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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 95 No. 7 (April 1), 2000:
pp. 2407-2412
PHAGOCYTES
Regulation of polymorphonuclear leukocyte phagocytosis by myosin
light chain kinase after activation of mitogen-activated protein kinase
Pamela J. Mansfield,
James A. Shayman, and
Laurence A. Boxer
From the Departments of Pediatrics and Internal Medicine, University
of Michigan, Ann Arbor, MI.
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Abstract |
Polymorphonuclear leukocyte (PMNL) phagocytosis
mediated by Fc RII proceeds in concert with activation of the
mitogen-activated protein (MAP) kinase, extracellular signal-regulated
kinase ERK2. We hypothesized that myosin light chain kinase (MLCK)
could be phosphorylated and activated by ERK, thereby
linking the MAP kinase pathway to the activation of cytoskeletal
components required for pseudopod formation. To explore this
potential linkage, PMNLs were challenged with
antibody-coated erythrocytes (EIgG). Peak MLCK activity, 3-fold
increased over controls, occurred at 4 to 6 minutes,
corresponding with the peak rate of target ingestion and ERK2
activity. The MLCK inhibitor ML-7 (10 µmol/L) inhibited both phagocytosis and MLCK activity to basal values, thereby providing further support for the linkage between the functional response and the
requirement for MLCK activation. The MAPK kinase (MEK) inhibitor
PD098059 inhibited phagocytosis, MLCK activity, and ERK2 activity
by 80% to 90%. To directly link ERK activation to MLCK activation,
ERK2 was immunoprecipitated from PMNLs after EIgG ingestion. The
isolated ERK2 was incubated with PMNL cytosol as a source of
unactivated MLCK and with MLCK substrate; under these conditions ERK2
activated MLCK, resulting in phosphorylation of the MLCK substrate or
of the myosin light chain itself. Because MLCK activates myosin,
we evaluated the effect of directly inhibiting myosin
adenosine triphosphatase using 2,3-butanedione monoxime (BDM) and
found that phagocytosis was inhibited by more than 90% but MLCK
activity remained unaffected. These results are consistent with the
interpretation that MEK activates ERK, ERK2 then activates MLCK, and
MLCK activates myosin. MLCK activation is a critical step in the
cytoskeletal changes resulting in pseudopod formation.
(Blood. 2000;95:2407-2412)
© 2000 by The American Society of Hematology.
| |
Introduction |
Polymorphonuclear leukocytes (PMNLs) are recruited by
chemoattractants to sites of inflammation where they ingest, or
phagocytose, opsonized particles. Two classes of PMNL receptors are
involved in phagocytosis: the Fc receptor and the complement
receptor.1 The sequence of signal transduction events from
FcRIIa receptor ligation leading to the ingestion of IgG-opsonized
particles is partially known. One of the first steps after ligand
binding to the Fc receptor is activation of several nonreceptor protein
tyrosine kinases, such as Syk, p55fgr, and
p59hck.2-6 Subsequently, phospholipase
D is activated during PMNL phagocytosis7,8 and generates
phosphatidic acid.9 Phosphatidate phosphohydrolase generates diglyceride from phosphatidic acid,10 thereby
activating protein kinase C (PKC).8 The
Ca++-independent PKC appears to be important to PMNL
phagocytosis. Our group has recently shown that PKC and Raf-1
translocate to the plasma membrane during the first few minutes of PMNL
phagocytosis.11 In most systems, Raf-1 then initiates the
mitogen-activated protein (MAP) kinase cascade.12 Raf-1
activates MEK, which then phosphorylates ERK.13,14
Previously we showed that activation of the MAP kinases ERK2 and ERK1,
as well as phospholipase D, are required for the ingestion of
IgG-opsonized particles.7,15 Others also have found ERK
activity to increase during both FcR- and CR3-mediated phagocytosis16 and the inhibition of the ERK pathway to
inhibit phagocytosis and the oxidative burst.17 The in vivo
substrate for ERK2 during PMNL phagocytosis has not been determined.
