1 Department of Neurology and Neurological Sciences, Stanford University School
of Medicine, Stanford, CA 94305, USA
2 Department of Molecular Pharmacology, Stanford University School of Medicine,
Stanford, CA 94305, USA
3 GRECC and Neurology Service, Veterans Affairs Palo Alto Heath Care System,
Palo Alto, CA 94304, USA
* Author for correspondence (e-mail: rando{at}stanford.edu )
Accepted 26 February 2002
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Summary |
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Key words: Integrin, PKC, Muscle, FAK, MARCKS, Fibronectin
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Introduction |
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The importance of integrin signaling in the survival of skeletal muscle
cells is demonstrated by the recent reports of muscular degenerative disorders
in mice with specific integrin deficiencies
(Mayer et al., 1997;
Taverna et al., 1998
). These
reports show that a deficiency in either
5 or
7 integrins causes
muscular dystrophies, indicating that the expression of each integrin is
necessary for long-term survival of myofibers. These results indicate that
integrins are true signaling molecules, transmitting information from the
extracellular milieu into the cell. However, the integrin-signaling cascade
that regulates this survival pathway in muscle cells remains to be
elucidated.
Several studies using different systems have highlighted the importance of
protein kinase C (PKC) in integrin-mediated cell adhesion and spreading
(Vuori and Ruoslahti, 1993;
Schlaepfer et al., 1994
;
Disatnik and Rando, 1999
), as
well as in cell migration, FAK phosphorylation and focal adhesion formation
(Woods and Couchman, 1992
;
Vuori and Ruoslahti, 1993
;
Haimovich et al., 1996
).
Different approaches have been used to study the specific role of PKC in
integrin signaling. Pharmacological activators of PKC have been reported to
enhance the adhesion and spreading of cells
(Mercurio and Shaw, 1988
;
Disatnik and Rando, 1999
).
Pharmacological inhibitors of PKC prevent not only focal adhesion formation
but also stress fiber formation in fibroblasts plated on FN
(Woods and Couchman, 1992
).
PKC also appears to be a key intermediate between integrins and FAK signaling
in muscle cells and other cell types
(Vuori and Ruoslahti, 1993
;
Disatnik and Rando, 1999
;
Miranti et al., 1999
). Several
studies have indicated that PKC activation is required for FAK phosphorylation
in cells plated on FN (Vuori and
Ruoslahti, 1993
; Haimovich et
al., 1996
; Disatnik and Rando,
1999
). Although PKC and FAK colocalize at focal adhesion sites
(Schaller et al., 1992
;
Liao and Jaken, 1993
), the
precise functional relationship between these two kinases is not known.
The PKC family of serine-threonine kinases can be classified into three
major families (Ron and Kazanietz,
1999). The classical PKCs consist of
, ßI, ßII
and
PKC, which are Ca2+/diacylglycerol-dependent kinases.
The novel PKCs,
,
,
and
PKC, are Ca2+
independent but require diacylglycerol for activation. The third family,
atypical PKCs, consists of
and
/
PKC, which are neither
Ca2+- nor diacylglycerol-dependent. The PKC isozymes responsible
for mediating cell attachment and spreading are unknown and may be tissue and
stimulus specific. The lack of isozyme-selective modulators has limited the
information available regarding the role of specific PKC isozymes in integrin
signaling. Studies in different cell types have demonstrated
,
and
PKC localization to focal adhesions
(Liao and Jaken, 1993
;
Haimovich et al., 1996
;
Haller et al., 1998
),
suggesting that all these isozymes may be linked to the integrin-signaling
pathway.
One of the most predominant intracellular substrates for PKC that may play
a role in cell spreading is the myristoylated alanine-rich C kinase substrate
protein (MARCKS) (Aderem,
1992b). MARCKS contains three highly conserved domains: an
N-terminal myristoylation domain, a region of conserved sequence at the single
intron splicing and an internal phosphorylation site domain (PSD), containing
serines phosphorylated by PKC. This domain also serves as the site of high
affinity calmodulin binding. Moreover, this region has also been shown to
crosslink actin filaments in vitro
(Hartwig et al., 1992
;
Swierczynski and Blackshear,
1995
). PKC-mediated phosphorylation of the PSD domain decreases
MARCKS affinity for the plasma membrane, calmodulin and actin, followed by its
translocation from the cell membrane to the cytosolic fraction. Several
reports have highlighted the potential role of MARCKS in cell attachment and
spreading, but the mechanism of action is still unknown
(Li et al., 1996
;
Manenti et al., 1997
;
Myat et al., 1997
;
Spizz and Blackshear,
2001
).
