Dephosphorylation of 21-kDa Protein

We previously observed that in differentiated HL-60 cells OZ induced dephosphorylation of a cytosolic 21-kDa phosphoprotein, which may play an important role in O production (15) . Two-dimensional electrophoresis of P-labeled proteins revealed that FMLP also caused remarkable dephosphorylation of the same protein (Fig. 1). This result seemed consistent with the previous observations on one-dimensional gels(11, 13, 14) . Then, we tested the effects of other stimulants on the phosphorylated state of the 21-kDa protein quantitatively. Fig. 2shows that the 21-kDa phosphoprotein was commonly dephosphorylated when the cells were stimulated with OZ, FMLP, or AA, well-known activators for O-generating respiratory burst oxidase. The reaction was rapid, and more than 70% of the incorporated P was lost within 1 min after the addition of stimulants. Therefore, together with the previous observation that the 21-kDa phosphoprotein could be coimmunoprecipitated with p67 and p47(15) , these results suggest that the dephosphorylation of the 21-kDa protein is a common event on the pathway leading to the activation of leukocytes. Phorbol ester, a potent artificial activator, did not induce such rapid dephosphorylation of the 21-kDa protein, probably because it could bypass dephosphorylation to provoke the respiratory burst (data not shown).


Figure 1: Dephosphorylation of the 21-kDa protein by FMLP. P(i)-loaded HL-60 cells were exposed to HBSS (A) or 0.5 µM FMLP (B) at 37 °C for 2 min. The cytosolic fraction derived from 1 10^6 cells was subjected to two-dimensional electrophoresis and autoradiography. The arrow in each photograph indicates the 21-kDa protein.




Figure 2: Time course of dephosphorylation of the 21-kDa protein. P(i)-loaded HL-60 cells were activated with FMLP (), AA (▴), or OZ (bullet) at 37 °C for the indicated time, and two-dimensional electrophoresis was performed as in Fig. 1. P incorporated into the 21-kDa protein was determined by radioluminography and expressed as percent of control (not activated) on the verticalaxis. The standard deviations were less than 4.7% of the control.



Protein Sequence of 21-kDa Protein

To characterize the 21-kDa protein, we tried to determine its amino acid sequence. First, the spots of 21-kDa protein on electroblotted membranes were collected and subjected to a protein sequencer. However, the amino-terminal sequencing was not successful, probably because the amino terminus was blocked. Then, the proteolytic fragments of the 21-kDa protein were prepared by in-gel digestion using lysyl endopeptidase, purified by high performance liquid chromatography, and amino acid sequences were determined. 12 individual peptides were found to align with human cofilin (25) (named non-muscle type(26) ), a widely distributed actin-binding protein(16, 17, 26) , by searching the protein sequence data base (Fig. 3).


Figure 3: Alignment of sequences of 12 peptides derived from the 21-kDa protein with the amino acid sequence of human cofilin (non-muscle type). The asterisks indicate the same amino acids between the peptides and human cofilin. X shows an unidentified amino acid. PIP(2) means phosphatidylinositol 4,5-bisphosphate.



Immunoblotting

To investigate whether the 21-kDa protein has the known characteristics of cofilin, immunoblotting was carried out using antibody to cofilin. According to earlier studies, a few spots of isotypes of cofilin had been expected to appear on the immunoblot(26, 27) . Actually, in the order of isoelectric points (from acidic to basic), three major spots corresponded to a phosphorylated form of non-muscle-type cofilin, an unphosphorylated form of muscle-type cofilin, and an unphosphorylated form of non-muscle-type cofilin, respectively. The most acidic spot of the three major spots coincided with the spot of the P-labeled 21-kDa protein on the autoradiogram (Fig. 4, panels1 and 2). By employing another pH-gradient gel that could separate the individual spots of cofilin more clearly, it was confirmed that the spot of the phosphorylated form of non-muscle-type cofilin was perfectly superimposed on the spot of the 21-kDa protein on the autoradiogram (Fig. 4, panels3 and 4). The more acidic minor spot of the phosphorylated form of muscle-type cofilin could also be detected by using more than 8 10^6 cells (Fig. 4, panel4), while it did not correspond with the 21-kDa protein. Other faint spots on the immunoblots were not identified. Other antibodies including anti-destrin (a cofilin-like protein), anti-Rac (a small G protein required for O production), anti-myosin light chain, and anti-nucleoside diphosphate kinase antibodies reacted with spots distinct from that of the 21-kDa protein on the immunoblots (not shown). Therefore, we concluded that the cytosolic 21-kDa phosphoprotein in differentiated HL-60 cells is a phosphorylated form of non-muscle-type cofilin.