The later part of the phagocytic signaling pathway involves activation
of the cytoskeletal components actin and myosin. Actin polymerization
is required for pseudopod extension and particle engulfment, whereas
myosin furnishes propulsive force by its interaction with actin. Actin
polymerization is initiated by many stimuli, including cross-linking of
FcRIIa.18 Myosin concentrates in the anterior pseudopod
of phagocytosing PMNLs,19 where it binds F-actin and
hydrolyzes adenosine triphosphate (ATP).20 Myosin converts
ATP hydrolysis energy to movement through conformational change of the
myosin molecule while it is bound to actin.21,22 Nonmuscle
myosin II ATPase activity is up-regulated by light chain phosphorylation, relieving the inhibitory effect of the light chains,22 and this phosphorylation is accomplished by
myosin light chain kinase (MLCK).23,24
PMNL chemotaxis and random migration involve MLCK activation and myosin
phosphorylation.25,26 Klemke et al27 used COS-7 cells transfected with constitutively active MEK to show that MLCK is
phosphorylated and activated by the MAP kinase ERK2, and these signals
are required for chemotaxis. Because the MAP kinase cascade and
cytoskeletal interactions are known to be critical to PMNL
phagocytosis, we conjectured that these were similarly linked in
phagocytic signaling. In this study, we demonstrate that ERK2,
activated by MEK, activates MLCK during PMNL phagocytosis and that this
is followed by the activation of myosin ATPase.
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Materials and methods |
Reagents
Peptide substrate specific for myosin light chain kinase,
corresponding to residues 11-23 of smooth muscle myosin light chain, was obtained from Biomol (Plymouth Meeting, PA). The myosin light chain
kinase inhibitor ML-7, (5-iodonaphthalene-1-sulfonyl) homopiperazine, was purchased from Calbiochem (San Diego, CA). 2,3-Butanedione 2-monoxime (BDM) was obtained from Sigma (St. Louis, MO). The MEK
inhibitor PD098059 (2'-amino-3'-methoxyflavone) was a gift from Alan R. Saltiel, Parke-Davis (Ann Arbor, MI). Polyclonal antibody
against ERK2 (p42; C-14) and secondary antibodies were obtained from
Santa Cruz Biotechnology (Santa Cruz, CA). Polyclonal anti-ERK
recognizing both p42 and p44 was a gift from Hua Yu, University of
Southern Florida (Tampa, FL). [-32P]ATP was purchased
from ICN Pharmaceuticals (Irvine, CA). Polyclonal antiplatelet myosin
was a kind gift from Dr Robert Adelstein's laboratory (National
Institutes of Health, Bethesda, MD).
Cells
PMNLs were isolated from peripheral venous blood from healthy
volunteers as previously described.28 PMNLs were pretreated with 5 mmol/L diisopropyl fluorophosphate on ice for 5 minutes, then
washed 3 times with phosphate-buffered saline (PBS). Incubation with
inhibitors was performed before phagocytosis as follows: PMNLs were
incubated with the indicated concentrations of ML-7 or BDM for 10 minutes at 37°C or with 50 µmol/L PD098059 for 30 minutes at
22°C.
Phagocytosis and preparation of PMNL lysates
Phagocytosis was conducted as previously described.7,29
Briefly, sheep erythrocytes (E) were opsonized with anti-sheep E
antibody (EIgG) (Cappel Organon Teknika, Durham, NC). PMNLs (2 × 106/mL) were warmed at 37°C for 3 minutes,
and EIgG was added to PMNLs at a ratio of 50:1. No priming agent was
used. Samples were removed at the indicated time points and microfuged
for 7 seconds, then noningested EIgG were lysed with double-distilled
water containing 1 mmol/L Na3VO4 and 50 mmol/L
NaF. PMNLs were returned to isotonicity by adding KCl, microfuged, and
suspended in MLCK kinase buffer (40 mmol/L HEPES pH 7.0, 5 mmol/L Mg
acetate, 0.55 mmol/L CaCl2, and 0.1% Tween-80) containing
freshly added 1 mmol/L Na3VO4, 50 mmol/L NaF, 1 mmol/L phenylmethylsulfonyl fluoride (PMSF), 10 µg/mL pepstatin, 10 µg/mL leupeptin, 10 µg/mL aprotinin, and 100 µg/mL soybean
trypsin inhibitor. Samples were probe sonicated on ice for 12 seconds 2 times and microfuged for 5 minutes. Supernatants were used for the MLCK
assay. Parallel samples were taken in each experiment to evaluate
phagocytosis, as described previously.15 Significant
differences in phagocytosis were assessed using 1-sample, 2-tailed
Student t tests.