In this report, we present evidence for the activation of distinct PKC
isozymes leading to the phosphorylation of FAK and mediating spreading of
skeletal muscle cells. In response to integrin engagement, there was a rapid
but transient activation of ,
and
PKC. Peptide modulators
of individual PKC isozymes have been recently developed that inhibit or
promote binding of individual PKC isozymes to their anchoring proteins, RACKs
(Receptors for Activated C Kinase)
(Mochly-Rosen, 1995
;
Souroujon and Mochly-Rosen,
1998
; Dorn et al.,
1999
; Hu et al.,
2000
; Mochly-Rosen et al.,
2000
). The function of these short peptides conjugated to a
cell-permeable peptide derived from the Antennapedia protein has been
previously described in a variety of cells, including cardiac myocytes
(Mochly-Rosen, 1995
;
Souroujon and Mochly-Rosen,
1998
; Dorn et al.,
1999
; Hu et al.,
2000
; Mochly-Rosen et al.,
2000
; Chen et al.,
2001
). The 6-10 amino acid peptides derived from individual PKC
isozymes were shown to act selectively on their corresponding isozymes by
inducing (for the activator peptides) or inhibiting (for the inhibitor
peptides) PKC translocation and cellular activity (for a review, see
Souroujon and Mochly-Rosen,
1998
). To assess further the role of individual PKC isozymes, we
used
,
and
PKC-selective activator and inhibitor peptides
and examined their effects on cell spreading and FAK phosphorylation. The
results of these studies suggest that
PKC activation is necessary to
promote muscle cell attachment with a concomitant activation of
and
PKC that mediate cell spreading. Our results further demonstrate that
MARCKS might be downstream of PKC in the integrin-signaling pathway that
mediates muscle cell spreading. MARCKS may be the intermediate signaling
molecule that lead to cell attachment and spreading. Taken together, these
results support the link between specific PKC isozymes, MARCKS and the
integrin-signaling pathway in muscle cell attachment and spreading.
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Materials and Methods |
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PKC peptides
Peptide activators are pseudo-RACK1 [amino acids 241-246 of PKC
(SVEIWD) (Ron and Mochly-Rosen,
1995
; Souroujon and
Mochly-Rosen, 1998
)], pseudo-
RACK [amino acids 74-81 of
PKC (MRAAEDPM) (Chen et al.,
2001
)], and pseudo-
RACK [amino acids 85-92 of
PKC
(HDAPIGYD) (Dorn et al.,
1999
)]. Peptide inhibitors are
C2-4 [amino acids 218-226 of
PKC (SLNPQWNET) (Souroujon and
Mochly-Rosen, 1998
)],
V1-1 [amino acids 8-17 of
PKC
(SFNSYELGSL) (Chen et al.,
2001
)] and
V1-2 [amino acids 14-21 of
PKC (EAVSLKPT)
(Dorn et al., 1999
)]. The
peptides were synthesized and purified (>95%) at the Stanford Protein and
Nucleic Acid Facility. The peptides were crosslinked via an N-terminal Cys-Cys
bond to the Drosophila Antennapedia homeodomain-derived carrier
peptide (CRQIKIWFQNRRMK-WKK) or carrier-carrier dimer as a control
(Derossi et al., 1994
;
Theodore et al., 1995
).
Adhesion and spreading
For assessment of cell adhesion and spreading, 60 mm dishes were coated
with FN (5 µg/ml; Gibco BRL) for 24 hours at room temperature. 1 hour
before plating, all dishes were coated with 1% bovine serum albumin (Sigma, St
Louis, MO). Cells were trypsinized and treated as indicated. In PKC activation
or inhibition experiments, the cells were treated in suspension with
respective peptides at 1 µM and then plated on FN for 30 minutes in the
presence of the peptides as indicated. The cultures were assessed and
photographed using a 40x phase contrast immersion objective on a Zeiss
Axioskop microscope.
Western blot analysis
After trypsinization, cells were plated on FN for 30 minutes. For PKC
activation, phorbol 12-myristate 13-acetate (PMA; Alexis Biochemicals, San
Diego, CA) or specific PKC peptides were added to the cells in suspension for
10 minutes at the indicated concentration. For PKC inhibition, respective
peptides were added to the cells in suspension for 20 minutes prior to
plating. After 30 minutes of plating, attached and unattached cells were
collected, spun and washed with cold PBS. The cells from both fractions were
pooled and lysed in RIPA buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.5%
deoxycholate, 1% Nonidet P-40) containing aprotinin (20 µg/ml), leupeptin
(20 µg/ml), phenylmethylsulfonyl fluoride (10 µ/ml), sodium
orthovanadate (1 mM), sodium pyrophosphate (10 mM) and sodium fluoride (100
mM). Proteins from total extracts were electrophoresed by 10%
SDS-polyacrylamide gel electrophoresis, transferred to nitrocellulose then
incubated for 2 hours in PBS containing 0.05% Tween and 5% non fat dry milk.
Phosphotyrosine-containing proteins were detected with a monoclonal
antiphosphotyrosine antibody, PY-99 (1:5000; Santa Cruz Biotechnology, Santa
Cruz, CA), as described previously
(Disatnik and Rando, 1999)
followed by a horseradish-peroxidase-coupled anti-mouse secondary antibody
(Amersham Corp., Arlington Heights, IL). Duplicate blots were also probed (or
blots were reprobed after stripping) with anti-FAK polyclonal antibodies
(1:1000; Santa Cruz Biotechnology) followed by a
horseradish-peroxidase-coupled anti-rabbit secondary antibody. Specific
antibody binding was detected by an enhanced chemiluminescence system
(Amersham Corp., Arlington Heights, IL). Where indicated, the bands were
quantitated using a Bio-Rad Fluor-S MultiImager (Bio-Rad, Hercules, CA).
Fractionation analysis
5-expressing or
5-deficient cells were trypsinized, and
3x106 cells were replated on FN-coated dishes. At different
time points after plating, non-adherent cells from duplicate dishes were
pooled and collected by centrifugation. Adherent cells were scraped from the
dish in homogenization buffer (50 mM Tris-HCl, 1 mM EDTA, 1 mM EGTA)
containing protease inhibitors and phosphatase inhibitors as indicated above.
