Department of Biochemistry, Hadassah Medical School The Hebrew
University, Jerusalem 91120, Israel
*
These authors contributed equally to this paper
Author for correspondence (e-mail:
ravid{at}md2.huji.ac.il
)
Accepted May 3, 2001
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SUMMARY |
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Key words: Myosin II, MHC localization, MHC phosphorylation, Protein kinase C
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INTRODUCTION |
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Myosin II light chain and MHC phosphorylation in vivo has been demonstrated
in a wide variety of organisms and cell types (Tan et al.,
1992). It has been shown in
Dictyostelium cells that phosphorylation of MHC plays a crucial role
in myosin II function by regulating its filament assembly state (Kuczmarski
and Spudich, 1980
; Pasternak
et al., 1989
; Ravid and
Spudich, 1992
; Abu-Elneel et
al., 1996
; Egelhoff et al.,
1993
; Kolman et al.,
1996
). Furthermore, our
laboratory has shown that a PKC specific to MHC plays a critical role in the
in vivo regulation of myosin II filament formation and is important for proper
chemotaxis of Dictyostelium cells (Abu-Elneel et al.,
1996
; Ravid and Spudich,
1992
).
Recent cloning, sequencing and biochemistry studies suggest that heavy
chains of all non-muscle myosin II have a PKC phosphorylation site within
their tail regions (Moussavi et al.,
1993). PKC phosphorylates MHC
from human platelets and rat basophil leukemic cells both in vivo and in vitro
(Kawamoto et al., 1989
;
Ludowyke et al., 1989
). Later,
this myosin II was identified as MHC-A and the PKC phosphorylation site is the
serine residue at position 1917 (Moussavi et al.,
1993
). An analogous site has
been shown to be present in MHC-B but, in this case, the phosphorylatable
residue is a threonine, not a serine, residue (Moussavi et al.,
1993
; Takahashi et al.,
1992
). Both sites are located
within the rod region of these MHCs. Using C-terminal fragment of MHC-B,
Murakami et al. found that a mixture of several PKC isoforms phosphorylate
this MHC-B fragment on serine residues in vitro (Murakami et al.,
1998
; Murakami et al.,
2000
; Murakami et al.,
1995
). They further showed
that the phosphorylation of MHC-A and MHC-B by PKC have different effects on
the assembly properties of these myosin II isoforms. Phosphorylation of MHC-B
by PKC resulted in inhibition of MHC-B assembly in vitro; by contrast, the
assembly properties of MHC-A were not affected by PKC phosphorylation
(Murakami et al., 1995
). These
results indicate that assembly of myosin II isoforms can be regulated via
phosphorylation of the heavy chain by PKC in an isoformspecific manner, which
might cause a rearrangement of myosin II, resulting in cell shape changes.
Cell motility and chemotaxis play a crucial role in physiological as well
as in pathological processes, but we know very little about the involvement
and the regulation of myosin II in these processes. To begin understanding the
role and regulation of myosin II in chemotaxis of mammalian cells, we used
prostate metastatic tumor cells (TSU-pr1) (Iizumi et al.,
1987). These cells
preferentially metastasize to bony sites and lymph nodes at a frequency in
excess than would be predicted by random tumor cell dissemination (Saitoh et
al., 1984
). The prostate tumor
cells spread to these sites owing to the presence of epidermal growth factor
(EGF) (Rajan et al., 1996
).
Furthermore, inhibition of EGF receptors (EGFRs) using anti-EGFR monoclonal
antibody inhibited the chemomigration of TSU-pr1 cells towards EGF (Zolfaghari
and Djakiew, 1996
).
In this study, we report that MHC-A and MHC-B in TSU-pr1 cells respond to EGF stimulation by different kinetics of MHC phosphorylation and cellular rearrangement. These results might indicate the involvement of myosin II in chemotaxis of prostate tumor cells towards EGF. Furthermore, we demonstrate that PKC is involved in the phosphorylation of MHC-A and MHC-B in response to EGF stimulation of TSU-pr1 cells.
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MATERIALS AND METHODS |
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Gel electrophoresis and western blot analyses
Gel electrophoresis was performed either by the method described by Kelley
et al. (Kelley et al., 1996)
or using the system of Laemmli (Laemmli,
1970
). Cells were washed twice
in ice-cold PBS, scraped off the plate and transferred to tubes. Cells were
counted and lysed in ice-cold 20 mM Tris (pH 7.5), 0.1% NP40, 1 mM DTT, 10 mM
EDTA and a mixture of protease inhibitors (Sigma). Western blots were blocked
with 5% milk-TBS and probed with affinity-purified specific polyclonal
antibodies against MHC-A and MHC-B (kindly provided by R. S. Adelstein,
Laboratory of Molecular Cardiology NIH/NHLBI, Bethesda, MD). Antibodies
specific for different PKC isoforms were purchased from Transduction
Laboratories and Santa Cruz Biotechnology. The blots were developed using a
horseradish peroxidase coupled to a secondary antibody (Jackson Immunoresearch
Laboratories). Electrochemiluminescence (ECL) was performed using a kit from
Amersham.