The binding of EIgG to PMNL was tested for possible effects of ML-7 and
BDM by preincubating with inhibitors as described above, then placing
the samples on ice. EIgG was added, and PMNLs were incubated for 30 minutes. No lysis step was performed; samples were fixed and evaluated
by counting the number of PMNLs with and without bound EIgG.
Kinase assay
Each PMNL lysate sample was divided into 2 equal aliquots, 1 to
receive substrate and 1 no substrate. A saturating concentration of
substrate, 300 µmol/L, was chosen for this assay. A reaction mixture
containing substrate peptide or buffer, 5 µCi [-32P]
ATP, and 0.5 mmol/L unlabeled ATP was added to each tube, and samples
were incubated for 10 minutes at 30°C. The reaction was terminated
by filtering through Whatman P81 paper (Clifton, NJ). Filters were
added to scintillation fluid and placed in a scintillation counter
(Wallac, Gaithersburg, MD). Blanks were assay samples run without
substrate. One-sample, 2-tailed Student t tests were used to
assess statistical significance of increases in kinase activity.
Orthophosphate labeling and immunoprecipitation of myosin
PMNLs were suspended at 108/mL in 30 mmol/L HEPES pH
7.4, 140 mmol/L NaCl, 5 mmol/L KCl, 1 mmol/L MgCl2, 10 mmol/L glucose, 2 mg/mL bovine serum albumin, and 1 mCi/mL
H3[32P]O4. They were incubated at
37°C for 30 minutes, then washed with PBS and suspended in PBS with
1 mmol/L Ca++ and 1 mmol/L Mg++. PMNLs were
allowed to phagocytose EIgG for 4 minutes, then samples were lysed with
buffer containing 1% NP-40, 250 mmol/L NaCl, 5 mmol/L EGTA, 20 mmol/L
Tris pH 8, 40 mmol/L 4-nitrophenyl phosphate, and phosphatase
inhibitors and protease inhibitors as described above. Lysates were
incubated overnight with antiplatelet myosin, then for 2 hours with
Protein A-Sepharose. Samples were subjected to 12% SDS-PAGE, and the
gels were dried and exposed to X-ray film.
Preparation of cytosol
PMNLs were isolated and DFP-treated as described above. They were
suspended at 108/mL in extraction buffer (50 mmol/L Tris pH
7.5, 2 mmol/L EGTA, 1 mmol/L PMSF, 1 µg/mL leupeptin, 10 µmol/L
benzamidine, 10 µmol/L pepstatin, and 0.2 µg/mL aprotinin) and were
probe sonicated for 12 seconds 2 times. Extracts were centrifuged for
10 minutes at 800g, and the supernatants were layered on 15%
sucrose in 10 mmol/L HEPES, pH 7.5. The samples were centrifuged in a
swinging bucket rotor at 150 000 × g for 30 minutes.
Cytosol was removed from the top half of the upper layer and used
within 2 hours.
