Integrin-mediated interaction with the
extracellular matrix plays a critical role in the function of
osteoclasts, the bone-resorbing cells. This study examines the role of
p130Cas (Crk-associated
substrate (Cas)) in actin organization in osteoclasts. Multinucleated osteoclast-like cells (OCLs) were obtained in a co-culture of murine bone marrow cells and primary osteoblasts. After
plating on culture dishes, OCLs formed a ringlike structure consisting
of F-actin dots at cell periphery (actin ring). The percentage of OCLs
with actin rings and its diameter increased with time and cell
spreading. Tyrosine phosphorylation of a protein (p130) increased with
actin ring formation. Treatment with cytochalasin D disrupted actin
rings and reduced tyrosine phosphorylation of p130. Using specific
antibodies, p130 was identified as Cas. By immunocytochemistry, Cas was
localized to the peripheral regions of OCLs and its distribution
overlapped that of F-actin. In OCLs derived from Src(
/
) mice, in
which osteoclast activity is severely compromised, tyrosine
phosphorylation of Cas was markedly reduced. Moreover, Cas was
diffusely distributed in the cytoplasm and actin ring formation is not
observed. These findings suggest that Src-dependent tyrosine phosphorylation of Cas is involved in the adhesion-induced actin organization associated with osteoclast activation.
 |
INTRODUCTION |
Integrins are a major family of cell surface receptors that play
crucial roles in cell-cell and cell-extracellular matrix (ECM)1 interactions (1).
Integrin/ECM protein interactions participate in a variety of
biological processes including embryonic development, wound healing,
tumor metastases, and immune responses (1, 2). It is now established
that, in addition to mediating cell adhesion, integrins activate
multiple signaling pathways. These include elevation of intracellular
Ca2+, lipid turnover, and tyrosine phosphorylation, leading
to cytoskeletal rearrangement and de novo gene expression
(3). The proteins that are tyrosine-phosphorylated by ECM-integrin
interactions include focal adhesion kinase (FAK) (4), paxillin (5),
tensin (6), and cortactin (7).
Osteoclasts, the bone-resorbing cells, play a critical role in bone
remodeling (8-10). Their adhesion to the bone surface induces the
cytoskeletal reorganization associated with activation. The recognition
of extracellular matrix components is, therefore, an important step in
the initiation of osteoclast function. Several studies have
demonstrated that
v
3 integrins play a
central role in osteoclast adhesion (11-16). Using murine
osteoclast-like multinucleated cells formed in vitro, we
have recently reported that integrin-mediated cell adhesion to ECM
molecules, such as vitronectin, fibronectin or type I collagen, induces
the formation of a ringlike structure of F-actin dots at the cell
periphery (17). This ringlike organization, the actin ring, is formed
by the assembly of podosomes that precedes the formation of the sealing
zone (18-20) and has been considered to be a marker of osteoclast
activation (17, 21, 22). Actually, various inhibitory agents of
osteoclast function disrupt this actin ring (23).
In this study, we examined protein-tyrosine phosphorylation occurring
during the adhesion-induced actin organization in osteoclasts, and
identified p130Cas (Crk-associated
substrate (Cas)) as a molecule that participates in the
signaling cascade of actin ring formation. Cas was originally described
as a major tyrosine-phosphorylated protein in cells transformed by
either v-src (24-26) or v-crk (27-29). The
recent molecular cloning of Cas has shown that Cas contains an
N-terminal SH3 domain, a substrate domain, a proline-rich region, and
several tyrosine residues near the C terminus (30, 31). The SH3 domain of Cas is known to bind to FAK (32, 33), FAK-related nonkinase (33),
and PTP1B (protein-tyrosine phosphatase 1B) (34). The substrate domain,
which has 15 potentially phosphorylated tyrosine residues, binds to
v-Crk (35, 36). The proline-rich region near the C terminus and Tyr-762
provide the binding sites for the SH3 and SH2 domains of Src kinase,
respectively (36). These structural characteristics indicate that Cas
is an adapter molecule, which can transmit cellular signals via
interaction with the SH2 and SH3 domains of various signaling
molecules. It has already been reported that Cas undergoes tyrosine
phosphorylation upon integrin-mediated cell adhesion in fibroblasts (7,
37, 38). Evidence presented here shows that tyrosine phosphorylation of Cas is involved in adhesion-induced actin organization in osteoclasts and is absent in the compromised osteoclasts of Src(
/
) mice.