In vivo phosphorylation of MHC
3x105 TSU-pr1 cells were grown on 60 mm plates for about
14 hours. Cells were washed twice in 2 ml prewarmed RPMI-H (RPMI 1640 medium
containing 12 mM HEPES (pH 7.4)) without phosphate. 1 ml of RPMI-H containing
50 µCi 32P orthophosphate was added to each plate and the plates
were incubated in a humidified atmosphere of 5% CO2 and 95% air at
37°C for 2.5 hours. The plates were washed three times with 1 ml prewarmed
RPMI-H without phosphate. 7 ng ml-1 EGF or 200 nM TPA or 100 nM
calphostin C (Kobayashi et al.,
1989) were then added to each
plate. The medium was removed at different times after incubation and the
cells were lysed in lysis buffer (LB) (60 mM Tris (pH 7.4), 200 mM NaCl, 100
mM sodium pyrophosphate, 200 mM sodium floride, 1 mM sodium vanadate, 20 mM
EGTA, 1.5% NP-40 and protease inhibitor mix (Sigma)). The cell extracts were
incubated on ice for 10 minutes and spun for 10 minutes at 4°C at 30,000
g. 300 µl H2O were added to the cell extracts,
which were transferred to tubes containing 200 µl of the complex
Staphylococcus A cells-MHC-A or MHC-B antibodies (prewashed in LB).
The mixture was incubated for 2 hours on a rotator at 4°C. The
immunoprecipitates were washed four times in LB and analyzed on 7% SDS-PAGE
gels. To determine the relative phosphorylation of the immunoprecipitated
MHC-A and MHC-B, the gels were scanned with a scanning laser densitometer and
the peak areas were evaluated using the Gelscan XL software program, which
integrates the area under the peak. The amounts of 32P incorporated
into MHC-A and MHC-B were determined using phosphorimaging with a Fujix
Bas2000 bioimage analyzer. Relative phosphorylation of MHC-A and MHC-B was
determined by dividing the values obtained with the PhosphorImager by the
values obtained by scanning of the Coomassie-Blue-stained gels.
Co-immunoprecipitation
For detection of the co-immunoprecipitation of MHC-A and MHC-B with the
different PKC isoforms, TSU-pr1 cells were grown, stimulated with EGF for 0
minutes, 1 minutes, 2 minutes, 4 minutes, 6 minutes and 10 minutes, after
which the cells were lysed, and MHC-A or MHC-B were immunoprecipitated as
described above. The immunoprecipitates were analyzed on western blots using
the different PKC isoforms antibodies (as listed below). In the reciprocal
experiments, the different PKC isoforms were immunoprecipitated as described
above and the immunoprecipitates were analyzed on western blots using the
polyclonal antibodies against MHC-A and MHC-B as described above.
Immunomicroscopy
For indirect immunofluorescent staining using MHC-A and MHC-B antibodies,
cells were grown to 30% confluence in six-well dishes in which cover slips
coated with 27 µg ml-1 rat tail collagen I were placed at the
bottom of the wells. Cells were washed twice in RPMI-H and cells were
incubated at 37°C for 2.5 hours. EGF stimulation was carried out by
placing 5 µl of 7 ng ml-1 EGF at a prelabeled corner of the
cover slip and incubating for 0 minutes, 1 minutes, 2 minutes, 4 minutes, 6
minutes and 10 minutes at 37°C. The cells were fixed in 3.7%
paraformaldehyde and incubated at RT for 10 minutes followed by three washes
with PBS. Triton solution (0.5% BSA and 0.2% Triton X-100 in PBS) was then
added to the cover slips and they were incubated at room temperature for 3
minutes followed by three washes with PBS. Polyclonal antibodies specific for
MHC-A and MHC-B were applied at 1:500 dilution and incubated for 45 minutes at
37°C. Cy5-conjugated, affinity-purified goat anti-rabbit IgG (Jackson) at
1:150 dilution was added to each cover slip and incubated for 45 minutes at
37°C. 8 µl of mounting solution (Vector Laboratories) was added to each
slide before mounting the cover slips. The labeled corner of each cover slip
was examined using a 40x objective under a Zeiss LSM 410 confocal laser
scanning system attached to the Zeiss Axiovert 135 M inverted microscope with
40x Apochromat oil immersion lens (Carl Zeiss, Thornwood, NY). The
system was equipped with a 25-mW air-cooled argon laser (488 nm excitation
line with 515 nm long pass barrier filter for the excitation of green
fluorescence). Red fluorescence was excited with the 633 nm internal helium
neon laser. Confocal images were converted to TIF format and transferred to a
Zeiss imaging workstation for pseudocolor representation.