The adherent and non-adherent cells were pooled, and the extract was passed
through a 26-gauge needle and then spun at 100,000 g for 40 minutes
at 4°C. The cytosolic fraction was collected, and the membrane fraction
was solubilized in RIPA buffer. 80 µg protein was loaded on a 7.5%
SDS-polyacrylamide gel. The level of MARCKS in each fraction was detected by
western blot analysis, as above, using a goat polyclonal MARCKS antibody at
1:100 dilution (Santa-Cruz Biotechnology) followed by a
horseradish-peroxidase-coupled anti-goat secondary antibody (1:15000; Pierce
Endogen, Rockford, IL). For a loading control, actin protein was analyzed
using a rabbit polyclonal antibody at 1:5000 dilution (Sigma).
Kinase assay and immunoprecipitation
5-expressing or
5-deficient cells were trypsinized, and
2x106 cells were replated on FN-coated dishes. At different
time points after plating, non-adherent cells from duplicate dishes were
pooled and collected by centrifugation. Adherent cells were lysed in 100 µl
RIPA buffer and combined with spun cells. The lysates were incubated on ice
for 1 hour, and insoluble proteins were then pelleted by centrifugation.
Protein estimation was done on the soluble fraction, and equal amounts of
protein were used for immunoprecipitation of
PKC,
PKC and
PKC using isozyme-specific antibodies (1:100; Santa Cruz Biotechnology).
PKC isozymes were immunoprecipitated for 3 hours at 4°C. After the
addition of protein G-agarose beads, the reaction was incubated for 1 hour.
Immunocomplexes were washed three times with RIPA buffer and once with binding
buffer (20 mM Tris-HCl, pH 7.5, 20 mM MgCl2, 1 mM DTT, and 25 µM
ATP). For inhibition experiments, chelerythrine (2 µM, Alexis Biochemicals)
was added 10 minutes before the kinase assay. The PKC activity of
immunoprecipitated fractions was assayed by adding 40 µl of binding buffer
containing 5 µCi [
32P]ATP (5000 Ci/mmole, Amersham) and
40 µg histone III-S (Sigma) or 10 µg myelin basic protein (MBP). After
15 minute incubations at 37°C, assays were terminated by adding sample
buffer. The samples were loaded on a 10% or 12% SDS acrylamide gel, and the
levels of phosphorylated histone or MBP were quantified either by cutting the
band and counting 32P incorporated into the substrates using
scintillation fluid or by exposing the gel to autoradiographic film and
quantifying the bands using a Bio-Rad Fluor-S MultiImager. After exposure, the
blots were probed with specific PKC isozyme antibodies (1:500) for
normalization of the immunoprecipitated material. The results from eight
separate experiments were analyzed.
MARCKS cDNA cloning and transfection
Poly(A)+ RNA was extracted from the C2C12 cell line using
MicroFastrack purification kit (Invitrogen). We generated the full-length
mouse MARCKS cDNA (GenBank accession number M60474) with Titanium one-step
RT-PCR Kit (Clontech) using primers
5'-CGTCGTTACACCAACCGAAGGCTCT-3' and
5'-GAATTGCGTGAGGGCTCTGGAGCTT-3' and following the protocol
outlined by the manufacturer. The product of 1 kb was then cloned into
pGEM-T-Easy vector (Promega) and fully sequenced to confirm its sequence.
MARCKS cDNA was then subcloned in the forward (sense) and reverse (antisense)
orientation in pcDNA3.1/hygro vector (Invitrogen). The MARCKS-sense, the
MARCKS-antisense or the vector alone was transfected into 5-expressing
cells with Lipofectamine 2000 (Invitrogen). Hygromycin-resistant colonies were
pooled, and clones were isolated by limiting dilution. Antisense and control
transfected cells were maintained in the presence of 200 µg/ml
hygromycin.
Immunocytochemistry
Myoblasts were plated on FN-coated chamber slides for different lengths of
time and then fixed with cold methanol followed by acetone or with 4%
paraformaldehyde. Non-specific binding was blocked for 1 hour with 1% normal
goat serum or normal rabbit serum in PBS containing 0.1% Triton X-100
(blocking solution) followed by overnight incubation with PKC isozyme-specific
antibodies at 1:100, polyclonal FAK antibodies at 1:1000, polyclonal skeletal
actin antibodies (Sigma) at 1:1000, polyclonal MARCKS antibodies (Santa-Cruz
Biotechnology) at 1:100 or monoclonal paxillin antibody at 1:1000 in blocking
solution containing 2 mg/ml bovine serum albumin. The cells were washed with
the blocking solution followed by 2 hour incubations with a
fluorescein-conjugated anti-rabbit IgG antibody (ICN Pharmaceuticals, Aurora,
OH, diluted at 1:1500), a rabbit anti-goat Alexa fluor 488 antibody (Molecular
Probes, Inc., Eugene, OR diluted at 1:500) or a goat anti-mouse Alexa fluor
546 (Molecular Probes at 1:500) in the presence, as indicated, of 1 µm/ml
TRITC-Phalloidin (Sigma), which binds to F-actin. The specificity of the PKC
staining was determined as described previously
(Disatnik et al., 1994). After
washing the cells three times with blocking solution, the slides were mounted
with Vectashield (Vector, Burlingame, CA) and viewed with a Zeiss Axioskop
microscope (Carl Zeiss, Inc., Thornwood, NY) using a 63x oil immersion
objective. Images were recorded on Kodak T160 film.
