From the Department of Cell Biology, Duke University
Medical Center, Durham, North Carolina 27710 and the ¶ Department
of Genetics, Cell Biology, and Development, University of Minnesota,
Minneapolis, Minnesota 55455
Received for publication, September 12, 2000
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ABSTRACT |
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Directed cell migration occurs in response to
extracellular cues. Following stimulation of a cell with
chemoattractant, a significant rearrangement of the actin cytoskeleton
is mediated by intracellular signaling pathways and results in
polarization of the cell and movement via pseudopod extension. Amoeboid
myosin Is play a critical role in regulating pseudopod formation in
Dictyostelium, and their activity is activated by heavy
chain phosphorylation. The effect of chemotactic stimulation on the
in vivo phosphorylation level of a
Dictyostelium myosin I, myoB, was tested. The myoB heavy
chain is phosphorylated in vivo on serine 322 (the myosin TEDS rule phosphorylation site) in chemotactically competent cells. The
level of myoB phosphorylation increases following stimulation of
starving cells with the chemoattractant cAMP. A 3-fold peak increase in
the level of phosphorylation is observed at 60 s following stimulation, a time at which the Dictyostelium cell
actively extends pseudopodia. These findings suggest that chemotactic
stimulation results in increased myoB activity via heavy chain
phosphorylation and contributes to the global extension of pseudopodia
that occurs prior to polarization and directed motility.
The directed movement of cells in response to attractive and
repulsive chemical cues is essential for correct development of
multicellular organisms and the survival of microorganisms (1).
Extracellular chemoattractants bind to G protein-coupled receptors
that, in turn, transmit signals to the actomyosin cytoskeleton that
generates biased motility (2). The intracellular signals that regulate
and coordinate these processes are beginning to be understood, but the
full pathway from the receptor to the cytoskeleton remains to be elucidated.
Dictyostelium has proven to be an invaluable model organism
for investigating the molecular basis of chemotaxis. Many, if not all,
of the components of the signaling cascade are shared with those found
in mammalian cells and the motile response of the cell is
indistinguishable from that of a leukocyte (1, 2). Stereotypical
transient morphological and cytoskeletal changes rapidly occur in
Dictyostelium amoebae following cAMP stimulation (3). Within
the first 20 s, the cell rounds up or "cringes" (4). The cell
then actively extends pseudopodia in random directions 60 s
following stimulation. Finally, by 90-120 s, the cell becomes
repolarized and moves in the direction of the stimulus (5). These
morphological changes are accompanied by alterations in the levels of
F-actin and the phosphorylation state of the myosin II heavy chain.
Concomitant with the cringe (0-20 s) is an increase in total F-actin
(with a peak at 10 s) (5). This is followed by an increase in the
level of myosin II heavy chain phosphorylation (that promotes
depolymerization of myosin II thick filaments (Ref. 6)), with a peak of
phosphorylation at 30-40 s (7, 8). Myosin II is also translocated to
the cortex, where it is thought to become more accessible to the heavy chain kinase (9, 10), along with the actin cross-linking protein,
ABP-120 (11). The F-actin levels then increase again, with a second
peak at 60 s, coincident with active pseudopod extension (5). This
sequence of molecular and morphological events provides a fundamental
framework for understanding how chemotactic stimulation results in
directed cell migration.
A key process in the chemotactic response is the orderly extension of
pseudopodia and work in Dictyostelium has revealed that class I myosins play an important role in controlling their location and number (12-14). The class I myosins are ubiquitous; they are expressed in organisms ranging from yeast to man (15). They all share a
conserved motor domain, a light chain binding domain, and a tail region
that contains a polybasic region that directly binds to membranes via
electrostatic interactions and also to actin (15-17). The amoeboid
subclass of myosin Is have two additional domains in their tails. The
first is a region rich in the amino acids glycine, serine, and alanine
(or glutamate or serine) that also constitutes an ATP-insensitive actin
binding site, and the second is a Src homology 3 (SH3)1 domain, a known
protein-protein interaction domain (18). The in vitro
activity of the class I myosins from lower eukaryotes requires
phosphorylation of a single serine or threonine residue in the motor
domain (referred to as the TEDS rule site; Refs. 18 and 19) by a G
protein-regulated myosin I heavy chain kinase (MIHCK) that is a member
of the p21-activated kinase (PAK) family of kinases (20,
21).