ERK2 immunoprecipitation and coupled kinase assay
PMNLs were allowed to phagocytose for 5 minutes as described above;
controls were incubated at 37°C for the same interval. PMNLs
(1 × 107) were lysed in 800 µL buffer containing
50 mmol/L HEPES (pH 7.5), 100 mmol/L NaCl, 2 mmol/L EDTA, 1% Nonidet
P-40, 1 µmol/L pepstatin, 1 µg/mL leupeptin, 0.2 mmol/L PMSF, 0.5 mmol/L sodium orthovanadate, 50 mmol/L NaF, 2 µg/mL aprotinin, and 40 mmol/L 4-nitrophenyl phosphate. Precleared lysates were incubated with
1 µg anti-ERK2 overnight at 4° C while rotating. Protein
A-Sepharose was added to each sample and incubated for 1 hour at
4°C while rotating. Beads were washed twice with cold lysis buffer,
then twice with cold MLCK kinase buffer. Cytosol from resting PMNLs, or
buffer, was added to samples as indicated (106 cell
equivalents/sample). Beads were then resuspended in MLCK kinase buffer
containing 500 µmol/L ATP, 5 µCi/sample [-32P]ATP,
and 300 µmol/L MLCK peptide substrate or buffer for blanks. Samples
were incubated for 10 minutes at 30°C and microfuged for 30 seconds. The reaction was terminated by filtering supernatants through
Whatman P81 paper. Filters were added to scintillation fluid and
counted in a scintillation counter. Counts were expressed as percentage
of controls from 22°C PMNLs. Paired, 2-tailed Student t
tests were used to assess differences between treatments.
These experiments were also conducted using a crude PMNL myosin,
prepared according to Boxer and Stossel,30 as substrate. After treatment with ERK2, the myosin was immunoprecipitated as described in "Orthophosphate Labeling and Immunoprecipitation of
Myosin." Incorporation of 32P was measured by
scintillation counting of the immunoprecipitate.
Immunoblotting
To ensure equal ERK2 was immunoprecipitated from each sample,
immunoblotting was performed on samples. Beads were combined with
sample buffer,31 boiled 5 minutes, and run on 10% SDS-PAGE mini-gels. Proteins were transferred to polyvinylidene fluoride (PVDF)
membrane (Schleicher and Schuell, Keene, NH) and then blocked with 5%
nonfat dry milk in Tris-buffered saline containing 0.2% Tween-20. The
membrane was probed with anti-ERK, which recognizes 42- and 44-kd MAP
kinases diluted 1:1000 in blocking buffer, and washed 3 times with
0.2% Tween-20 in TBS. The membrane was then incubated with horseradish
peroxide-conjugated sheep antirabbit antibody (Santa Cruz), diluted
1:10 000 in blocking buffer, and again washed 3 times. Phosphorylated
bands were visualized using enhanced chemiluminescence (ECL) detection
reagents (Amersham, Arlington Heights, IL) and exposing the membrane to
Hyperfilm ECL (Amersham). Immunoblotting for tyrosine-phosphorylated
Syk was performed as described previously.32
Intracellular calcium
PMNLs were loaded with the fluorescent calcium indicator fluo-3 as
described previously.33 PMNLs were incubated with 10 µmol/L ML-7, 10 mmol/L BDM, or buffer for 10 minutes at 37°C, then stimulated with 100 nM
N-formyl-methionyl-leucyl-phenylalanine (fMLP) and fluorescence
monitored continuously.
| |
Results |
MLCK activity was measured using lysates from phagocytosing PMNL and
peptide substrate specific for MLCK to determine the kinetics of MLCK
activation. The peptide substrate was previously characterized to have
an apparent Km of 7.5 µmol/L,34 comparable to
the Km of 8.6 µmol/L for myosin light
chain.35 MLCK activity increased more than 3-fold during
phagocytosis, in contrast to control PMNLs (maintained for up to 10 minutes without phagocytic targets) which showed no increase in
activity (Figure 1A). Unopsonized erythrocytes were not ingested by PMNL and did not stimulate MLCK activity (data not shown). The MLCK activity peaked at 4 to 6 minutes,
corresponding to EIgG ingestion, which also reaches a maximum rate
within 5 minutes of initiating phagocytosis.7 Paralleling
the phagocytic rate, MLCK activity decreased to baseline by 10 minutes.