 |
EXPERIMENTAL PROCEDURES |
Animals--
Heterozygote Src(+/
) mice were obtained from the
Jackson Laboratory (Bar Harbor, ME). A quarter of their littermates are expected to be Src(
/
). Homozygote Src(
/
) mice were
phenotypically distinguished from their Src(+/?) siblings by the lack
of tooth eruption. All animals were cared and housed under conditions
as stated in the Institutional Animal Care and Use Committee
(IACUC) Guide for the Care and Use of Laboratory Animals, and the
studies were reviewed and approved by the Merck Research Laboratories Institutional Animal Care and Use Committee.
Cell Culture--
Murine osteoclast-like multinucleated cells
(OCLs) were obtained from co-cultures of primary osteoblasts and bone
marrow cells from ddY mice in the presence of 10 nM
1
,25-dihydroxyvitamin D3 (Wako Pure Chemical Co., Osaka,
Japan) (crude preparations of OCLs) and purified by 0.001% Pronase
(Calbiochem Co., La Jolla, CA) (purified preparations of OCLs)
(39-41). Src(
/
) and Src(+/?) OCLs were also obtained from
co-cultures of primary osteoblasts derived from normal murine calvaria
and spleen cells from either c-Src-deficient mice or their normal
littermates and purified as described above.
Immunofluorescent Analyses--
Cells were cultured for 2 h
on glass coverslips and fixed for 15 min at room temperature with 4%
paraformaldehyde. Cells were then washed with PBS and treated for 10 min with 0.2% Triton X-100 to permeate cell membranes. After
incubating for 30 min with 5% skim milk to block nonspecific binding,
the cells were incubated for 30 min at 37 °C with mouse anti-Cas
monoclonal antibodies (Transduction Laboratories, Lexington, KY) or
rabbit anti-Cas polyclonal antibodies (Santa Cruz Biotechnology, Santa
Cruz, CA) diluted 1:100 with 5% skim milk. Negative control cells were
incubated with non-immune mouse or rabbit serum (1:100 dilution with
5% skim milk). The cells were washed with PBS and incubated for 30 min
at 37 °C with a second antibody (fluorescein
isothiocyanate-conjugated goat anti-mouse immunoglobulins or
Cy3-conjugated goat anti-rabbit immunoglobulins). For identification of
F-actin, rhodamine-conjugated phalloidin or fluorescein
isothiocyanate-conjugated phalloidin (Molecular Probes, Inc., Eugene,
OR) diluted to 1:100 with 5% skim milk was added to the second
antibody solution.
Double staining for tartrate-resistant acid phosphatase (TRAP), a
marker enzyme for osteoclasts and F-actin, was performed as described
previously (17).
Western Blot Analyses--
Cells on culture plates were washed
twice with ice-cold PBS and lysed with 1× sample buffer for SDS-PAGE.
Samples were denatured by boiling for 5 min and electrophoresed on
7.5% SDS-polyacrylamide gels. Proteins were transferred onto
Immobilon-P (Millipore Co., Bedford, MA), and nonspecific binding sites
on the membrane were blocked by incubating at 4 °C overnight in 2%
bovine serum albumin in Tris-buffered saline containing 0.1% Tween 20 (TBS-T). The membranes were then probed for 2 h with
anti-phosphotyrosine (Tyr(P)) monoclonal antibody (Upstate
Biotechnology Inc., Lake Placid, NY) in 2% bovine serum albumin at a
dilution of 1:1000, washed with TBS-T three times, and incubated for
1 h with horseradish peroxidase-conjugated sheep anti-mouse
immunoglobulins. After washing with TBS-T, the membranes were developed
using enhanced chemiluminescence (ECL, Amersham International plc.,
Amersham Place, United Kingdom).
The blots were stripped for 40 min at 55 °C in 62.5 mM
Tris-HCl (pH 6.7), 2% SDS, and 100 mM 2-mercaptoethanol,
re-equilibrated in TBS-T, blocked, and reprobed separately with
anti-Cas monoclonal antibody at a dilution of 1:500, anti-c-Cbl
polyclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA) at a
dilution of 1:300, or anti-Src (mAb327) monoclonal antibody (Oncogene
Science Inc., Manhasset, NY) at a dilution of 1:1000 in the manner
described above.