Several of the Dictyostelium class I myosins play a role in
motility (12, 13, 22). Two of these myosins, myoA and myoB, have been
shown to regulate the number, timing, and placement of pseudopodia
(12-14). Consistent with its role in motility, myoB has been shown to
be localized to the leading edge of chemotactic cells (23), and its
level of expression is significantly increased during aggregation
(i.e. when Dictyostelium cells become highly chemotactic) (24). The activity of the amoeboid myosin Is is likely to
be strictly regulated in vivo by heavy chain
phosphorylation. This has been demonstrated by the finding that
mutation of the TEDS rule site at residue 332 from serine to alanine
renders this motor inactive in vivo (25-28). Understanding
the basis of myosin I functions in vivo requires the
identification of the signals and conditions that change its
phosphorylation state. Although recent progress has been made in the
identification and characterization of MIHCKs in both
Acanthamoeba and Dictyostelium, virtually nothing is known about the kinase that acts on Dictyostelium myoB,
an important regulator of pseudopod formation in
Dictyostelium (12, 20, 21, 29, 30). The ability to
synchronize populations of Dictyostelium during chemotactic
stimulation was, therefore, exploited to directly examine changes in
the levels of myoB heavy chain phosphorylation in vivo to
determine whether increased myoB activity is correlated with active
pseudopod extension.
Maintenance of Strains--
The Dictyostelium
discoideum Ax3 axenic strain and the myoB-strain
HTD3-4 (31) were maintained in HL5, a nutrient medium for axenic
stains (32), in suspension at 150 rpm. Strains expressing mutant forms
of myoB were maintained in HL5 supplemented with 20 µg/ml blasticidin
(Calbiochem Chemical Corp., San Diego, CA). The S332A-myoB and
myoB/SH3 Starvation and cAMP Stimulation of Dictyostelium--
Prior to
all experiments, log-phase cells were collected by centrifugation and
washed twice in starvation buffer (20 mM MES, pH 6.8, 0.2 mM CaCl2, 2 mM MgSO4).
Cells were resuspended to a density of 2-4 × 107
cell/ml and shaken at 150 rpm for 3.5 h at room temperature. The
onset of starvation initiates the Dictyostelium
developmental program, and cells prepared in this manner are referred
to as aggregation-competent throughout this paper. In some experiments the cells were pulsed with 50 nM cAMP every 6 min for
2.5 h after the first hour of starvation to ensure initiation of
the early developmental program.
Aggregation-competent cells were collected by centrifugation and
resuspended in 0.5 ml of starvation buffer. A 100-µl sample was taken
for the time 0 sample and lysed immediately by the addition of an equal
volume of sodum dodecyl sulfate (SDS) lysis buffer (50 mM
Tris, pH 7.5, 100 mM NaCl, 0.4% SDS, 2 mM
EDTA, 50 mM NaF, 50 mM sodium pyrophosphate,
fresh 40 mM Radiolabeling of Dictyostelium Proteins--
One ml of cells at
a density of 1-2 × 107 cells/ml were labeled with
35S-amino acids by incubation at room temperature in
starvation buffer containing 0.6 mCi/ml of Tran35S-label
(ICN Biomedicals, Irvine, CA) while shaking at 150 rpm for 3.5 h.
Labeled cells were harvested by centrifugation at 510 × g for 5 min and the pellet washed two times with starvation buffer.
Cells were labeled with 32P to detect phosphorylated
proteins by incubating them in the presence of
[32P]orthophosphate during the final stages of
starvation. A total of 4-8 × 107 aggregation-stage
cells were collected at 3.5 h and resuspended in 0.5 ml of
starvation buffer containing 0.2-0.4 mCi/ml
[32P]orthophosphate (ICN Biomedical, Irvine, CA) and 20 mM DTT (8). The cells were then incubated with shaking at
190 rpm for 30 min at room temperature. Some experiments included the
addition of 10 mM DTT to inhibit extracellular
phosphodiesterase following 3 h of pulsing to suppress spontaneous
cAMP oscillations.