Cloning and site-directed mutagenesis of MHC-A and MHC-B tail
domains
All DNA manipulations were carried out using standard methods (Sambrook et
al., 1989). We used the
expression vector pET21 (Novagene), which allows the production of proteins
carrying an N-terminal His tag. pET21-MHC-A and pET21-MHC-B were constructed
as follows. First, mRNA was isolated from TSU-pr1 cells using an mRNA capture
kit (Boehringer Mannheim). The mRNA was used for reverse transcriptase (RT)
PCR (Boehringer Mannheim) as described by the supplier. Primers for MHC-A
were: 29-mer, upstream sequence containing an EcoRI site (underlined)
5'-GGAATTCCATGGACCAGATCAACGCCGAC-3'; 24-mer, downstream
sequence containing HindIII site (underlined),
5'-AAGCTTTTCGGCAGGTTTGGCCTC-3' corresponding to the
human MHC-A (accession no. M31013) sequences 3140-3157 and 3725-3742,
respectively. Primers for MHC-B: 29-mer, upstream sequence containing
EcoRI site (underlined)
5'-GGAATTCCATGAAGTCTAAGTTCAAGGCC-3'; 24-mer, downstream
sequence containing HindIII site (underlined),
5'-CCCAAGCTTCTCTGACTGGGGTGG-3' corresponding to the
human MHC-B (accession no. M69181) sequences 5504-5522 and 5993-6006,
respectively. The RT-PCR products (MHC-A 602 bp and MHC-B 502 bp) were
subcloned into pGEM kit (Promega) and sequenced. The RT-PCR products were
digested with EcoRI and HindIII, isolated, and ligated into
EcoRI and HindIII sites in the plasmid pET21.
The MHC-A and MHC-B RT-PCR products containing the previously mapped PKC
phosphorylation sites MHC-A Ser1917 and MHC-B Thr1923
(Moussavi et al., 1993) were
replaced with alanine residues (MHC-A S/A and MHC-B T/A, respectively) using
site-directed mutagenesis as described (Deng and Nickoloff,
1992
). The mutagenesis primers
used were: 5'-TTGGCGCTTCAGTCGgCgGATTTCTTGTTCGAG-3' for MHC-A S/A
and 5'-TCGGCGCTCCAGTCTgCgCTTTTCTTGGCCGAC-3' for MHC-B T/A. The
mutagenesis was verified by sequencing. The mutagenized RT-PCR products were
subcloned into pET21 as described above.
Bacterial expression and purification of recombinant MHC-A and MHC-B
tail domains
Expression and purification of the MHC-A, MHC-B, MHC-A S/A and MHC-B T/A
were carried out essentially as described (O'Halloran et al.,
1990). Briefly, bacteria
containing the expression vectors were grown to an optical density of 0.5,
then 2 mM IPTG was added and the cells were grown for an additional 3 hours.
The bacteria were pelleted, lysed using sonication and the cell debris removed
by centrifugation. These supernatants were boiled and cleared using
centrifugation. These supernatants contained the different MHC tail domains
with about 90% purity and were used for myosin heavy chain kinase (MHCK)
assays.
Partial purification of MHC-A and MHC-B kinases
2x106 TUS-pr1 cells were grown in 100 mm Petri dishes for
14-18 hours. Cells were washed twice in RPMI-H and incubated in RPMI-H at
37°C for 2 hours. 7 ng ml-1 EGF was added to the cells that
were incubated for 1 minute or 4 minute, after which 1 ml lysis buffer (50 mM
Tris (pH 7.4), 50 mM NaCl, 5 mM EDTA, 0.2% Triton X-100 and protease inhibitor
mix (Sigma)) was added to the plate. The cell extracts were transferred to
fresh tubes, incubated on ice for 10 minutes and spun for 15 minutes at 30,000
g at 4°C. To purify the MHC-A kinase, the supernatants
obtained from cells that were stimulated for 1 minute with EGF were loaded on
MHC-A tail fragment affinity columns. To purify the MHC-B kinase, the
supernatants obtained from cells that were stimulated for 4 minutes with EGF
were loaded on MHC-B tail fragment affinity column. The columns were prepared
as described (Ravid and Spudich,
1989). After several washes
with lysis buffer the kinases were eluted in a step wise manner with a lysis
buffer containing 100 mM, 150 mM, 200 mM or 250 mM NaCl. These fractions were
assayed for MHC-A and MHC-B kinase activities as described below.
MHC-A and MHC-B kinase assay
4x105 TUS-pr1 cells were grown in 60 mm Petri dishes for
14-18 hours. Cells were washed twice in RPMI-H and incubated in RPMI-H at
37°C for 2 hours. 7 ng ml-1 EGF or 200 nM TPA or 7 ng
ml-1 EGF in the presence of 100 nM calphostin C (Kobayashi et al.,
1989) were added to the cells,
and 400 µl of lysis buffer (50 mM Tris (pH 7.4), 50 mM NaCl, 5 mM EDTA,
0.2% Triton X-100 and protease inhibitor mix (Sigma)) were added to the plate
at intervals indicated below. The cell extracts were transferred to fresh
tubes, incubated on ice for 10 minutes and spun for 15 minutes at 30,000
g at 4°C. The supernatants or fractions obtained from the
MHC-A and MHC-B tail fragment columns described above were used for MHCK assay
described below.