Statistical analysis
The results presented are from three to eight separate experiments, as
indicated. Data are presented as means ±s.d. Student's paired
t-tests were used for comparisons. P<0.05 was considered
statistically significant.
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Results |
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|
Since FAK localizes to focal adhesion sites in cells plated on FN
(Schaller et al., 1992;
Hanks et al., 1992
), we
analyzed the cells for focal adhesion formation and FAK localization at these
sites as a function of time after plating. Within 15 minutes of plating on FN,
FAK was localized to the nucleus and diffusely in the cytosol of skeletal
muscle cells with faint staining at the periphery of the cells
(Fig. 1B). At 30 minutes,
predominant punctate FAK staining was detected at cell edges, accentuating
adhesion contacts with the substrate. Distinct focal adhesion sites were
identified after 60 minutes on FN (Fig.
1B). In addition to FAK translocation, we measured the
phosphorylation of FAK by western blot analysis in cells plated on FN for
different period of time (Disatnik and
Rando, 1999
). FAK phosphorylation is necessary for
integrin-mediated cell attachment and spreading
(Kornberg et al., 1992
;
Hanks et al., 1992
;
Richardson and Parsons, 1996
).
Our results show that there is phosphorylation of FAK within 5 minutes of
plating (Fig. 1C), at a time
when cells have just begun the process of attachment and spreading. FAK
phosphorylation increases with time after plating, as cell spreading, stress
fiber formation and focal adhesion formation proceed. The phosphorylation of
FAK reaches a maximum at 60 minutes when cell spreading is complete
(Fig. 1C), and it does not
change thereafter.
Our previous results demonstrated that PKC activation promotes muscle cell
spreading on FN and that this activation is necessary for the interaction of
5 integrin with FN to promote cell spreading and FAK phosphorylation
(Disatnik and Rando, 1999
). To
determine which of the PKC isozymes are involved in this integrin-signaling
pathway in muscle cells, we first determined which isozymes are expressed in
skeletal muscle cells by western blot analysis using isozyme-specific
antibodies (Fig. 2A). We found
that only
,
and
PKC were expressed at detectable levels.
Surprisingly,
PKC, which is known as a muscle specific isozyme
(Osada et al., 1992
), was not
expressed in cultured myoblasts nor in myotubes. The same pattern of PKC
isozyme expression was found in differentiated myotubes in culture (data not
shown).
|
Previously, we found that inhibition of PKC with either calphostin C or
bisindolylmaleimide I completely prevented integrin-mediated cell spreading
and FAK phosphorylation (Disatnik and
Rando, 1999). To determine which of the PKC isozymes were
activated upon myoblast attachment to FN, cells were plated on FN-coated
dishes for various lengths of time, and individual PKC isozymes were
immunoprecipitated from total cell lysates. The activation of the PKC isozymes
was determined by measuring the kinase activity in immunoprecipitates using
histone III-S as a substrate. The incorporation of phosphate into histone was
quantified, and the results for
,
and
PKC activity are
shown in Fig. 2B. The temporal
pattern of activation differed among these three isozymes, suggesting a
different role for each isozyme in cell attachment and spreading.
PKC
was activated upon cell attachment to FN and reached a maximum activation
after 15 minutes, followed by a rapid decline. We found that
PKC was
activated during the first 15 minutes of plating, although the magnitude of
the increase was less than that of
and
PKC.
PKC
activation declined back to baseline levels over the next 15-30 minutes. By
contrast,
PKC was highly activated as early as 2.5 minutes after plating
and remained activated for at least 30 minutes. To determine the importance of
the
5 integrin signaling pathway in PKC isozyme activation, we repeated
this experiment using
5-deficient cells, which were described
previously to fail to spread on FN
(Disatnik and Rando, 1999
).
There was no activation of any of these isozymes when
5-deficient cells
were plated on FN (Fig. 2C),
providing further evidence that PKC activation is a downstream effector
pathway for integrin signaling.
Increasingly, immunoprecipitation-based kinase assays are being used to
evaluate the activity of individual PKC isozymes
(Zang et al., 1997;
Reyland et al., 1999
;
Bahr et al., 2000
). However, to
exclude the possibility that an unknown kinase that coimmunoprecipitates with
PKC was responsible for histone phosphorylation, and also to show that
immunoprecipitated PKC remained active, we performed a similar assay when
PKC was immunoprecipitated from untreated myoblasts and from myoblasts
treated with 100 nM PMA for 5 minutes. We compared the ability of the
immunoprecipitated material to phosphorylate histone and MBP in the presence
and absence of chelerythrine, a specific inhibitor of PKC
(Herbert et al., 1990
).
Fig. 3 shows that chelerythrine
blocked the phosphorylation of these substrates, demonstrating that the kinase
remains active following immunoprecipitation and that the activity is indeed
caused by immunoprecipitated PKC.
|
To complement the biochemical studies of PKC isozyme activation
(Fig. 2), we compared the
subcellular localization of ,
and
PKC by
immunocytochemistry in myoblasts plated on FN over time
(Fig. 4). After the cells had
been plated on FN for 15 minutes, we were able to assess the localization of
PKC isozymes. Prior to this time point, the rounded morphology of most cells
prevented any reliable assessment of isozyme localization by microscopic
examination. Since, on the basis of the data from the kinase assay, we knew
that PKC activation returns to basal levels by 1 hour after plating, we
assessed cells for PKC isozyme localization between 15 minutes and 1 hour of
plating on FN to correlate cellular localization with biochemical activation.