Immunoprecipitation and Analysis of Phosphorylation
Levels--
Triton X-100 was added to the cooled cell lysates to a
final concentration of 0.2%. Two to three µl of undiluted rabbit
polyclonal anti-myoB (31) or rabbit polyclonal anti-mhcA antibody (an
antibody specific for the conventional myosin heavy chain; kind gift of Dr. J. Spudich, Stanford University, Stanford, CA (Ref. 8)) were added
and the mixtures incubated with gentle shaking for at least 1.5 h
at room temperature. Fifty µl of Protein A-Sepharose bead slurry
(Amersham Pharmacia Biotech) were added and the sample mixed by gentle
shaking at room temperature for 1 h. In some experiments, the
Protein-A-Sepharose beads were pre-cleared by incubation with a
myoB
The immunoprecipitated gel samples were briefly spun to pellet the
beads and equal portions of each supernatant applied to a 7.5%
SDS-PAGE gel. Following electrophoresis, the gel was either stained
briefly with Coomassie Brilliant Blue R and dried on filter paper or
immediately transferred to either PVDF or nitrocellulose membrane
(Millipore, Bedford, MA) (33). The phosphorylated proteins were
detected by exposing the dried gel or membrane to Kodak XAR5 film
(Eastman Kodak Co.) for 6 - 8 days in the initial experiments. The
films were scanned using an Epson ES1200C color scanner (Epson, Pittsburgh, PA) and the amount of signal determined by densitometry of
the scanned film using NIH Image 1.54 software. In later experiments, the dried gels or membranes were exposed to a phosphorimage storage screen for 24-48 h and radioactive bands detected using a
phosphorimager system (either the Fuji MacBas 1000 phosphorimaging
system (Fuji Medical Systems, Stanford, CT) or the Molecular Dynamics
PhosphorImager 400). Manufacturer's software was used to quantify the
signal in each lane. Following autoradiography, the levels of myoB
heavy chain on the blotted membranes were determined. The membrane was incubated with the myoB antibody followed by a goat anti-rabbit secondary antibody conjugated to horseradish peroxidase. Detection was
performed determined with an ECL kit per manufacturer's instructions (Amersham Pharmacia Biotech). Multiple exposures of the immunoblot films were taken to ensure that the bands were in the linear range, and
these were scanned using an Epson scanner. The amount of protein was
determined by densitometry of the scanned film using NIH Image 1.54 software. The relative level of phosphorylation was determined by
dividing the amount of phosphorylation determined by autoradiography by
the amount of protein at that time point as determined by scans of the
Western blots.
Phosphoamino Acid Determination--
A total of 2-4 × 107 32P-labeled aggregation-competent cells in
a 2-ml volume were stimulated with chemoattractant by adding 1 × 10 In Vitro Phosphorylation of myoB--
First, a clarified high
speed supernatant was made from aggregation-competent
Dictyostelium (see above). Cells were collected after 4 h of starvation and resuspended to a final density of 1 ml/g of cells
in 50 mM HEPES, pH 7.5, 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 30% sucrose, 1 mM DTT, 5 mM benzamidine-HCl, 40 µg/ml TLCK,
1 mM phenylmethylsulfonyl fluoride, and CLAP. Cells were drop-frozen in liquid nitrogen and stored at
In vitro kinase assays of myoB were carried out by
incubating 200 µl of S2 plus 1 µl of a 1:1:1 mixture of saturated
phenylmethylsulfonyl fluoride, 1 M DTT, and CLAP with 10 µCi of [ The phosphorylation state of a protein of interest in
vivo can be determined by immunoprecipitation from labeled cell
lysates. This method also allows one to test for various cellular
conditions that could alter the protein's phosphorylation state. A
specific rabbit polyclonal antibody generated against a denatured
Dictyostelium myoB tail fusion protein (31) was tested for
its ability to immunoprecipitate myoB from a cell extract. Because the
myoB heavy chain is not a highly abundant protein, cells were removed
from nutrient media and starved for 4 h to increase endogenous
levels (24). The cells were labeled with 35S-amino acids
and aliquots lysed with detergent, boiled, and incubated with the
antibody of interest. The antibody-antigen complex was then isolated
using Protein A-Sepharose beads. Following electrophoresis of the
samples and autoradiography of the dried gel, a single band at
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
strains were generated by transformation of the
myoB
null strain with an expression plasmid
containing altered forms of the myoB gene (27). The
S332A-myoB heavy chain carries an alteration in the TEDS rule site; the
serine residue at position 332 is changed to alanine. The
myoB/SH3
heavy chain is a truncated form of myoB that
lacks the C-terminal SH3 domain.