To demonstrate phosphorylation of myosin light chain, we labeled PMNLs
with 32P and immunoprecipitated myosin from detergent
lysates using antiplatelet myosin. We observed a single band at
approximately 20 kd whose intensity was increased in samples from
phagocytosing PMNLs compared with controls (Figure 1B). This band was
most likely myosin light chain based on its relative mobility and its
recognition by antiplatelet myosin. In prior studies we observed ERK2
activity to peak by 3 minutes,15 consistent with the
hypothesis that ERK2 resides upstream of MLCK in the phagocytic
signaling pathway.
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| Fig 1.
MLCK activation during PMNL phagocytosis.
(A) Kinase activity. PMNLs (2 × 106/mL) were
challenged with EIgG (1 × 108/mL) (circles) or
incubated with no EIgG (triangles) at 37°C for the indicated times.
PMNL lysates were prepared by probe sonication as described in
"Materials and methods." MLCK was assayed in the lysates by
phosphorylation of substrate peptide (myosin light chain, amino acids
11-23) in the presence of [-32P]ATP for 10 minutes at
30°C. Data represent the mean ± SEM of 4 experiments.
Significantly different from time 0 sample; P < .05. (B)
Phosphorylation of endogenous myosin light chain. PMNLs were labeled
with 32P, challenged with EIgG (P) or not stimulated (U)
for 4 minutes, and lysed. Samples were immunoprecipitated with
antiplatelet myosin and subjected to 12% SDS-PAGE followed by
autoradiography.
|
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Because the time course of MLCK activity corresponded to the rate of
phagocytosis, we next determined whether the inhibition of MLCK would
block phagocytosis. PMNLs were incubated for 10 minutes with ML-7, a
MLCK inhibitor,36 before assaying for phagocytosis and MLCK
activity. Phagocytosis was inhibited partially (50%) using 3 µmol/L
ML-7 and almost completely at 10 µmol/L ML-7 (Figure 2). Similarly, MLCK activity was also
inhibited to 13% of control rates in the presence of 10 µmol/L ML-7
(Figure 2). The observed inhibition of phagocytosis supports the notion
that MLCK activation is linked to the functional response of particle
ingestion. To test the specificity of ML-7 for phagocytosis and its
link to MLCK, we conducted assays of fMLP-stimulated (100 nmol/L)
intracellular Ca++ transients and phagocytosis-stimulated
tyrosine phosphorylation of Syk. Neither response was significantly
blocked by ML-7 (Table 1). In addition, the
binding of EIgG to PMNLs, a preliminary step for phagocytosis, was
tested at 4°C (to preclude ingestion). ML-7 had no effect on EIgG
binding to PMNLs (Table 1). These data are consistent with the
selective inhibition of MLCK activity by ML-7.
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| Fig 2.
Phagocytosis and MLCK activity inhibition by MLCK
inhibitor ML-7.
PMNLs were incubated for 10 minutes at 37°C with the indicated
concentrations of ML-7, then challenged with EIgG for 30 minutes
(phagocytosis) or 5 minutes (MLCK activity). EIgG that were not
ingested were hypotonically lysed, and phagocytosis samples were fixed
with glutaraldehyde for microscopic quantitation. MLCK activity was
measured as described for Figure 1. Data points represent the
mean ± SEM of 4 experiments. *Significantly different from
control (no inhibitor); P < .05. **Significantly different
from control; P < .005.
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Previously we and others have found that ERK2 and ERK1 are activated by
MEK during PMNL phagocytosis and in other cell
systems.15-17,37 Activation of ERK2 is 20-fold higher than
ERK1 during PMNL phagocytosis,15 leading us to believe that
ERK2 is more relevant to phagocytosis. We also reported that the MEK
inhibitor PD09805938 inhibits phagocytosis by 50% and the
ERK2 activation required for phagocytosis by 90% in fMLP-primed
PMNLs.15 Using nonprimed PMNLs, pretreatment with the MEK
inhibitor PD098059 decreased EIgG phagocytosis and ERK2 activation by
80.1% ± 3.0% and 91.9% ± 2.7%, respectively (mean ± SEM of 3 experiments). MLCK activity was also inhibited by 82.1% ± 7.8% when PMNLs were pretreated with the MEK
inhibitor before incubation with phagocytic targets. These results are
consistent with the hypothesis that MLCK acts downstream of MEK and
ERK2 activation.