Fig. 4 shows the differential
localization of
,
and
PKC at 15 minutes and 1 hour. There
was little difference in the localization of any of the isozymes between 15
and 30 minutes after plating, suggesting that most of the cellular
translocation occurred between 30 and 60 minutes after plating. We found that
PKC was localized at focal adhesion sites 15 minutes after plating
cells on FN whereas, at later time points,
PKC was distributed more
diffusely in the cytosol (Fig.
4).
PKC was found predominantly in a perinuclear,
Golgi-like distribution after 15 minutes. After 1 hour on FN,
PKC was
found in the cytosol in a punctate staining pattern
(Fig. 4).
PKC was
initially localized to the nucleus and to perinuclear regions. At 1 hour,
PKC was found diffusely in the cytosol as well as in the nucleus
(Fig. 4). Therefore, these
three different isozymes translocate to distinct locations in the cell after
integrin activation, which may further indicate distinct roles for these PKC
isozymes in cell attachment and spreading.
|
We previously demonstrated that activation of PKC was necessary for
integrin-mediated cell spreading and FAK phosphorylation in muscle cells
plated on FN and that PKC activation promoted 5-deficient cell
spreading on FN, indicating that PKC signaling is a downstream effector
pathway for integrin signaling (Disatnik
and Rando, 1999
). To test which individual PKC isozyme, when
activated, is sufficient to promote spreading and FAK phosphorylation in
5-deficient cells, we used peptide activators (see Materials and
Methods) that have been shown to activate individual PKC isozymes
(Ron and Mochly-Rosen, 1995
;
Souroujon and Mochly-Rosen,
1998
; Dorn et al.,
1999
; Chen et al.,
2001
). These small peptides were conjugated to a cell-permeable
peptide derived from the Antennapedia protein
(Dorn et al., 1999
). It was
previously shown that about 10% of the applied peptide enters into greater
than 95% of the cells (Souroujon and
Mochly-Rosen, 1998
). We observed no toxic effects on the cells
with peptide concentrations up to 1 µM. To test the propensity of
individual PKC isozymes to promote muscle cell attachment and spreading,
5-deficient cells were plated on FN in the presence or absence of the
individual activator peptides. Activation of
PKC with pseudo-RACK1
peptide led to
5-deficient cell spreading on FN nearly as well as that
seen in cells treated with 3 nM PMA, a general PKC activator
(Fig. 5A). Although
pseudo-RACK1 activates all classical PKC isozymes
(Ron and Mochly-Rosen, 1995
),
we could use it as a selective
PKC activator in the cells, since none
of the other classical isozymes are expressed
(Fig. 2A). In response to the
activation of
PKC, approximately 80% of the cells were spread 30
minutes after plating.
PKC activation with pseudo-
RACK
(Chen et al., 2001
) promoted
cell attachment and the formation of distinct lamellipodia in 90% of the cells
within 30 minutes of plating, indicating the beginning of cell spreading. The
activation of
PKC with the
PKC-selective agonist pseudo-
RACK
(Dorn et al., 1999
) promoted
cell attachment very effectively (Fig.
5). Within 30 minutes of plating, 100% of the cells were attached
but revealed a rounded morphology. The subsequent phases of cell spreading
were not as effectively promoted by the activation of
PKC, suggesting
perhaps that activation of
PKC and
PKC may be important for the
progression from attachment to spreading. In the presence of all the
activators together, the process of cell attachment and spreading was
comparable to that seen in the presence of PMA
(Fig. 5A). Fewer rounded cells
(i.e. attached but not spread) were observed under this condition when
compared with the cells plated in presence of individual isozyme activators
alone.
|
Since FAK phosphorylation is such a critical determinant of cell attachment
and spreading (Hanks et al.,
1992; Burridge et al.,
1992
; Disatnik and Rando,
1999
), we induced cell spreading using PKC activators as described
above and assessed FAK phosphorylation by western blot analysis. Indeed, cell
attachment and spreading induced by the PKC activators
(Fig. 5A) correlated with
increases in FAK phosphorylation (Fig.
5B). The increased progression from cell attachment to cell
spreading promoted by activation of
PKC and
PKC as compared with
PKC is reflected in the somewhat greater FAK phosphorylation after 30
minutes of plating in cells treated with the respective activators.
To confirm the specificity of ,
and
PKC activators, we
tested whether these processes could be blocke by isozyme-specific PKC
inhibitors. We tested the ability of isozyme-specific activators to promote
5-deficient cell spreading and FAK phosphorylation in the presence or
absence of their respective isozyme-specific inhibitor peptides,
C2-4
for
PKC (Souroujon and
Mochly-Rosen, 1998
),
V1-1 for
PKC
(Chen et al., 2001
) and
V1-2 for
PKC (Dorn et al.,
1999
). The promotion of cell spreading and phosphorylation of FAK
were nearly completely inhibited when cells were treated with the specific
inhibitor of the isozyme that was being activated
(Fig. 5C). None of the
inhibitors had any effect on cells treated with activators of other
isozymes.