-glycerol phosphate, 1 mM sodium
orthovanadate, 2 mM ATP, 2 mM dithiothreitol
(DTT), and 10 µg/ml each of chymostatin, leupeptin, antipain, and
pepstatin (CLAP)) and stored on ice until further processing. A 1 × 10
6 M pulse of cAMP (final
concentration) was added to the sample with rapid mixing, and 100-µl
samples were added to an equal volume of ice-cold SDS lysis buffer at
various times point up to 90 s. The samples were then heated at
100 °C for 3 min, cooled to room temperature, and the myoB heavy
chain immunoprecipitated as described below.
cell lysate prepared in a similar manner
for 1 h and then washed extensively in the lysis buffer. The beads
were collected by a brief centrifugation (10 s) in a microcentrifuge
and gently washed three times with phosphate-buffered saline followed
by two washes with lithium buffer (0.5 M LiCl, 0.1 M Tris, pH 7.4) to remove nonspecifically bound proteins.
The washed pellets were then resuspended in 40 µl of 2× SDS-PAGE
sample buffer and boiled for 3 min.
6 M cAMP, as described above.
Sixty seconds after the addition of chemoattractant, the cells were
lysed and the myoB heavy chain immunoprecipitated. The entire sample
was run on a 6% SDS-PAGE curtain gel, electrotransferred to PVDF, and
exposed to a phosphorimager screen. The position of the 125 kDa
phosphorylated myoB heavy chain was confirmed by immunodetection. The
major band that correlated with a band of radioactivity was excised
from the membrane, rinsed briefly in methanol, then water, and
incubated in 6 M HCl under N2 gas at 100 °C
for 1 h in a screw-top vial (34). The hydrolysate was collected
and counted by Cerenkov counting to confirm the recovery of
32P. The phosphoamino acids were separated by high voltage
electrophoresis on a thin layer chromatography (TLC) plate in parallel
with phosphoamino acid standards as described in (35). The pH 1.9 electrophoresis buffer was a 50:156:1794 (v/v/v) mixture of 88% formic
acid-glacial acetic acid-distilled H2O mixture. Following
electrophoresis, the TLC plate was sprayed with ninhydrin to visualize
the position of the phosphoamino acid standards and then exposed to a
phosphorimager plate overnight.
80 °C. Cells were thawed by dilution to 20% sucrose with ice cold 100 mM
HEPES, pH 7.4, 0.5 mM EDTA, 5 mM
N-
-p-tosyl-L-arginine methylester
(TAME), 50 µg/ml TLCK, 5 mM benzamidine-HCl, and
CLAP, then lysed with three passes through a tightly fitting Dounce
homogenizer (Wheaton Scientific, Millville, NJ). DTT was added to a
final concentration of 1 mM and the lysate clarified by
centrifugation at 10,000 × g for 20 min. The
supernatant was collected and centrifuged at 50,000 × g for 3 h at 4 °C to obtain supernatant "S2."
Ammonium sulfate was added to a final concentration 80% for storage at
20 °C. Small samples were dialyzed into HEMK (20 mM
HEPES, 1 mM EGTA, 5 mM MgCl2, 50 mM KCl, pH 7.4) buffer when needed.