For MHCK assay, 10 µl of the solubilized kinase was incubated with 15
µl of 2x MHCK assay mix (50 mM Tris-HCl (pH 7.4), 20 mM
MgCl2 and 2 mM DTT, and the expressed MHC tail domain (0.5-1 mg
ml-1)). The reaction was initiated by adding 5 µl ATP mix (50 mM
Tris (pH 7.4), 1.2 mM 32P-ATP), after which tubes were
incubated for 20 minutes at 37°C (the reaction was linear up to 40
minutes). The reactions were stopped by the addition of an equal volume of
ice-cold 10% trichloroacetic acid (TCA). To determine the amount of phosphate
incorporated into the MHC tail domains, we used one of the following
techniques. In the first, the TCA-treated samples were pelleted in a
microcentrifuge after incubation for 15 minutes on ice, washed twice with 5%
TCA, resuspended in 25 µl SDS-PAGE sample buffer and electrophoresed on 12%
SDS-PAGE gels. To determine the amount of 32P incorporated into the
MHC tail domains, the gels were stained and destained, the bands were cut out
of the gels and counted in a scintillation counter in 5 ml of scintillation
fluid. In the second technique, the TCA-treated samples were filtered through
nitrocellulose filters, washed three times with 5% TCA and the filters were
counted in scintillation counter as described above. Protein concentration was
determined using the Bradford assay (Bradford,
1976
) to calculate the MHCK
specific activity.
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RESULTS |
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Response to EGF: a transient increase in in vivo phosphorylation
It has been shown in lower eukaryotes that, during chemotaxis, myosin II
undergoes reorganization that is regulated by MHC phosphorylation (Abu-Elneel
et al., 1996; Berlot et al.,
1987
; Berlot et al.,
1985
; Egelhoff et al.,
1993
; Kolman et al.,
1996
; Nachmias et al.,
1989
; Soll et al.,
1990
; Yumura and Fukui,
1985
). However, this
phenomenon had not been described for mammalian cells. It was therefore of
interest to find out whether MHC-A and MHC-B undergo phosphorylation in
response to EGF stimulation. For this purpose, TSU-pr1 cells were incubated in
the presence of 32P orthophosphate and then stimulated with EGF.
MHC-A and MHC-B were immunoprecipitated using MHC-A- and MHC-B-specific
antibodies (see Materials and Methods). The phosphorylation levels of MHC-A
and MHC-B were analyzed using SDS-PAGE and PhosphorImager (see Materials and
Methods). We first determined the EGF concentration that resulted in maximal
phosphorylation of MHC-A and MHC-B. This was 7 ng ml-1 for both
MHC-A and MHC-B (data not shown). We therefore used this concentration to
study the EGF-dependent phosphorylation of MHC-A and MHC-B in TSU-pr1
cells.
The EGF stimulation of the TSU-pr1 cells resulted in transient increases of
both MHC-A and MHC-B phosphorylation with quite different kinetics and to
different extents (Fig. 2).
Quantification of the EGF-dependent relative phosphorylation showed that the
peak of MHC-A phosphorylation occurred 2 minutes after EGF stimulation,
whereas the peak of MHC-B phosphorylation occurred 6 minutes after
stimulation. Furthermore, EGF stimulation of TSU-pr1 cells resulted in an
45% increase in MHC-A phosphorylation and an
30% increase in MHC-B
phosphorylation (Fig. 2).
Interestingly, the increase in phosphorylation of both MHC-A and MHC-B was
followed by a decrease in their phosphorylation, which might indicate that a
MHC phosphatase(s) was activated.
|
EGF-dependent translocation to the cell cortex
To gain an insight into the EGF-dependent localization of MHC-A and MHC-B
in TSU-pr1 cells, and to begin identifying the myosin II isoform involved in
TSU-pr1 chemotaxis, we subjected these cells to EGF stimulation, followed at
various times after stimulation by indirect immunofluorescence analysis using
the MHC-A and MHC-B specific antibodies (Figs
3,
4). In unstimulated TSU-pr1
cells (Fig. 3; 0 minutes),
MHC-A was localized mainly in the cytoplasm. By 1 minute after EGF
stimulation, most of MHC-A translocated to the cell cortex
(Fig. 3). The amount of MHC-A
in the cell cortex continued to increase with the time after EGF stimulation
(Fig. 3) until, 6 minutes after
EGF stimulation, the amount of MHC-A in the cell cortex approached saturation
(Fig. 3).
|
|
The kinetics of the EGF-dependent MHC-B translocation to the cell cortex
and the patterns of MHC-B staining were very different from those of MHC-A
(Figs 3,
4). In unstimulated TSU-pr1
cells, most of the MHC-B was diffusely distributed throughout the cytoplasm
(Fig. 4; 0 minutes). Similar
results were reported for Xenopus cells (Kelley et al.,
1996). 1 minute and 2 minutes
after EGF stimulation, MHC-B gradually translocated to the cell cortex
(Fig. 4). 4 minutes after EGF
stimulation, most of MHC-B localized to the cell cortex
(Fig. 4). Interestingly, 6
minutes after EGF stimulation, most of the MHC-B returned to the cytoplasm. 10
minutes after EGF stimulation, there is another peak of MHC-B localization to
the cell cortex.