Immunoprecipitation--
All procedures were performed at
4 °C. Cells were washed twice with ice-cold PBS, then lysed in cold
lysis buffer containing 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 5 mM EDTA, 1% Nonidet P-40, 10 mg/ml
aprotinin, 2 mM Na3VO4, and 1 mM phenylmethylsulfonyl fluoride. Lysates were clarified by
centrifugation at 12,000 × g for 20 min and precleared
by incubation with protein G- or protein A-Sepharose beads
(Zymed Laboratories Inc.) for 1 h. Proteins were
immunoprecipitated by incubation with anti-Cas, anti-Tyr(P), anti-Src,
or anti-paxillin monoclonal antibody (Transduction Laboratories) for
2 h, followed by addition of protein G- or protein A-Sepharose beads, and incubated for another 1 h. Immunoprecipitates were washed five times with lysis buffer, extracted in 2× SDS sample buffer, then separated using 7.5% SDS-polyacrylamide gels and analyzed
by Western blotting with anti-Tyr(P) antibody, followed by reblotting
with anti-Cas, anti-Src, or anti-paxillin antibody.
 |
RESULTS |
Time Course of Actin Ring Formation in OCLs Induced by Cell
Adhesion--
When crude preparations of OCLs were plated on culture
plates in the presence of 10% fetal bovine serum, OCLs began to form actin rings at the cell periphery within 10 min (Fig.
1, a and b). The
percentage of OCLs with actin rings and the diameter of the rings
increased with time (Fig. 1, c-g), peaking 2 h after cells were plated. At this time, more than 80% of OCLs had formed actin rings, the average diameter of which attained 175 µm (Fig. 1,
h and i). This state of actin ring formation was
maintained at least for another 10 h.

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Fig. 1.
Time course of actin ring formation in
OCLs. Crude preparations of OCLs were placed on culture plates in
the presence of 10% fetal bovine serum. After culture for 10 min
(a and b), 30 min (c), 1 h
(d), 2 h (e), 7 h (f), and
12 h (g), cells were fixed and stained with
rhodamine-conjugated phalloidin (b-g). TRAP staining was
added to identify OCLs (a). Bars = 40 µm.
h, the diameter of the actin rings of the OCLs was measured
after culture for indicated periods. Data are expressed as the
means ± S.E. of 60 rings. i, the percentage of
TRAP-positive OCLs having actin rings relative to the total number of
TRAP-positive OCLs was determined after culturing the cells for the
indicated periods. Data are expressed as the means ± S.D. of four
cultures. 60 OCLs were evaluated in each group.
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|
We next examined whether actin rings of OCLs were affected by the
removal of osteoblasts. One hour after osteoblasts were removed, almost
all purified OCLs had actin rings (Fig.
2). A similar number of OCLs had actin
rings after 3 h (data not shown).

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Fig. 2.
Actin ring formation in purified OCLs.
After crude preparations of OCLs were cultured for 6 h,
osteoblasts were removed with 0.001% Pronase to obtain purified OCLs
as described under "Experimental Procedures." After culture for
another 1 h, purified OCLs were fixed and stained with
rhodamine-conjugated phalloidin (b), and then subjected to
TRAP staining (a). Bars = 100 µm.
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|
Involvement of Protein-tyrosine Phosphorylation in Actin Ring
Formation--
Tyrosine kinases, such as c-Src, were shown to be
involved in osteoclastic bone resorption (42-44). We examined,
therefore, by Western blot analysis the general pattern of tyrosine
phosphorylation during actin ring formation. In crude preparations of
OCLs, tyrosine phosphorylation of several proteins was enhanced in a
time-dependent manner after plating (Fig.
3a, lanes 1-5) for
equal gel loading (Fig. 3b, lanes 1-5). This
time-dependent increase in tyrosine phosphorylation
correlated with adhesion-induced actin ring formation in OCLs (Fig. 1).
To determine which tyrosine-phosphorylated proteins were derived from
OCLs, total cell lysates from purified OCL preparations were examined.
In purified OCLs cultured for 3 and 7 h, we detected four highly
tyrosine-phosphorylated proteins with molecular mass values of around
130, 89, 85, and 74 kDa (p130, p89, p85, p74) (Fig. 3a,
lanes 6 and 7, arrowheads). The
tyrosine phosphorylation of these four proteins was much less
pronounced in the crude OCL preparation kept in suspension (Fig.