-32P]ATP for various times at room
temperature. Samples also included 10 µl of phosphatase inhibitors
(200 mM NaF, 50 mM sodium phosphate, 250 mM
-glycerol phosphate, 0.1 nM okadaic acid,
50 µM cypermethrin and deltamethrin) in the reaction
mixture. Phosphorylation reactions were stopped by adding lysis buffer
without SDS and heating at 100 °C for 3 min. The samples were then
cooled, and the myoB heavy chain was immunoprecipitated as described
above. SDS-PAGE samples were applied to a 6% gel, transferred to PVDF
membrane, and subjected to autoradiography and immunodetection.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
125
kDa was observed in immunoprecipitates with the myoB antibody but not
when the preimmune sera is used (Fig.
1A, lanes 1 and 2). An antibody that recognizes the myosin
II heavy chain was used as a control for the immunoprecipitation method
(8), and it was found to precipitate the expected 240-kDa band (Fig. 1A, lane 3).
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Fig. 1.
Phosphorylation of the myoB heavy chain
in vivo and in vitro.
A, specificity of the myoB antibody. Antibodies directed
against either the myoB heavy chain (lane 1) or
the myosin II heavy chain (lane 3) were added to
radiolabeled lysates from starved Ax-3 cells and the antibody-antigen
complex immunoisolated. myoB preimmune sera was used in a parallel
control experiment (lane 2). Samples were
subjected to gel electrophoresis and autoradiography. The positions of
the 125-kDa myoB heavy chain and 240-kDa myosin II heavy chain are
indicated on the right side of the gel. B,
in vivo phosphorylation of myoB. Starved Ax-3
(lane 1) or myoB
(lane 2) cells were harvested and incubated with
shaking in the presence of [32P]orthophosphate. The myoB
antibody was incubated with the labeled cell lysates and precipitated
with Protein A-Sepharose beads. Samples were subjected to gel
electrophoresis, transferred to nitrocellulose and analyzed by
autoradiography (phos) and then Western blotting with the
anti-myoB antibody (
-myoB). Only the region of the gel
containing the myoB heavy chain is shown. C, in
vitro phosphorylation of the myoB heavy chain. A high speed
supernatant containing myoB was incubated with
[
-32P]ATP, myoB immunoprecipitated, and assayed for
the level of phosphorylation over time. The 15-min time point marked
with an asterisk (*) is a sample that was incubated for 15 min without the addition of phosphatase inhibitors. Inset,
autoradiogram of a myoB immunoprecipitate from a high speed supernatant
incubated with [
-32P]ATP. Lane
1, 15-min time point with phosphatase inhibitors.
Lane 2, 15-min time point without phosphatase
inhibitors. Only the region of the gel containing the myoB heavy chain
is shown.
The amoeboid myosin Is are phosphorylated in vitro by a
specific MIHCK (29, 36) and the TEDS rule serine is essential for
in vivo function of the lower eukaryotic myosin Is (25-28). It was therefore predicted that the Dictyostelium myoB heavy
chain would be phosphorylated in vivo. The in
vivo phosphorylation of myoB was examined by incubating
aggregation-competent cells with [32P]orthophosphate
followed by immunoprecipitation, electrophoresis, blotting, and
autoradiography of the myoB heavy chain. Immunodetection was used to
verify the presence of the myoB heavy chain as Coomassie staining of
the gel did not always reveal the presence of a prominent band at 125 kDa. The immunoprecipitated myoB heavy chain was phosphorylated (Fig.
1B, lane 1). A control experiment that
employed a myoB null mutant (myoB) strain was
performed in parallel. No 125-kDa band was visible either in the
immunoblot or in the autoradiogram (Fig. 1B, lane 2), again demonstrating the specificity of the antibody and
the immunoprecipitation protocol.
The MIHCKs from Acanthamoeba and Dictyostelium
are present in high speed supernatants (29, 37), suggesting that a
significant proportion of these enzymes may be readily solubilized.
Since the myoB heavy chain has been shown to be a poor substrate for the Dictyostelium MIHCK (29), an extract was tested for a
kinase activity that might phosphorylate the myoB heavy chain. A high speed supernatant from aggregation-competent cells enriched for myoB
was incubated at room temperature in a reaction buffer containing [-32P]ATP and a mixture of phosphatase inhibitors. The
myoB heavy chain was immunoprecipitated from the reaction at various
times following the addition of ATP, and the level of phosphorylation quantified. A steady increase in the level of myoB phosphorylation was
observed over the 30-min time course (Fig. 1C). Little or no
phosphorylation was detected in a sample that did not contain phosphatase inhibitors (Fig. 1C).