These results indicate that the kinetics of the EGF-dependent MHC-A and MHC-B subcellular localization are clearly very differently, possibly suggesting different roles in cell motility and chemotaxis for these myosin II isoforms. Furthermore, the EGF-dependent MHC-B and MHC-A translocation to the cell cortex correlated with the in vivo kinetics of MHC-B phosphorylation but not of MHC-A phosphorylation. The peak of MHC-B localization at the cell cortex (Fig. 4, 4 minutes) took place when MHC-B is almost unphosphorylated (Fig. 2). By contrast, 6 minutes after EGF stimulation, when MHC-B was at the peak of its phosphorylation (Fig. 2), most of the MHC-B returned to the cytoplasm (Fig. 4). These results might indicate that the EGF-dependent phosphorylation of MHC-B plays a role in the EGF-dependent MHC-B cellular localization. By contrast, there is no correlation between the EGF-dependent phosphorylation and the cellular localization of MHC-A (Figs 2, 3). Once the cells were stimulated with EGF, MHC-A localized to the cell cortex and resided there regardless of the extent of the EGF stimulation (Fig. 3).
Both the different kinetics and extent of EGF-dependent phosphorylation of
MHC-A and MHC-B (Fig. 2), and
the different kinetics of the EGF-dependent MHC-A and MHC-B subcellular
localization (Figs 3,
4) suggest different modes of
regulation and different roles for these myosin II isoforms. These results are
consistent with recent observation that the filament formation of the 47 kDa
C-terminal fragment of MHC-B but not of MHC-A is regulated by phosphorylation
(Murakami et al., 2000).
PKC is involved in EGF-dependent phosphorylation
It has been shown that MHC-A and MHC-B are phosphorylated in vivo and in
vitro by several kinases, including PKC (Moussavi et al.,
1993; Murakami et al.,
1998
; Murakami et al.,
1995
). PKC phosphorylates a
single serine residue (Ser1917) in MHC-A (Moussavi et al.,
1993
). Sequence comparison of
MHC-A and MHC-B has shown that there is an analogous site in MHC-B, but, in
this case, the phosphorylatable residue is a threonine (Thr1923),
not a serine (Moussavi et al.,
1993
; Takahashi et al.,
1992
). This analysis might
suggest that Thr1923 is the PKC site on MHC-B (Moussavi et al.,
1993
). However, Murakami et
al., found that a fragment of 47 kDa from the C terminus of MHC-B expressed in
Escherichia coli can be phosphorylated by a mix of PKC isoforms on
serine residues (Murakami et al.,
1998
; Murakami et al.,
1995
).
To determine whether the external EGF signal is transmitted to MHC-A and MHC-B via the activation of PKC, we exposed TSU-pr1 cells to the phorbol ester PMA (phorbol myristate) and compared the resulting changes in MHC-A and MHC-B phosphorylation with those caused by EGF stimulation (Fig. 5). Phosphorylation levels of MHC-A and MHC-B were measured 2 minutes and 6 minutes after EGF or PMA stimulation, respectively (see Materials and Methods). Exposure of TSU-pr1 cells to PMA caused increases of MHC-A and MHC-B phosphorylation by 52% and 36% respectively (data not shown). Similar results were obtained for EGF-stimulated TSU-pr1 cells (Figs 2, 5).
|
To explore the involvement of PKC in the EGF-dependent MHC-A and MHC-B
phosphorylation further, we used the PKC-specific inhibitor calphostin C
(Kobayashi et al., 1989).
TSU-pr1 cells were incubated with 32P in the presence of 100 nM
calphostin C and stimulated with EGF. MHC-A and MHC-B were immunoprecipitated
2 minutes and 6 minutes after stimulation, respectively, and analyzed as
described in Materials and Methods. Exposure of TSU-pr1 cells to calphostin C
caused decreases of MHC-A and MHC-B phosphorylation by 68% and 80%,
respectively (Fig. 5). The
above results together provide strong evidence that PKC is involved in
EGF-dependent MHC-A and MHC-B phosphorylation.
In an attempt to discover whether these PKC(s) had been previously
described, we used antibodies against different PKC isoforms (,
ßI, ßII,
,
,
,
,
,
,
) and found that TSU-pr1 cells express PKC isoforms ßII,
,
,
and
(R.S. and S.R., unpublished). We further found
that the addition of 32P-
-ATP to MHC-A or MHC-B
immunoprecipitated from EGF-stimulated TSU-pr1 cells resulted in the
phosphorylation of these myosin II isoforms (data not shown). These results
indicate that the MHC-A and MHC-B kinase(s) are associated with these myosin
II isoforms. To discover whether PKC isoforms ßII,
,
,
,
are associated with these myosin II isoforms and might
therefore be involved in the EGF-dependent phosphorylation, we
immunoprecipitated MHC-A and MHC-B and analyzed them on a western blot using
antibodies for the above PKC isoforms. The reciprocal experiment was also
performed as described in Materials and Methods. None of the PKC isoforms that
are produced by TSU-pr1 co-imunoprecipitated with MHC-A or MHC-B (R.S. and
S.R., unpublished). Seemingly, these results indicate that MHC-A and MHC-B are
not phosphorylated directly by PKC. However, because the antibodies against
other known PKCs were not available to us, it is still possible that MHC-A
and/or MHC-B are phosphorylated by PKC.
