(Received for publication, April 18, 1995)
From the
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.
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), ()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 p47
was 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.
All other chemicals used were commercial preparations of the highest purity.
Figure 1:
Dephosphorylation of the
21-kDa protein by FMLP. P
-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
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
-loaded HL-60 cells were
activated with FMLP (
), AA (▴), or OZ (
) 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.
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 means phosphatidylinositol
4,5-bisphosphate.
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
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.
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.
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.
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.