The identity of the in vivo phosphorylated residue on the
myoB heavy chain was determined by phosphoamino acid analysis. The phosphorylated myoB heavy chain was isolated following transfer to PVDF
membrane and hydrolyzed by acid. The hydrolysate was then separated by
high voltage one-dimensional electrophoresis at pH 1.9. The
phosphoamino acid(s) were visualized by autoradiography and the
position compared with known standards. Serine was the sole
phosphoamino acid detected (Fig. 2). To
determine whether serine 332 (the TEDS rule site) was the site of
phosphorylation, the S332A-myoB heavy chain was immunoprecipitated from
cells labeled with [32P]orthophosphate. Following
immunoprecipitation, autoradiography revealed that the S332A-myoB heavy
chain was not phosphorylated while the control wild type myoB heavy
chain was (Fig. 3A). Thus, the
sole site of myoB heavy chain phosphorylation in vivo is
serine 332, the TEDS rule site.
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The Acanthamoeba and Dictyostelium MIHCKs contain
a PXXP motif that has the potential to bind to SH3 domains
(29, 38, 39). The amoeboid myosin Is have C-terminal SH3 domains and deletion of the myoB SH3 domain renders this motor nonfunctional in vivo without altering the localization of this myosin
(25, 27). One explanation for the inactivity of the
myoB/SH3 heavy chain (a truncated myoB heavy chain that
lacks the C-terminal SH3 domain) is that the kinase can not bind to the
myosin I via its SH3 domain and phosphorylate the TEDS rule site
in vivo. The in vivo phosphorylation of the
myoB/SH3
heavy chain was examined to test this
possibility. Both the wild type and truncated myoB heavy chain are
phosphorylated in vivo (Fig. 3B). Therefore, loss
of the SH3 domain does not affect the ability of the MIHCK to
phosphorylate the myoB heavy chain in vivo. This result is
consistent with a recent analysis of Acanthamoeba myosin IC
tail mutants demonstrating that deletion of the SH3 domain does not
affect either the Km or Vmax
for phosphorylation by MIHCK (17).
Cells lacking myoB exhibit defects in pseudopod formation (12),
indicating that myosin I activity is required for efficient motility.
The effect of chemotactic stimulation on the level of myoB
phosphorylation was tested. Aggregation-competent
Dictyostelium were stimulated with a physiological dose of
cAMP as described for the analysis of myosin II phosphorylation
in vivo (8). Cells were incubated either in the presence or
absence of caffeine (this inhibits adenylcyclase, blocking the
production of intracellular cAMP) and [32P]orthophosphate
and then subjected to a single, physiological, 1 × 106 M pulse of cAMP. Samples were
taken every 20-30 s and the myoB heavy chain immunoprecipitated and
analyzed for phosphorylation. The level of myoB heavy chain
phosphorylation appeared to increase either in the presence (Fig.
4A) or absence (Fig.
4B) of caffeine, with a peak at 60 s. This indicates
that the increased phosphorylation was not due to an intercellular
increase in cAMP but is due to signaling via the cAMP receptor. The
relative increase in the level of heavy chain phosphorylation was
determined by normalizing the levels of phosphorylation to the amount
of myoB in each sample in three independent experiments. The overall
level of phosphorylation increased 3-fold by 60 s (Fig.
5), after which time it returned to
resting levels.
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DISCUSSION |
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Chemotactic stimulation of Dictyostelium results in a significant increase in myoB heavy chain phosphorylation at the TEDS rule site (Figs. 3-5). Higher levels of heavy chain phosphorylation most likely result in increased myosin I activity (18). The observed peak of phosphorylation 60 s (Fig. 5), a time at which active pseudopod formation takes place, is consistent with a role for myoB in pseudopod formation and cell motility (12). This work reveals that chemotactic signaling through the cAMP receptor results in increases in myosin I activity either by activating MIHCK or by making myosin I more available for phosphorylation.