3a, lane 1). Moreover, cytochalasin D, an
inhibitor of actin polymerization, also disrupted the actin rings of
OCLs (Fig. 4, a and
b) and markedly reduced tyrosine phosphorylation of p130 in
purified OCLs (Fig. 4, c and d). Tyrosine phosphorylation of other proteins was less affected by cytochalasin D. These results suggest that tyrosine phosphorylation of p130 is closely
associated with actin organization in osteoclasts.

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Fig. 3.
Time course for the general pattern of
tyrosine phosphorylation in OCLs during actin ring formation.
a, crude preparations of OCLs were kept in suspension
(lane 1) or plated on culture dishes (lanes
2-5). After culture for 0 min (lane 1), 30 min
(lane 2), 1 h (lane 3), 2 h (lane
4), and 7 h (lane 5), total cell lysates were
collected. Purified preparations of OCLs were obtained from crude OCL
preparations by removing osteoblastic cells after culturing the cells
for 2 h (lane 6) or 6 h (lane 7). After
culture for another 1 h, total cell lysates were collected from
the purified OCLs. Total cell lysates were separated by 7.5% SDS-PAGE,
transferred onto Immobilon-P, and probed with anti-phosphotyrosine
antibody. The molecular masses of marker proteins are indicated in
kilodaltons on the left. Arrowheads show the
positions of highly tyrosine-phosphorylated proteins. b,
total proteins were stained with Coomassie Brilliant Blue to confirm
equal loading.
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Fig. 4.
Effects of cytochalasin D on actin rings and
general pattern of tyrosine phosphorylation in OCLs. After crude
preparations of OCLs were cultured for 2 h, cells were treated
with (b) or without (a) 5 µM
cytochalasin D for 20 min. Cells were fixed and stained with
rhodamine-conjugated phalloidin. Bars = 40 µm.
c, purified preparations of OCLs were obtained from crude
OCL preparations by removing osteoblastic cells after culture for
2 h. After culture for another 1 h, purified OCLs were
treated with cytochalasin D at a concentration of 0 µM
(lane 1), 0.5 µM (lane 2), or 5 µM (lane 3) for 20 min. Total cell lysates
were separated by 7.5% SDS-PAGE, transferred onto Immobilon-P, and
probed with anti-phosphotyrosine antibody. The molecular masses of
marker proteins are indicated in kilodaltons on the left.
The arrowhead shows the position of a 130 kDa-tyrosine-phosphorylated protein. d, total proteins were
stained with Coomassie Brilliant Blue to confirm equal loading.
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|
Identification of Tyrosine-phosphorylated p130 in Actin Ring
Formation--
To identify the tyrosine-phosphorylated p130, total
cell lysates from purified OCLs were blotted with anti-Tyr(P) antibody (Fig. 5, lanes 1,
2, 5, and 6), then reprobed with
several antibodies containing anti-c-Cbl (Fig. 5, lanes 3 and 4) and anti-Cas (Fig. 5, lanes 7 and
8) antibodies. A band was recognized by anti-c-Cbl antibody
(Fig. 5, arrowhead), but its molecular weight differed from
that of tyrosine-phosphorylated p130. c-Cbl, the product of the
c-cbl proto-oncogene, has been reported to be a
tyrosine-phosphorylated c-Src substrate in OCLs (45). In this study,
c-Cbl was also tyrosine-phosphorylated in c-Cbl immunoprecipitates from
purified OCLs (data not shown), although we could not detect tyrosine
phosphorylation of c-Cbl in total cell lysates. Positive bands were
also recognized during reblotting with anti-FAK and with anti-Src
substrate p120 antibodies, but neither were identical to the
tyrosine-phosphorylated p130 (data not shown). On the other hand, two
bands (Cas A and Cas B) were recognized by anti-Cas antibody, and one
(Cas B) was the same as that of the tyrosine-phosphorylated p130 (Fig.
5, lanes 5-8, arrows). It has been reported
that, in normal fibroblasts, Cas is detected as two bands at 125 and
130 kDa (Cas A and Cas B, respectively) (31). These results suggest
that Cas is a candidate for the 130-kDa tyrosine-phosphorylated
protein. Moreover, in immunoprecipitates with anti-Cas antibody from
total cell lysates of purified OCLs, Cas B was tyrosine-phosphorylated
(Fig. 6, lanes 1-4). Cas B
was also detected in immunoprecipitates with anti-Tyr(P) antibody (Fig.