To further assess the linkage between ERK2 and MLCK, a coupled reaction
was performed to determine whether activated ERK2 could activate MLCK,
thereby leading to phosphorylation of the peptide substrate. Using
ERK2-specific antibody, ERK2 was immunoprecipitated from control and
phagocytosing PMNLs to obtain inactive and active ERK2, respectively.
An immunoblot with an ERK antibody that recognizes both ERK1 and ERK2
demonstrated that ERK2 was present in approximately equal amounts in
our samples from control and phagocytosing PMNLs, while no ERK1 was
observed (data not shown; consistent with our previous
reports).15 The cytosol of resting PMNL was used as a
source of inactive MLCK; though MLCK is found in association with the
cytoskeleton,39 it is also distributed in cytosol, especially in resting cells.40,41 We combined the ERK2,
PMNL cytosol, ATP, and peptide substrate to assay for MLCK activity. Four times as much activity was seen using active ERK2,
immunoprecipitated from phagocytosing PMNLs, as using ERK2 derived from
resting cells (Figure
3). This result
demonstrated that activation of ERK2 leads to activation of MLCK. To
verify that the observed kinase activity was cytosolic, active ERK2
from phagocytosing PMNLs was incubated with peptide substrate in the
absence of cytosol. MLCK activity under these conditions (Figure 3,
fourth bar) was not significantly different from controls that
contained cytosol and ERK2 from resting PMNLs (Figure 3, first bar).
This observation confirms the specificity of the substrate for MLCK
because active ERK2 was unable to directly phosphorylate the peptide
substrate. Preincubating the cytosol with 10 µmol/L of the MLCK
inhibitor ML-7 prevented MLCK from activation by active ERK2 (Figure 3,
fifth bar). Similar results were obtained using exogenously added PMNL
myosin instead of substrate peptide. In these experiments, activated
ERK2 led to the phosphorylation of immunoprecipitated myosin that was
610% of unactivated ERK2 (control) (Figure 3, third bar); ML-7
inhibited phosphorylation to 133% of control (Figure 3, sixth bar).
These data demonstrated that ERK2 activated during PMNL phagocytosis
leads to activation of MLCK.
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| Fig 3.
Activation of MLCK by ERK2.
PMNLs were allowed to undergo phagocytosis of EIgG for 5 minutes or
incubated without EIgG, then lysed with NP-40. ERK2 was
immunoprecipitated from lysates to obtain activated and unactivated
ERK2, respectively. Cytosol isolated from resting PMNLs or buffer was
added to samples along with [-32P]ATP and peptide
substrate (plain bars), and kinase activity was quantitated as
described for Figure 1, or PMNL myosin replaced peptide substrate
(cross-hatched bars) and was immunoprecipitated. R, ERK2
immunoprecipitated from resting PMNLs incubated at 37°C; P, ERK2
immunoprecipitated from phagocytosing PMNLs. +, cytosol present;
 , no cytosol; +ML-7, cytosol incubated before kinase assay with
10 µmol/L ML-7. Data represent the mean ± SEM of 3 experiments.
MLCK activity in PMNLs at 22°C = 100%. *Significantly different
from control (left bar); P < .005. Other data are not
significantly different from each other.