The activation and translocation patterns of each of these isozymes suggest
divergent roles in integrin-mediated muscle cell spreading. The results with
the activators indicate that the activation of each isozyme is sufficient, at
least partially, to promote cell attachment, spreading and FAK
phosphorylation. To test for the necessity of individual isozyme activation in
integrin-mediated muscle cell spreading, we plated 5-expressing cells
on FN in the presence or absence of
,
and
PKC inhibitors
and measured the level of FAK phosphorylation. Similarly, we treated
5-deficient cells with PMA and then plated them on FN in the presence
or absence of
,
and
PKC inhibitors and measured the level
of FAK phosphorylation (Fig.
6).
PKC inhibition by
V1-2 peptide reduced FAK
phosphorylation in both cell populations. In contrast, selective inhibition of
PKC and
PKC did not affect the level of FAK phosphorylation or
cell spreading (Fig. 6).
However, treatment with either inhibitor did result in a delay in the
progression from attachment to spreading (data not shown). Together with the
results on isozyme activation and translocation, these data indicate that
PKC activation is sufficient to promote cell attachment and necessary to
promote cell spreading and FAK phosphorylation in cultured skeletal muscle
cells. Neither
PKC nor
PKC appears to be necessary,
individually, for muscle cell spreading. However, each is capable of promoting
attachment and spreading when activated. These data suggest that the early
activation of
PKC is required and that the later activation of one
additional isozyme may be necessary for the progression from attachment to
spreading, but that there may be redundancy in the effects of
PKC and
PKC activation for this process.
|
Some earlier studies had suggested that the promotion of cell spreading by
PKC activation (Vuori and Ruoslahti,
1993) may be via its effects on the regulation of actin dynamics
and stress fiber formation (Rosen et al.,
1990
). We first examined the reorganization of actin and formation
of stress fibers in muscle cells plated on FN.
Fig. 7 shows that actin is
localized at focal contacts only at the periphery of the cells after 15
minutes on FN. After 1 hour, punctate focal contacts were visible, distributed
more uniformly across the cell/substratum surface rather than just at the
periphery (Fig. 7). This
reorganization of actin is accompanied by the disassembly of stress fibers,
which was also reported after PMA treatment
(Rosen et al., 1990
) and is
consistent with reports of the disassembly, reorganization and reassembly of
the actin network associated with cell attachment and spreading
(Aderem, 1992a
;
Stossel, 1993
;
Defilippi et al., 1999
). We
also examined the dynamics of actin in stress-fiber formation associated with
cell adhesion and spreading. Soon after plating (15 minutes), no stress fiber
formation was evident (Fig. 7).
By 30 minutes, and increasing up to 1 hour, stress fibers were found at the
cell periphery and predominantly around the nucleus. This stress fiber
reorganization parallels the changes in focal contact distribution regulated
by actin cytoskeletal dynamics.
|
Among the many known substrates of PKC, MARCKS is one that is known to play
a critical role in the regulation of actin dynamics
(Aderem, 1992a). MARCKS has
been postulated to be involved in signaling initiated by interactions between
cells and ECM, and MARCKS has been localized at focal contacts in macrophages
(Rosen et al., 1990
). To
determine the localization of MARCKS in muscle cells,
5-expressing
cells were plated on FN for 30 minutes then stained with anti-MARCKS antibody
(Fig. 8A). After 30 minutes on
FN, MARCKS was recruited to cellular structures resembling focal adhesions. To
confirm that MARCKS is indeed at focal adhesion sites in muscle cells upon
spreading, we co-stained cells with antibodies to MARCKS and to paxillin and
found that they localized to the same sites after the cells were plated on FN
for 30 minutes (Fig. 8A).
Fig. 8B shows the localization
of MARCKS in myoblasts plated for various times on FN. Soon after adhesion (15
minutes), MARCKS was found in punctate structures typical of focal adhesion
throughout the cell. After 30 minutes and 1 hour on FN, MARCKS was observed
more diffusely in the cytosol in most of the cells, although localization at
focal contacts was still evident and very predominant at 30 minutes
(Fig. 8A). At all time points
MARCKS staining was also observed in perinuclear region.
|
To determine whether MARCKS activation is indeed downstream of integrin
signaling, we examined MARCKS localization in 5-expressing and
5-deficient cells plated on FN. We observed MARCKS translocation from
the membrane to the cytosol in
5-expressing cells plated on FN as early
as 15 minutes (Fig. 8C). This
translocation was as rapid and complete as that seen when the cells were
treated in suspension with PMA, which is known to cause MARCKS translocation
in other cell types. In
5-deficient cells, which fail to spread on FN
even 3 hours after plating, no translocation of MARCKS was observed over this
time course (Fig. 8C). These
data support the hypothesis that MARCKS translocation is mediated by integrin
signaling.
To assess directly the importance of MARCKS in muscle cell spreading, we
transfected 5-expressing cells with MARCKS cDNA in the antisense
orientation or either with vector alone or MARCKS cDNA in the sense
orientation as a control (both controls showed similar results). Transfected
clones were selected and expanded, although clonal populations in which
spreading was impaired (see below) were much more difficult to expand. Control
transfected clones appeared to be normal in assays of cell spreading and
expressed normal levels of MARCKS protein
(Fig. 9). Clones transfected
with the antisense vector, by contrast, displayed variable capacities to
spread on FN. When these clones were analyzed by western blot analysis, there
was a direct correlation between the reduction of MARCKS protein levels and
the inhibition of cell spreading. Fig.