EGF and PMA cause a transient increase in MHC-A and MHC-B kinase
activities
The results presented so far suggest that the EGF signal is transmitted to
MHC-A and MHC-B by the activation of PKC
(Fig. 5). Several studies have
also shown that extracellular stimulation of cells increases PKC activity
(Mochly-Rosen et al., 1990;
Nakamura and Nishizuka, 1994
).
Therefore, we next tested whether EGF and PMA stimulation of TSU-pr1 cells
increased MHC-A and MHC-B kinase activities, and the addition of calphostin C
inhibited these activities. For this purpose, we expressed tail domains of
MHC-A and MHC-B in E. coli that served as substrates for kinase
assays. These tail domains contain the MHC-A Ser1917 PKC
phosphorylation site, the MHC-B Thr1923 putative PKC
phosphorylation site and the peptides to which the specific antibodies of
MHC-A and MHC-B were made. The expressed proteins were purified by a simple
technique based on boiling the E. coli cell extracts and clearing the
denatured proteins (see Materials and Methods). Analysis of the supernatant of
the boiled cell extracts indicated that the MHC-A and MHC-B were >90% pure
(Fig. 6). The expressed
proteins were also analyzed by western blotting with the MHC-A- and
MHC-B-specific antibodies, and it was found that the expressed proteins are
the MHC-A and MHC-B tail fragments (Fig.
6).
|
To study the MHC-A and MHC-B kinase activities, TSU-pr1 cells were
stimulated with EGF, PMA or EGF in the presence of calphostin C, and the cells
were lysed and subjected to MHCK assay at different times after incubation
(see Materials and Methods). The MHC-A or MHC-B tail domains were used as
substrates (Fig. 6). Addition
of EGF or PMA to TSU-pr1 cells resulted in a transient increase in MHC-A
kinase activity with a peak at 1 minute after stimulation
(Fig. 7). By contrast,
stimulation of TSU-pr1 cells with EGF in the presence of calphostin C
abolished the MHC-A kinase activity (Fig.
7). TSU-pr1 cells stimulated with either EGF or PMA and tested for
MHC-B kinase activity showed a transient increase in MHC-B kinase with a peak
at 4 minutes (Fig. 7);
addition of calphostin C abolished this activity
(Fig. 7). These results
together with those in Fig. 5
strongly indicate that the EGF signal is transmitted to MHC-A and MHC-B via
PKC. The peak of activity of the MHC-A and MHC-B kinase(s) coincided with the
localization of MHC-A and MHC-B at the cell cortex, but it preceded the peak
of maximum EGF-dependent in vivo phosphorylation of MHC-A and MHC-B. A
plausible explanation of these findings is that EGF stimulation of TSU-pr1
cells leads to translocation and activation of MHC-A and MHC-B kinases to the
cell cortex, as well as to translocation of MHC-A and MHC-B, leading to
phosphorylation of these myosin II isoforms.
|
Replacement of the MHC-A and MHC-B PKC phosphorylation sites with
alanine
To determine whether the EGF-activated MHC-A and MHC-B kinase(s) are
members of the PKC family, we produced in E. coli MHC-A and MHC-B
tail domains in which the previously reported PKC phosphorylation site MHC-A
Ser1917 and the putative PKC phosphorylation site MHC-B
Thr1923 (Moussavi et al.,
1993) were replaced with
alanine residues (MHC-A S/A and MHC-B T/A, respectively). To test whether
MHC-A S/A and MHC-B T/A were effective substrates for the EGF-activated MHC-A
and MHC-B kinases, we stimulated TSU-pr1 cells with EGF and subjected them to
MHCK assay at different times after incubation using the above mutated MHC
tail domains (see Materials and Methods). As shown in
Fig. 8, in contrast to MHC-A
and MHC-B tail domains, the mutated MHC-A S/A and MHC-B T/A tail domains were
inefficient substrates for EGF-activated MHCKs. Using the MHC-A and MHC-B tail
domains as substrates in the EGF-activated MHCK assay provided similar results
to those shown in Fig. 7.
However, using MHC-A S/A and MHC-B T/A tail domains as substrates in this
assay resulted only in a basal level of activity. These observations indicate
that the EGF-activated MHC-A and MHC-B kinases phosphorylate the previously
mapped PKC site on MHC-A or the putative PKC site on MHC-B, and further
suggest that these kinases are members of the PKC family. In addition, these
results indicate that, in response to EGF stimulation of TSU-pr1 cells, there
is an activation of PKC(s) that is involved in MHC-A and MHC-B
phosphorylation.
|
As mentioned above, Murakami et al. reported that the C-terminal 47 kDa
fragment of brain-type MHC-B can be phosphorylated on serine residues by a mix
of PKC isoforms (Murakami et al.,
1998; Murakami et al.,
2000
). These serine residues
are within the MHC-B tail domain described here. These observations are
inconsistent with the results described above, in which a threonine residue is
phosphorylated by cell extract obtained from EGF-stimulated TSU-pr1. A
plausible explanation for this discrepancy is that the basal level of
phosphorylation of MHC-B T/A (Fig.