Phosphorylation of myosin I heavy chains has been reported for Acanthamoeba and brush border myosin Is (40-43). Like Dictyostelium myosin Is, the Acanthamoeba myosin Is are regulated by phosphorylation at the TEDS rule site (18). Immunoelectron microscopy using antibodies specific for the phosphorylated forms of Acanthamoeba myosin IA, IB, and IC revealed that each of these myosin Is are phosphorylated in vivo (43). The fraction of each myosin I that was phosphorylated in vivo varied for each myosin I, but the phosphomyosin Is were concentrated in actively motile regions. The activity of brush border myosin I (BBMI) is, in contrast, regulated by calcium-calmodulin (15). However, the BBMI heavy chain is phosphorylated in cytoskeleton preparations (40, 41) and in vitro experiments revealed that BBMI can be phosphorylated by protein kinase C on both serine and threonine when BBMI is associated with acidic phospholipids (42). The site of phosphorylation resides in the C-terminal tail region, but the role of phosphorylation in BBMI function is unknown (42).
A single MIHCK has been identified Dictyostelium (21, 29). It is highly homologous to the Acanthamoeba MIHCK and is a member of the PAK family of small G protein-regulated kinases (21, 30). The MIHCKs are activated by the non-Dictyostelium small G proteins Cdc42 and Rac1, autophosphorylation, and acidic phospholipids (20, 44). The Dictyostelium G protein that activates MIHCK in vivo or the exchange and activating factors that act upon it have not yet been identified. However, a number of small G proteins are expressed in aggregation-competent cells, and it will be of interest to determine which plays a role in the activation of MIHCK and the signaling pathways that activate it.
The purified Dictyostelium MIHCK does not appear to be
capable of phosphorylating the myoB heavy chain (29). Instead, it can
phosphorylate the closely related myoD heavy chain (29), a myosin I not
known to play a major role in cell motility based on mutant analyses
(45). Further underscoring the physiological distinction between myoB
and myoD, the overall levels of the myoB heavy chain increase
significantly during development (up to 7-fold 8.75 h after the
onset of development) but those of the myoD heavy chain remain
unchanged (24). It is, therefore, quite likely that the myoB heavy
chain is phosphorylated by a distinct MIHCK, perhaps one that is
developmentally regulated or contains regulatory sequences that respond
to signals from the cAMP receptor. The identification of myoB heavy
chain kinase activity in cell extracts (Fig. 1C) suggests
that a kinase required for activating this myosin I is present during
chemotaxis. Purification of this kinase and identification the
mechanism of its activation should help to elucidate how stimulation of
cells by chemoattractants results in efficient, directed motility.
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ACKNOWLEDGEMENT |
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We thank Dr. Katherine Swenson (Duke University) for generously sharing her methods and expertise in protein phosphorylation and for many stimulating discussions.
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FOOTNOTES |
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* The work was supported by National Institutes of Health Grants F32-GM16090 (to N. R. G.) and GM46486 (to M. A. T.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Present address: Universal Imaging Corp., 502 Brandywine Pkwy., West Chester, PA 19380.
To whom all correspondence should be addressed: Dept. of
Genetics, Cell Biology and Development, 6-160 Jackson Hall, 321 Church St. S.E., University of Minnesota, Minneapolis, MN 55455. Tel.: 612-625-8498; Fax: 612-624-8118; E-mail:
titus@lenti.med.umn.edu.
Published, JBC Papers in Press, November 17, 2000, DOI 10.1074/jbc.M008319200
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ABBREVIATIONS |
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The abbreviations used are:
SH3, Src homology
domain 3;
DTT, dithiothreitol;
PAGE, polyacrylamide gel
electrophoresis;
PVDF, polyvinylidene difluoride;
TLCK, N-p-tosyl-L-lysine
chloromethyl ketone;
MIHCK, myosin I heavy chain kinase;
MES, 4-morpholineethanesulfonic acid;
BBMI, brush border myosin I;
CLAP, chymostatin, leupeptin, antipain, and pepstatin.
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REFERENCES |
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