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After phosphorylation of myosin light chains by MLCK, both the
association of myosin with actin and the actin-stimulated adenosine triphosphatase activity of myosin increase.23,24 To
determine whether myosin activation is required for phagocytosis, we
used BDM, a known inhibitor of nonmuscle myosin II and myosin V
adenosine triphosphatases including those of platelets42
and macrophages.43 BDM has no effect on actin filaments in
PtK2 cells42 or on the formation of actin-rich phagocytic
cups in macrophages,43 implying it would not affect PMNL
actin. PMNL phagocytosis was completely inhibited by 10 mmol/L BDM,
whereas MLCK activity remained unaffected (Figure 4).
This result indicated that the myosin ATPase activity lies downstream
of MLCK and precluded nonspecific effects of BDM upstream of MLCK. To
further evaluate the effects of BDM, we tested Ca++, the
phosphorylation of Syk, and the binding of EIgG to PMNL in the presence
of BDM. None of these processes were significantly inhibited (Table 1),
demonstrating the relative specificity of BDM. The inhibition of
phagocytosis by BDM was consistent with the interpretation that myosin
ATPase activity is required for phagocytosis, supplying the energy to
extend the pseudopod and to permit ingestion of the particles.
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| Fig 4.
Inhibition of phagocytosis and MLCK activity by myosin
adenosine triphosphatase inhibitor BDM.
PMNLs were incubated for 10 minutes at 37°C with the indicated
concentrations of BDM, then challenged with EIgG for 30 minutes
(phagocytosis) or 5 minutes (MLCK activity). EIgG that were not
ingested were hypotonically lysed, and phagocytosis samples were fixed
with glutaraldehyde for microscopic quantitation. MLCK activity was
measured as described for Figure 1. Data points represent the
mean ± SEM of 4 experiments. *Significantly different from
control (100%); P < .05. **Significantly different from
control (100%); P < .005. ***Significantly different from
control (100%); P < .001.
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| |
Discussion |
This study demonstrated that the activation of MLCK during
PMNL phagocytosis is a result of ERK2 activation. Both the increase in
phosphorylation of a specific MLCK substrate during phagocytosis and
the concurrent inhibition of this phosphorylation and phagocytosis by
an inhibitor of MLCK implicate MLCK activation as a requirement for the
ingestion of IgG-opsonized particles. The ability of ERK2 immunoprecipitated from phagocytosing, but not resting, PMNLs to activate cytosolic MLCK demonstrated that ERK2 activation is necessary for MLCK activation. However, our data did not rule out the
presence of a cytosolic intermediate between ERK2 and MLCK.
A proposed model of PMNL signaling during FcR-mediated phagocytosis is
shown in Figure 5. We and others have investigated several aspects of this model. Our observations suggest that most of
the signaling activity occurs in the first few minutes of phagocytosis, corresponding with the maximum rate of ingestion. Phospholipase D
activity rises by 1 minute after initiation of ingestion and remains
elevated for 30 minutes.7 Translocation of PKC and Raf-1
occurs at 3 minutes.11 ERK2 phosphorylation rises at 1 to 5 minutes and ERK2 activity peaks at 3 to 5 minutes.15 Syk phosphorylation similarly peaks at 3 to 5 minutes.32
Translocation of PKC and Raf-1 and the phosphorylation of ERK2 and
Syk all decrease by 10 minutes into phagocytosis, a time at which the ingestion rate has steeply declined. Consistent with these
observations, in the current study MLCK activity peaked at 4 to 6 minutes and decreased to baseline by 10 minutes. The temporal proximity
of activation of these kinases suggested that they were closely linked signaling steps in the phagocytic signaling cascade.
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| Fig 5.
Proposed model for PMNL signaling through the MAP kinase
pathway during FcR-mediated phagocytosis.
Receptor ligation directly or indirectly initiates tyrosine
phosphorylation, followed by activation of phospholipase D. Phosphatidic phosphohydrolase generates diglyceride, a cofactor for
PKC. PKC and Raf-1 are known to translocate to the plasma membrane
during phagocytosis, and the MEK-MAP kinase pathway is activated. In
this study we show that activation of ERK2 leads to activation of MLCK,
which then phosphorylates myosin, and myosin ATPase activation is
required for phagocytosis.