9A shows three clones with varying levels of MARCKS protein
expression, showing the range of inhibition of protein expression by the
antisense vector. The clones with the highest level of MARCKS expression
(although still reduced compared to controls) showed mild impairment of
spreading, whereas those with the lowest levels of MARCKS expression showed
the most severe impairment of spreading
(Fig. 9B). The clone in which
MARCKS protein was undetectable by western blot analysis (clone 02) displayed
almost no spreading on FN (Fig.
9B). To confirm that cell spreading is mediated by the activation
of MARCKS by PKC, clone 02 was treated with PMA (100 nM) then plated on FN.
PMA treatment did not activate clone 02 spreading on FN
(Fig. 9C), demonstrating that
cell spreading is mediated by MARCKS through PKC activation. These data
suggest that MARCKS is essential for muscle cell spreading and, together with
the data in previous figures, support the model that MARCKS is a key target of
PKC phosphorylation in the regulation of the integrin-mediated process.
|
![]() |
Discussion |
---|
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---|
We previously demonstrated using pharmacological and genetic approaches
that PKC is involved in 5ß1 integrin `outside-in' and `inside-out'
signaling pathways, which lead to cell spreading and cell survival
(Taverna et al., 1998
;
Disatnik and Rando, 1999
). We
showed that integrin engagement leads to FAK phosphorylation via a PKC
signaling pathway and that PKC activation mediates a crosstalk between
5ß1 and
4ß1 integrin that induces muscle cell
spreading on FN (Disatnik and Rando,
1999
). PKC appears to be one of the key intermediates in
integrin-mediated signaling in many cells
(Juliano and Haskill, 1993
;
Clark and Brugge, 1995
), and
several reports have demonstrated that cell spreading is induced by PKC
activation (Haller et al.,
1998
). Previous studies reported that the activation of PKC may
result from the increase in phospholipase C activity induced following
integrin engagement (Cybulsky et al.,
1993
; Plopper et al.,
1995
; Banno et al.,
1996
; Zhang et al.,
1999
). For example, in vascular smooth muscle cells,
diacylglycerol content increases as early as 10 minutes after plating on FN
(Haller et al., 1998
). The
activation of PKC by PMA also promotes cell attachment and spreading on
extracellular matrix proteins (Mercurio
and Shaw, 1988
; Vuori and
Ruoslahti, 1993
; Disatnik and
Rando, 1999
; Miranti et al.,
1999
). PKC activation has been found to precede the morphological
changes that are characteristic of cell spreading
(Woods and Couchman, 1992
;
Vuori and Ruoslahti, 1993
),
suggesting that a target of PKC activity may be important in regulating those
morphological changes.
Our results have focused on the role of individual PKC isozymes in
integrin-mediated muscle cell adhesion and spreading. The specific substrates
of these isozymes are still not known. As focal adhesion formation is
integrally linked to cell spreading
(Kornberg et al., 1992;
Hanks et al., 1992
), the
proteins that constitute these sites are obvious candidates as targets of PKC
activity. Indeed, components of the cytoskeleton as well as focal adhesion
proteins were reported to be regulated by PKC signaling
(Woods and Couchman, 1992
;
De Nichilo and Yamada, 1996
;
Emkey and Kahn, 1997
;
Adams et al., 1999
). The
localization of
PKC at focal adhesion sites that we describe here and
as has been reported by others (Liao and
Jaken, 1993
) may phosphorylate proteins at these sites. The focal
adhesion protein, paxillin, is phosphorylated on serine and threonine and has
been shown to shuttle from focal adhesions to a trans-Golgi-endosomal network,
accompanied by vinculin (Brown et al.,
1998
; Norman et al.,
1998
). Our finding, in this report, of
and
PKC at
the Golgi apparatus and around the nucleus suggests that these isozymes may
likewise be involved in the regulation of focal adhesion proteins.
MARCKS is a PKC substrate that cycles on and off membranes by a mechanism
termed the myristoyl-electrostatic switch
(Aderem, 1992b). It is a
protein known to crosslink actin filaments regulated by PKC and therefore is
important in the stabilization of the cytoskeletal structure
(Hartwig et al., 1992
). MARCKS
has been reported to be involved in cell spreading in several systems
(Rosen et al., 1990
;
Li et al., 1996
;
Manenti et al., 1997
;
Myat et al., 1997
;
Spizz and Blackshear, 2001
).
Myat et al. (Myat et al.,
1997
) demonstrated that MARCKS regulates membrane ruffling and
fibroblast cell spreading. They reported that fibroblast spreading is
inhibited in cells expressing a MARCKS mutant that fails to translocate upon
PKC activation. To support these data, a recent report by Spizz and Blackshear
(Spizz and Blackshear, 2001
)
demonstrated that the localization of MARCKS at the membrane may inhibit
cellular adhesion. Myat et al. (Myat et
al., 1997
) reported direct evidence that PKC-dependent MARCKS
activation regulates actin-dependent membrane ruffling and fibroblast
adhesion. Actin crosslinking increases the viscosity and stiffness of the
actin filament network, stabilizing the actin-rich cell cortex. The spreading
mechanism of the cells requires that stress fibers are rapidly disassembled
and filopodia and lamellipodia are extended at the leading edge of moving
cells to make contact with the matrix
(Haimovich et al., 1996
). The
rigidity caused by actin polymerization is probably a negative regulator of
cell spreading (Aderem, 1992a
).