Figure 4: Coincidence of the 21-kDa protein with the phosphorylated form of human cofilin (non-muscle type). The proteins in the cytosolic fractions derived from 8 10^6 cells were two-dimensionally separated as in Fig. 1(panels1 and 2) or by using one-dimensional gels with a different pH-gradient (panels3 and 4) and electroblotted on polyvinylidene difluoride membranes. Panels1 and 3, autoradiograms of P-labeled phosphoproteins. The arrow indicates the 21-kDa phosphoprotein. Panels2 and 4, immunoblots using anti-cofilin antibody on the electroblotted membranes of panels1 and 3, respectively. The arrow in each photograph indicates the phosphorylated form of non-muscle-type cofilin. Details are described in the text.



Subcellular Distribution of Cofilin

The cytosolic components (p47, p67, and Rac protein) of respiratory burst oxidase translocate from cytosol to plasma membranes to form an active complex of the oxidase when neutrophils are activated (8, 28) . It has also been reported that the intracellular localization of cofilin changes in response to specific stimuli in cultured fibroblasts(18) , T-cells(29) , and thyroid cells(30) . Therefore, we investigated whether the intracellular distribution of cofilin was changed during the activation of the neutrophil-like HL-60 cells. The resting and activated cells were disrupted by nitrogen cavitation, and subcellular fractionation was performed by successive centrifugations. Plasma membranes were contained both in the membranous fraction and in the phagosome and nucleus fraction. As shown in Fig. 5A, the amount of p47 in the membranous fraction was increased by the treatment with FMLP, AA, or OZ, consistent with the previous observations(8, 28) . With activation, the amount of cofilin in the membranous fraction was also increased significantly (1.3-1.5-fold) (Fig. 5B). The activation-induced increase of cofilin was the most remarkable in the phagosome and nucleus fractions (Fig. 5C). Among the stimulators tested, OZ caused the highest rise (4.0-5.0-fold) in cofilin in the fraction, probably because OZ could form the high dense phagosomes. The results of subcellular fractionation suggest that when the cells were activated a significant portion of cofilin translocated from the cytosol to particulate organelles, including the plasma membranes. However, the association of cofilin with the membranous organelles seemed not so tight because sonication for cell disruption caused the release of cofilin from the membranes (not shown).


Figure 5: Activation-dependent increase in p47 and cofilin in particulate fractions. The cells were exposed with HBSS (1), FMLP (2), AA (3), or OZ (4), and subcellular fractionation was carried out. An aliquot of each fraction was subjected to SDS-polyacrylamide gel electrophoresis, and p47 and cofilin were detected by immunoblotting. Details are described under ``Experimental Procedures.'' A, p47 in membranous fraction; B, cofilin in membranous fraction; C, cofilin in phagosomal and nuclear fraction.



Fluorescence Staining of Cofilin in the Cells

To visualize the changes in cofilin localization upon activation, the intracellular cofilin was indirectly stained with fluorescence-labeled antibody and observed by confocal laser scanning microscopy. As shown in Fig. 6, in the unstimulated neutrophilic HL-60 cells, cofilin was diffusely distributed in the cytosol and nuclear regions. Similar observations have been reported on cultured fibroblasts (18) and thyroid cells(30) . Stimulation with AA or FMLP caused remarkable morphological changes in the cells, and a large proportion of cofilin appeared along certain areas of the cell periphery, which are defined as ruffled membranes. OZ, a particulate activator, was taken by the phagocytes, and a large amount of cofilin was collected around the forming endocytic vesicles. The translocation of cofilin was rapid (0.5-1.0 min), and similar localization of cofilin was also observed when the cells were exposed to the activators for 5 min. These results are consistent with the data of subcellular fractionation (Fig. 5). Taken together, the results indicated that upon activation a large portion of cofilin translocated along the dynamically shape-changing plasma membranes, where O should be generated.


Figure 6: Confocal laser scanning microscopy of differentiated HL-60 cells stained with anti-cofilin antibody and dichlorotriazinyl amino fluorescein-conjugated second antibody. The cells were incubated with the following activators or vehicle at 37 °C for 0.5 or 1.0 min. A, HBSS, 1 min; B, 0.5 µM FMLP, 0.5 min; C, 20 µM AA, 1 min; D, OZ, 0.5 min.




DISCUSSION

In this study, we examined a phosphoprotein that was rapidly and commonly dephosphorylated upon activation of differentiated HL-60 cells by FMLP, AA, and OZ. Although similar dephosphorylation has been described(11, 12, 13, 14, 15) , this is the first report on the characteristics of the phosphoprotein. We identified the phosphoprotein as cofilin, a widely distributed actin-binding protein, based on the following reasons. 1) The amino acid sequences of the proteolytic fragments of the phosphoprotein coincided with that of cofilin of human placenta (25) . 2) The apparent molecular mass of the phosphoprotein was estimated to be 21,000 daltons on SDS-polyacrylamide gel electrophoresis, like that of cofilin(16) . 3) The spots of the 21-kDa phosphoprotein on autoradiograms were perfectly superimposed on the spots of phosphorylated cofilin (non-muscle type(26) ) on immunoblots. 4) Phosphoserine was exclusively detected as a phosphorylated amino acid of the 21-kDa protein(15) , as reported for cofilin(27) .