7) represents phosphorylation on serine residues reported earlier
(Murakami et al., 1998
).
However, EGF stimulation of TSU-pr1 cells results in the activation of a PKC
that phosphorylates Thr1923 on MHC-B. Therefore, replacing this
threonine with alanine abolished the EGF-dependent increases in MHC-B T/A
phosphorylation but, nevertheless, the basal level of phosphorylation of this
protein is similar to that of MHC-B (Fig.
7). To explore this possibility, we performed EGF-dependent in
vivo phosphorylation of MHC-B but, instead of using 32P to detect
the phosphorylated form of MHC-B, we used anti-phosphothreonine antibody. We
found that EGF stimulation of TSU-pr1 cells resulted in transient increases in
MHC-B phosphorylation on threonine residue(s) (A. Ben-Ya'acov and S.R.,
unpublished). These results strongly suggest that the EGF-dependent increases
in MHC-B phosphorylation occurred on threonine residue(s).
EGF-dependent in vivo phosphorylation is carried out by two different
kinases
To characterize further the kinase(s) that phosphorylate MHC-A and MHC-B in
response to EGF stimulation, we partially purified these kinases using MHC-A
and MHC-B tail fragment affinity columns (columns A and B, respectively) as
described in Materials and Methods. To investigate the elution profile of
MHC-A and MHC-B kinase(s) from the columns, the fractions obtained from these
columns were subjected to MHCK assay as described in Materials and Methods. As
shown in Fig. 9, the maximum
activity of the kinase purified on column A (MHC-A kinase) and assayed using
MHC-A tail fragment as a substrate was eluted with 100 mM NaCl
(Fig. 9; `A-A'). However, the
maximum activity of the kinase purified on column B (MHC-B kinase) and assayed
using MHC-B tail fragment as a substrate was eluted with 200 mM NaCl
(Fig. 9; `B-B'). The different
elution profiles of MHC-A and MHC-B kinases might indicate that they are
different kinases. To explore this possibility further, we tested whether the
MHC-A kinase is competent to phosphorylate the MHC-B tail fragment
(Fig. 9; `A-B') and whether
MHC-B kinase is competent to phosphorylate MHC-A tail fragment
(Fig. 9; `B-A'). As shown in
Fig. 9, MHC-A kinase did not
phosphorylate the MHC-B tail fragment (Fig.
9; `A-B') but, by contrast, MHC-B kinase did phosphorylate the
MHC-A and MHC-B tail fragments to the same extent
(Fig. 9; `B-A' and `B-B').
These results further indicate that MHC-A and MHC-B kinases are different.
Furthermore, it is plausible that the EGF-dependent MHC-A and MHC-B
phosphorylation is carried out by two different kinases, which might explain
the different phosphorylation kinetics of these two myosin II isoforms.
|
![]() |
DISCUSSION |
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---|
EGF-induced cell motility requires the presence of a phosphotyrosine motif
in the intracellular regulatory region of the EGFR (Chen et al.,
1994). This suggests that the
immediate downstream effector molecule in the motogenic pathway is activated
by SH2-domain interactions. Numerous SH2-domain-containing effector molecules
interact with, and are activated by, EGFR (Carpenter,
1992
), and at least three of
these pathways can be linked to cell motility. Activation of a small
GTP-binding protein of the Rho subfamily leads to the formation of filopodia,
lamellipodia and focal adhesions, which are required for cell motility and
chemotaxis (Burridge, 1999
;
Horwitz and Parsons, 1999
;
Van-Aelst and D'Souza-Schorey,
1997
). A second pathway
involves the kinase phosphatidylinositol 3' kinase, whose activation is
required for chemotaxis mediated by the PDGFß receptor (Kundra et al.,
1994
; Wennstrom et al.,
1994
). A third signaling
pathway, which involves PLC
, might also promote cell motility.
PLC
hydrolysis of PIP2 releases actin-severing and
-sequestering proteins, which lead to the dissolution of stress fibers and
focal adhesions, enabling a cell to move (Banno et al.,
1992
; Goldschmit-Clermont et
al., 1991
; Stossel,
1993
). In addition, activation
of PLC
is required for chemotaxis (Kundra et al.,
1994
; Wennstrom et al.,
1994
) or is associated with
PDGFß-receptor-mediated chemotaxis (Bornfeldt et al.,
1994
). Chen et al. have shown
that enhanced inositol phosphate production was observed only in cell lines
demonstrating EGFR-mediated cell movement (Chen et al.,
1994
). This correlation
between the biochemical and biological responses suggested that PLC
was
the immediate downstream effector. To identify PLC
definitively as a
necessary intermediary, Chen et al. downregulated this enzyme activity using
antisense oligonucleotides (Chen et al.,
1994
). This treatment
partially abrogated both PLC
activity and EGF-induced motility. These
experiments indicate that PLC
is required for EGFR-mediated movement
and place PLC
directly downstream of the EGFR.