6, lanes 5 and 6). In addition, Cas B
immunoprecipitated from purified OCLs pretreated with cytochalasin D
was not tyrosine-phosphorylated (Fig. 6, lanes 7-10). Taken
together, these findings indicate that the p130, which is
tyrosine-phosphorylated during adhesion-induced actin rearrangement in
OCLs, is indeed Cas.

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Fig. 5.
Candidates for the p130 protein that is
tyrosine-phosphorylated during actin ring formation in OCLs.
Purified OCL preparations were obtained from crude OCL preparations by
removing osteoblasts after culture for 2 h. After culture for
another 1 h, total cell lysates were collected, separated by 7.5%
SDS-PAGE, transferred onto Immobilon-P, and probed with
anti-phosphotyrosine antibody (lanes 1, 2,
5, and 6). The same membrane was stripped and reprobed
with anti-c-Cbl antibody (lanes 3 and 4) or
anti-Cas antibody (lanes 7 and 8). The molecular
masses of marker proteins are indicated in kilodaltons on the
left. The arrowhead and arrows show
the positions of c-Cbl and Cas, respectively.
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Fig. 6.
Identification of p130 as
tyrosine-phosphorylated protein during actin ring formation in
OCLs. Purified OCL preparations were obtained from crude OCL
preparations by removing osteoblastic cells after culture for 2 h.
After culture for another 1 h, purified OCLs were treated with
(lanes 8 and 10) or without (lanes
1-7 and 9) 5 µM cytochalasin D for 20 min. Proteins immunoprecipitated from total cell lysates of purified
OCLs using anti-Cas antibody (lanes 1, 2,
7, and 8) or anti-phosphotyrosine antibody
(lanes 5 and 6) were separated by 7.5% SDS-PAGE,
transferred onto Immobilon-P, and probed with anti-Cas antibody
(lanes 5 and 6) or anti-phosphotyrosine antibody
(lanes 1, 2, 7, and 8). The
same membrane was stripped and reprobed with anti-Cas antibody
(lanes 3, 4, 9, and 10).
The molecular masses of marker proteins are indicated in kilodaltons on
the left. CD, IP, IB, and
Ig stand for cytochalasin D, immunoprecipitation,
immunoblotting, and immunoglobulins, respectively.
Arrowheads show the position of Cas.
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|
Intracellular Localization of Cas in OCLs--
By
immunohistochemistry, Cas was localized at perinuclear and peripheral
regions in OCLs (Fig. 7a), the
later distribution of which overlaps exactly with that of F-actin at
the cell periphery (Fig. 7, b and c). When OCLs
were treated with cytochalasin D at 5 µM, Cas was
re-distributed throughout the cytoplasm (Fig. 7d). No
immunolabeling was detected, with a nonspecific immunoglobulins used as
the first antibody (Fig. 7e). These findings suggest that Cas may play a role in actin ring formation or maintenance in OCLs.

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Fig. 7.
Intracellular localization of Cas in
OCLs. After crude OCL preparations were cultured for 2 h,
cells were treated with (d) or without (a-c and
e) 5 µM cytochalasin D for 20 min and fixed.
a, cells were stained with anti-Cas monoclonal antibody. In
OCLs (arrow), Cas is present in the peripheral and
perinuclear regions. b, the same field as in a,
double-stained with rhodamine-conjugated phalloidin to visualize
F-actin. c, the overlaid image of a and
b. Note that Cas (green) and F-actin
(red) overlap in the peripheral region, appearing as yellow
structures. d, cells treated with cytochalasin D were
stained with anti-Cas monoclonal antibody. Note that Cas is distributed
throughout the cytoplasm in an OCL treated with cytochalasin D
(arrow). e, when non-immune mouse serum was used
as the first antibody, no specific immunolabeling was detected. In
b, d, and e, the position of the
nuclei is indicated by DAPI staining. Bars = 20 µm.
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Tyrosine Phosphorylation of Cas and Actin Ring Formation in
Src(
/
) OCLs--
Recently, several lines of evidence have shown
that c-Src is involved in Cas tyrosine phosphorylation in the
integrin-mediated signaling pathway (46-49). We examined, therefore,
the relationship of c-Src and Cas to actin ring formation in
osteoclasts. For this purpose, we prepared Src(
/
) OCLs using the
co-culture of Src(
/
) spleen cells and normal primary osteoblasts.