We also investigated stimuli-triggered changes in the localization of cofilin. It was observed that in resting cells cofilin existed diffusely in cytosolic and nuclear regions, while in activated cells it translocated to the plasma membrane area including ruffled membranes and phagocytic vesicles. Cofilin is known to depolymerize actin filament (16) and bind phosphatidylinositol 4,5-bisphosphate specifically on the actin-binding site(31, 32) . Therefore, some possible roles of cofilin can be considered in the activation of phagocytes. First, cofilin may play an important role in the membrane movement of the activated cells, resulting in the formation of ruffled membranes or phagocytic vesicles through depolymerization of actin and remodeling of the cytoskeleton. Second, cofilin may be involved in stimuli-triggered O production for the following reasons. 1) Like cytosolic components of respiratory burst oxidase(28) , cofilin, in response to the stimuli, translocated to plasma membranes, where O should be generated. 2) According to the results of immunoprecipitation, at least a portion of cofilin associated with p67 and p47(15) . 3) Cofilin may regulate the cytoskeleton that associate with the components of the superoxide-generating enzyme(33, 34, 35, 36) . 4) Okadaic acid enhanced the OZ-provoked respiratory burst at non-inhibitory concentrations (less than 1 µM) for the dephosphorylation of cofilin, while at inhibitory concentrations (more than 2 µM) for the dephosphorylation of cofilin it inhibited the O production(15) . 5) Phosphatidylinositol 4,5-bisphosphate bound to profilin (a cofilin-like protein) can be a substrate for only phosphorylated active phospholipase C(37) , and phosphatidylinositol 4,5-bisphosphate released from cofilin can activate phospholipase D directly(38) . Both reactions can yield diacylglycerol, which is a possible physiological activator for O production(39, 40) . Third, the respiratory burst might be affected by the formation of membrane ruffles or endocytic vesicles, and cofilin may modulate superoxide generation indirectly through controlling the shape of plasma membranes.

Although the actual physiological roles of phosphorylation/dephosphorylation of cofilin remain unclear, several recent papers have suggested the importance of its dephosphorylation. Morgan et al. (41) reported that an unphosphorylated form of chick actin depolymerizing factor, a cofilin-like protein, was able to bind and depolymerize actin, while the phosphorylated form was not. If that is true of mammalian cofilin, generation of additional unphosphorylated cofilin could cause depolymerization of actin filaments and reorganization of the cytoskeleton. It has been described in fibroblasts (27) and T cells (29) that cofilin is dephosphorylated in response to exogenous stimuli and translocated to nuclei, resulting in the formation of actin/cofilin rods, whose functions are unknown. In this work, however, we did not observe any actin/cofilin rods in nuclei while cofilin was dephosphorylated. Dephosphorylation of cofilin may not always be linked directly to nuclear translocation of cofilin and formation of actin/cofilin rods. In the dog thyroid cells, thyrotropin also provoked cofilin dephosphorylation uncorrelated with its nuclear accumulation(30) . In those cells, each dephosphorylation reaction was much slower than in our study. On the other hand, Davidson and Haslam (42) recently reported that stimulation of human platelets by thrombin led to rapid dephosphorylation of cofilin. Although the time course of dephosphorylation is similar to our data, okadaic acid did not inhibit the dephosphorylation of cofilin in the activated platelets, unlike our results on the differentiated HL-60 cells(15) . Therefore, cofilin may play stimuli-responsive roles in various cells to make the cells express their particular functions. Our studies suggest that in leukocytes, dephosphorylation and translocation of cofilin play crucial roles in the host defense functions of the phagocytes. Further studies are required on the detailed mechanisms of phosphorylation/dephosphorylation of cofilin and on the functions of cofilin translocated to membranes.


FOOTNOTES

*
This study was performed in part by research grants provided by the Japan Health Sciences Foundation and by the Environment Agency of Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom all correspondence should be addressed.

(^1)
The abbreviations used are: OZ, opsonized zymosan; FMLP, N-formyl-methionyl-leucyl-phenylalanine; AA, arachidonic acid; PBS, phosphate-buffered saline; HBSS, Hanks' balanced salt solution.


ACKNOWLEDGEMENTS

We thank Drs. Ichiro Yahara and Kazuko Iida (Tokyo Metropolitan Institute of Medical Science) for providing affinity-purified rabbit anti-cofilin antibody and demonstrating how to prepare the fluorescence-labeled cells for microscopic observation. We also thank Drs. Takashi Obinata and Hiroshi Abe (Chiba University, Japan) for providing the monoclonal anti-cofilin antibody MAB22 and for useful discussion on this work.


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