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Inhibition of phagocytosis, ERK2 activation, and MLCK activity by a
specific MEK inhibitor demonstrates that these phenomena are regulated
by MEK. Immunoprecipitated ERK2, which was activated during PMNL
phagocytosis, could activate cytosolic MLCK, and this activity was
inhibitable by ML-7. Our results implicate a pathway similar to that
described for COS-7 cells by Klemke et al,27 who showed
that MEK-dependent ERK2 activation led to the activation of MLCK to
phosphorylate myosin light chain. Furthermore, Klemke et
al27 demonstrated direct phosphorylation of MLCK by ERK2.
Some studies have suggested possible mediators of MLCK activity or
myosin light chain phosphorylation other than ERK2. A cyclic adenosine
monophosphate (cAMP)-dependent protein kinase phosphorylates and
inactivates MLCK in fibroblasts.39 However, results from our laboratory suggest that cAMP has no significant role in PMNL phagocytosis of EIgG (data not shown). PI 3-kinase is activated during
phagocytosis.44,45 Recent work in our laboratory suggests that PI 3-kinase activation, though essential for PMNL phagocytosis, acts downstream of the MAP kinase cascade,32 making it a
possible activator of MLCK. However, in macrophages PI
3-kinase is involved in the closure of phagosomes into intracellular
vesicles and not in pseudopod extension,46 thus placing it
downstream of MLCK as well. Protein kinase C may phosphorylate myosin
light chain directly, but it does so on sites not included within the
peptide we used as MLCK substrate.47 More recently, the
small GTPase Rho has been shown to mediate myosin light chain
phosphorylation.48,49 However, when PMNLs were treated with
Clostridium difficile toxin B to inhibit Rho, phosphorylation
of myosin light chain still occurred during phagocytosis (data not shown).
Formation and movement of the pseudopod by actin-myosin
interaction occur during various forms of cell movement, including phagocytosis and chemotaxis. MLCK is involved in granulocyte macrophage colony-stimulating factor-stimulated random migration and
fMLP-stimulated chemotaxis of PMNLs.26 Activation of
platelets by thrombin, leading to aggregation and granule
release, results in diphosphorylation of myosin light chain by
MLCK.50 Phosphorylation of myosin II light chain does not
increase during phagocytosis of antibody-opsonized yeast particles by
macrophage-like J774 cells51; however, HeLa cell
phagocytosis appears to involve myosin II.52 In addition, myosin is phosphorylated during T-lymphocyte receptor
capping,53 implicating MLCK. Thus MLCK activation may be
dependent on cell type and function.
During PMNL phagocytosis, as part of the ERK2 pathway MLCK is
activated to phosphorylate and activate myosin. Inhibition of phagocytosis by a myosin ATPase inhibitor revealed phagocytosis depends
not only on MLCK but on the subsequent myosin-actin interaction arising from the stimulation of myosin ATPase activity. It is highly
likely that the myosin ATPase activity translates into propulsive force
to form the pseudopod, thereby permitting PMNLs to surround and ingest
foreign particles.
| |
Footnotes |
Submitted March 5, 1999; accepted December 13, 1999.
Supported by National Institutes of Health grants AI20065 (L.A.B.) and
DK41487 and DK39255 (J.A.S.).
Reprints: Laurence A. Boxer, Department of Pediatrics,
Hematology/Oncology, University of Michigan, L2110 Women's
Hospital, Box 0238, 1500 E. Medical Center Drive, Ann Arbor, MI 48109;
e-mail: laboxer{at}umich.edu.
The publication costs of this
article were defrayed in part by
page charge payment. Therefore,
and solely to indicate this fact,
this article is hereby marked
"advertisement"
in accordance with 18 U.S.C.
section 1734.
| |
References |
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Silverstein SC, Greenberg S, Di Virgilio F, Steinberg TH.
Phagocytosis. In:
William PE, ed.
Fundamental Immunology. New York: Raven Press; 1989:703.
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Abstract
Introduction