In Src(
/
) OCLs, tyrosine phosphorylation of Cas was markedly
reduced (Fig. 8b, top
panel). The expression of Cas protein in Src(
/
) OCLs was actually elevated, as shown by Western blotting (Fig. 8, a,
and b, top panel), but the mechanism for this
change is not known. A similar phenomenon was reported in Src(
/
)
fibroblasts (50). Importantly, the reduced phosphorylation of Cas in
Src(
/
) OCLs appears to be protein specific, because the tyrosine
phosphorylation of paxillin was not significantly different between
Src(+/?) and Src(
/
) OCLs (Fig. 8b, middle
panel). Moreover, in Src(
/
) OCLs, Cas was diffusely
distributed in the cytoplasm and actin rings were not formed (Fig.
9, b and d-f).
Whereas 83.1% of Src(+/?) OCLs (167 out of 201) formed actin rings,
none of Src(
/
) OCLs (0 out of 203) did. Instead of actin rings,
small focal adhesion contacts were formed around the cell periphery and
underneath the nuclei (Fig. 9d). The number of TRAP-positive
OCLs formed was not significantly different between Src(
/
) and
Src(+/?) culture (data not shown). These findings suggest that
Src-dependent tyrosine phosphorylation of Cas is involved
in actin ring formation and in the localization of Cas in actin
rings.

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Fig. 8.
Tyrosine phosphorylation of Cas in Src( / )
OCLs. Src( / ) and Src(+/?) OCLs were obtained from co-cultures
and purified as described under "Experimental Procedures."
a, expression of Cas in Src(+/?) and Src( / ) OCLs. Total
cell lysates were separated by 12% SDS-PAGE, transferred onto
Immobilon-P, and probed with both anti-Cas and anti-Src antibodies. The
molecular masses of marker proteins are indicated in kilodaltons on the
left. b, tyrosine phosphorylation of Cas in
Src( / ) OCLs. Total cell lysates from purified Src( / ) and
Src(+/?) OCLs were immunoprecipitated with anti-Cas, anti-paxillin, and
anti-Src antibodies, separated on 12% SDS-PAGE, transferred onto
Immobilon-P, and probed with anti-phosphotyrosine antibody (left
panels). The same membranes were stripped and reprobed with
anti-Cas (right upper panel), anti-paxillin (right
middle panel), and anti-Src (right lower panel)
antibodies.
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Fig. 9.
Localization of F-Actin and Cas in Src( / )
OCLs. a and b, Src(+/?) (a) and
Src( / ) OCLs (b) were stained with rhodamine-conjugated
phalloidin. Note that Src( / ) OCLs do not form actin rings
(b, arrow), whereas Src(+/?) OCLs form actin
rings (a, arrows). c, the same field
as in b, double-stained for TRAP, a marker enzyme for
osteoclasts to identify osteoclasts (arrow). d-f,
Src( / ) OCLs were double-stained with fluorescein
isothiocyanate-conjugated phalloidin to visualize F-actin
(d) and with anti-Cas polyclonal antibody (e).
f, overlaid image of d and e.
Bars = 10 µm.
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|
 |
DISCUSSION |
We have reported previously that actin ring formation in
osteoclasts is dependent on the interaction of integrins and matrix proteins (17). In this study, we examined the involvement of protein-tyrosine phosphorylation in the formation of actin rings in
osteoclasts. The findings clearly show that tyrosine phosphorylation of
Cas, a novel adaptor molecule, is involved in adhesion-induced actin
organization. Cas was originally identified as a highly tyrosine-phosphorylated protein during cellular transformation by v-Src
(24-26) or v-Crk (27-29), and was shown to form stable complexes with
these oncoproteins. Recent molecular cloning of Cas identified it as a
novel SH3-containing signaling molecule with a cluster of multiple
putative SH2-binding motifs (31). Moreover, several studies have
reported that tyrosine phosphorylation of Cas is induced by cell
adhesion (7, 37, 38), and that Cas is localized to focal adhesions (33,
38).