Indeed, it is known that PKC increases cell spreading and concomitantly
inhibits stress-fiber formation and causes reorganization of actin filaments.
We found that, in skeletal muscle cells, MARCKS is initially localized to
focal adhesion sites and quickly translocates to the cytosol upon integrin
activation, suggesting that MARCKS activation is an early event in cell
attachment and spreading. Poussard et al.
(Poussard et al., 2001
)
provided evidence of a MARCKS-PKC
complex in skeletal muscle by
chromatography, consistent with our data showing MARCKS and PKC
at
focal adhesion sites. In this report, we demonstrate that MARCKS is essential
for muscle cell spreading. Muscle cells that do not express MARCKS protein
failed to spread on FN. Together, these results indicate that MARCKS is a
component of the integrin pathway, downstream of PKC, that mediates skeletal
muscle cell spreading.
We have previously demonstrated that inhibition of PKC blocks FAK
phosphorylation and muscle cell spreading on FN
(Disatnik and Rando, 1999),
which is comparable to similar responses in other cells
(Woods and Couchman, 1992
;
Haimovich et al., 1996
).
However, the roles of respective PKC isozymes have not been widely studied.
Haller et al. found that inhibition of specific PKC isozymes with
pharmacological agents or antisense oligonucleotides resulted in a significant
decrease in cell adhesion and spreading
(Haller et al., 1998
). In this
report, we show that
,
and
PKC are expressed in cultured
skeletal muscle cells, and we demonstrate that an increase in activity of
these isozymes is detectable after cell plating on FN. Activation of
PKC,
PKC and
PKC following cellular adhesion has been
previously reported (Chun et al.,
1996
; Miranti et al.,
1999
). In C2C12 muscle cells,
,
and
PKC are
expressed, but only
PKC was found to be activated upon cell adhesion to
FN (Adams et al., 1999
).
Activated
PKC has been shown to restore spreading of cells in which
integrin signaling had been disrupted
(Berrier et al., 2000
). Chun et
al. reported that
PKC becomes activated upon cell attachment
(Chun et al., 1996
). These
studies and our present report reveal that
PKC plays an important role
in cell attachment and spreading. The early activation of
PKC in
response to integrin binding (Fig.
2) seems to be a critical early event in integrin signaling that
promotes cell spreading and cell survival. However, it is also clear that
other PKC isozymes are necessary in the signaling cascade, and the later
activation of both
and
PKC
(Fig. 2) as well as their
ability to promote muscle cell spreading
(Fig. 5) suggest that they
have, perhaps, overlapping functions in the downstream signaling cascade.
The specific function of the homologous PKC isozymes is determined by their
subcellular localization (Disatnik et al.,
1994; Ron and Kazanietz,
1999
; Dempsey et al.,
2000
). Upon activation, each PKC isozyme translocates to a
specific site where it is anchored by a specific RACK, a receptor for
activated C kinase (Mochly-Rosen,
1995
). Wrenn and Herman (Wrenn
and Herman, 1995
) have recently demonstrated PKC isozyme
translocation upon integrin occupation. Other investigators have demonstrated
PKC localization at focal adhesion sites and have suggested that this
isozyme may mediate cell spreading by targeting an unknown substrate at this
site (Liao and Jaken, 1993
;
Haller et al., 1998
). Our
results also reveal that
PKC is found at focal adhesion sites at a time
when cells are beginning to spread. Other studies of the cellular localization
of PKC isozymes have suggested an important role in integrin function
(Chun et al., 1996
). In
mammary epithelial cells,
PKC and ß1 integrin were found to
colocalize, and
PKC was shown to regulate the cellular distribution of
ß1 integrin, demonstrating a critical role for
PKC in dynamic
control of integrin function (Ng et al.,
1999
). Liliental and Chang
(Liliental and Chang, 1998
)
recently reported a direct association of RACK1 with the integrin ß
subunit cytoplasmic domain. They showed that the interaction of RACK1 with
integrins in vivo requires activation of PKC, which promotes cell spreading
and integrin-dependent cell adhesion. These results suggest a direct linkage
between integrins and PKC through RACK1 and further implicate PKC in
integrin-mediated cell signaling.
We previously proposed a model suggesting a positive feedback loop of
integrin engagement, signaling and activation in which we showed the role of
PKC (Disatnik and Rando,
1999). The results in this report support the model that integrin
engagement with FN mediates the activation of
PKC, which leads to cell
attachment and spreading, and these processes involve the organization of
actin stress fibers as well as the recruitment of focal adhesion proteins to
focal adhesion sites. We show that cell attachment initiates a low level of
FAK phosphorylation and a transient activation of
PKC and
PKC.
FAK and PKC signaling pathways induce an `inside-out' signaling, creating a
positive feedback loop (Disatnik and
Rando, 1999
). Further activation of integrins promotes an increase
of FAK phosphorylation and finally cell spreading. Here, we demonstrate the
importance of
PKC activation in cell attachment that precedes a
downstream cascade of biochemical changes involving the activation of
and
PKC. Furthermore the data presented here link the upstream
initiators of integrin and PKC signaling with the downstream processes of
focal adhesion formation, stress fiber dynamics and finally cell spreading. We
provide evidence that MARCKS is a key signaling molecule downstream of the PKC
pathway that may mediate these cellular changes. These results add our
understanding of the cellular components involved in the integrin-signaling
cascade, regulating cellular adhesion, attachment and spreading.
![]() |
Acknowledgments |
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