PLC activation by EGFR produces diacylglycerol (DAG) and inositol
trisphosphate, which activate members of the PKC family (Margolis et al.,
1990
). This is a large family
of at least 12 isoforms differing in their structure, tissue distribution,
subcellular localization, mode of activation and substrate specificity (Dekker
and Parker, 1994
). PKCs
phosphorylate a wide variety of substrates including proteins involved in
signal transduction (including Ras, GAP and Raf) (Hug and Sarre,
1993
), as well as
motility-associated cytoskeletal modulators (including Fak, profilin and
MARCKS) (Aderem, 1992
; Hansson
et al., 1988
). In addition, a
unique PKC phosphorylates MHC from Dictyostelium and plays an
important role in chemotaxis of this organism (Abu-Elneel et al.,
1996
; Ravid and Spudich,
1992
). All these studies
provide a strong evidence for the involvement of PKC isoforms in mediating and
regulating chemotaxis and cell motility. Although much is known about the
pathway that begins with the EGF receptor and continues through PLC
and
PKC, we know very little of how EGF mediates cell motility. Our results
provide the first indication of the mechanism linking the EGF receptor to cell
motility. Furthermore, we found that of the two vertebrate myosin II isoforms,
MHC-B is the one that is most likely to play a role in chemotaxis; EGF
stimulation of TSU-pr1 cells resulted in transient subcellular organization of
MHC-B but not of MHC-A. Such a transient response is expected from a
cytoskeletal protein that is involved in chemotaxis towards EGF.
Our results are consistent with several recent reports indicating that
myosin II plays a role in such cellular processes. Moores et al. have found
that green fluorescent protein-myosin II concentration increases in the tips
of retracting pseudopodia (Moores et al.,
1996). This suggests that
myosin II might play an important role in the dynamics of pseudopodia as well
as filopodia, lamellipodia and other cellular protrusions. Kelley et al.
reported that the lamellipodium in highly polarized, rapidly migrating cells
was dramatically enriched for MHC-B (Kelley et al.,
1996
), suggesting a possible
involvement of MHC-B-based contraction in leading extension and/or
retraction.
The time course of EGF-mediated MHC-B phosphorylation subcellular
localization fits that of EGF-mediated lamellipodium extension. Recently,
Segall et al. reported that the addition of EGF to mammary adenocarcinoma cell
line MTLn3 cells stopped ruffling and resulted in extension of hyaline
lamellipodia containing increased amounts of F-actin at the growing edge.
Lamellipodium extension was maximal within 5 minutes, followed by retraction
and resumption of ruffling (Segall et al.,
1996). Interestingly, the peak
of MHC-B associated with the cell cortex occurs at 4 minutes after EGF
stimulation (Fig. 4), at that
time point MHC-B is almost unphosphorylated
(Fig. 2). However, at the peak
of MHC-B phosphorylation (Fig.
2,
6 minutes), most of the MHC-B
translocated back to the cytoplasm (Fig.
4). It is therefore, plausible that MHC phosphorylation leads to
MHC-B filament dissociation such that, when the MHC-B is not phosphorylated,
it forms filaments that localized to the cell cortex. By contrast,
phosphorylation of MHC-B at the cell cortex possibly by PKC leads to filament
dissociation that translocate to the cytoplasm. Phosphorylation of MHC-B might
help to destabilize the MHC-B filaments. Such a function is also suggested by
the localization of the site of phosphorylation in MHC-B, which is in the
-helical portion of the rod just N terminal to the non-helical tail
(Moussavi et al., 1993
). This
part of MHC-B molecule has been implicated in filament formation (Hodge et
al., 1992
). Thus, the role of
MHC-B phosphorylation is to cause a major cellular rearrangement of this
myosin II that is required for MHC-B to function as a motor protein. The MHC-B
cellular rearrangement might result in a change in cell shape and, perhaps, in
the case of TSU-pr1 cells, in chemotaxis towards EGF. Recently, it has been
shown that the single Drosophila MHC can be phosphorylated by PKC (Su
and Kiehart, 2001
). However,
similar to mammalian MHC-A this phosphorylation has no effect on filament
assembly (Murakami et al.,
1998
; Su and Kiehart,
2001
). These results further
indicate that phosphorylation of MHC-B, for which effects on assembly have
been observed in vitro, might be more important for different function in the
organism that expresses them.
EGF and PMA stimulation of TSU-pr1 cells increased MHC-A and MHC-B kinase
activities. These findings, together with the finding that calphostin C
inhibited these kinase activities, provide strong evidence that these kinases
belong to the PKC family. Further support for this notion was provided by the
findings that replacing the previously mapped PKC phosphorylation site of
MHC-A and the putative PKC site of MHC-B with alanine residues resulted in
tail fragment that cannot be phosphorylated by EGF-stimulated TSU-pr1 cell
extract. Furthermore, we found that the increase in the in vivo EGF-dependent
MHC-B phosphorylation occurs on threonine residue(s) and not serine residues
(A. Ben-Ya'acov and S.R., unpublished), as was previously reported (Murakami
et al., 1998). It is
therefore, possible that the putative PKC phosphorylation site on MHC-B
(Thr1923) is indeed a PKC site.
![]() |
ACKNOWLEDGMENTS |
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