As shown in this study, Cas is also tyrosine-phosphorylated in OCLs
after cell adhesion, and co-localizes with F-actin at the cell
periphery as a ringlike structure, "the actin ring," Because the
actin ring is considered to be an assembly of podosomes (19-21), our
results suggest that tyrosine phosphorylation of Cas plays a role in
podosome formation. Moreover, treatment of OCLs with cytochalasin D
disturbed the intracellular localization of Cas and reduced Cas
tyrosine phosphorylation (Figs. 6 and 7). Possibly, cytosolic
protein-tyrosine phosphatases (PTP), such as PTP-1B (34) and PTP-PEST
(51), may play a role in the dephosphorylation of Cas that is
translocated to the cytoplasm by treatment with cytochalasin D. We also
reported that the disruption of cytoskeletal organization by
cytochalasin D treatment induced the inhibition of osteoclast function
(52). These findings suggest the close relationship between Cas
phosphorylation and actin organization, which is related to osteoclast
activity.
In normal fibroblastic cells from rats (3Y1) or mice (NIH3T3), Cas has
been detected as two bands, Cas A (125 kDa) and Cas B (130 kDa),
respectively (31). On the other hand, when cells are transformed by
v-Crk or v-Src, Cas A is decreased and another broad band of Cas C
(130-135 kDa) appears. Because tyrosine phosphorylation is found
mostly in Cas C, Cas C may be a modified form of Cas A or Cas B with a
retarded gel mobility, secondary to phosphorylation at multiple sites
(31). In OCLs, however, Cas C was not detected, and the
tyrosine-phosphorylated Cas was identified as Cas B (Fig. 6). This
might be a result of the fact that osteoclasts are non-transformed cells.
The next question is: which tyrosine kinase(s) phosphorylates Cas?
Focal adhesion kinase (FAK) is one of the candidates, as it is
phosphorylated upon adhesion with similar kinetics to those of Cas (7,
37, 38). Moreover, recent reports indicate that FAK can bind to the SH3
domain of Cas in vivo (32, 33). On the other hand, the
C-terminal portion of Cas can also bind directly the SH2 and SH3
domains of Src kinase (36). In addition, two lines of evidence have
demonstrated that the deficiency of c-Src, but not of FAK, completely
abrogated integrin-mediated Cas phosphorylation in fibroblasts (47,
48). These results suggest that tyrosine phosphorylation of Cas by
integrin engagement is mediated by Src family kinases, especially
c-Src, and FAK itself might not be necessary for Cas phosphorylation.
This is supported by the findings of this study, which show that
tyrosine phosphorylation of Cas was markedly reduced in Src(
/
)
OCLs, whereas the expression of Cas was not suppressed (Fig. 8).
Moreover, actin rings did not form in Src(
/
) OCLs and Cas was
localized throughout the cytoplasm (Fig. 9), suggesting that, in
osteoclasts, c-Src plays an important role in Cas phosphorylation and
in its localization. Considering that Cas phosphorylation is tightly
associated with actin ring formation, Src may participate in the
control of actin organization in osteoclasts via tyrosine
phosphorylation of Cas. In Src(
/
) mice, osteoclast activity is
severely compromised, resulting in osteopetrosis (42, 43) and, as shown
here, Src(
/
) OCLs do not form actin rings. Thus, the lack of
Src-dependent Cas phosphorylation may be one of the causes
for osteoclast inactivation in Src(
/
) mice. To prove this
hypothesis, rescue experiments of Src(
/
) mice using Cas or Cas
related molecules are required, and are now in progress.
Recently, Nakamoto et al. (53) reported that the association
of Cas not only with Src kinase but also with FAK family kinases plays
a pivotal role in the localization of Cas to focal adhesions in
fibroblasts. FAK, which can bind to both Cas and Src family kinases,
might recruit Src family kinases to phosphorylated Cas. Considering
that Cas has multiple SH2-binding sites, a SH3 region, and a
proline-rich region, Cas is likely to associate with various molecules
such as Src family kinases, FAK family kinases, paxillin, tensin, and
c-Crk, and thus might play a central role in podosome formation in
osteoclasts. Further studies are required to determine the hierarchy
among these molecules in the osteoclast polarization process. In
conclusion, Src-dependent tyrosine phosphorylation of Cas
appears essential for the signal transduction initiated by cell
adhesion, leading to the formation of actin organization in
osteoclasts.
We are grateful to Drs. Yasuhisa Fukui and
Sayoko Ihara (University of Tokyo) for their help in Western blot
analyses and to Lorraine Lipfert (Merck Research Laboratories) for
critical reading and fruitful discussion.