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INTRODUCTION |
Adhesion of cells to the extracellular matrix
(ECM)1 initiates signaling
pathways that lead to cellular spreading and migration as well as to
modulation of growth and differentiation. Integrins belong to a major
class of adhesion receptors that mediate these cellular functions.
Integrin engagement induces a cascade of tyrosine phosphorylation and
the recruitment of structural and signaling molecules to multimeric
complexes associated with the actin cytoskeleton, called focal adhesion
contacts (1, 2).
Proline-rich tyrosine kinase 2 (PYK2, also known as RAFTK and CAK
)
(3-5) is related to FAK, known to play an important role in cell
adhesion (6). Similar to FAK, PYK2 lacks a transmembrane region and SH2
and SH3 domains but has two proline-rich regions in its C terminus.
PYK2 is highly expressed in brain and various hematopoietic cells (3).
In PC12 cells, PYK2 tyrosine phosphorylation and activation are
stimulated by neuronal stimuli and stress signals, leading to
modulation of a potassium channel and activation of the JNK signaling
pathway (4, 7, 8). In addition, stimulation of G-protein-coupled
receptors induces tyrosine phosphorylation of PYK2 and complex
formation between PYK2 and Src via the SH2 domain of Src, leading to
activation of the MAP kinase signaling pathway (9). Similar to FAK,
PYK2 is tyrosine-phosphorylated and is activated by adhesion-mediated
signaling in platelets and B cells (10, 11). In addition, PYK2
interacts with and phosphorylates the focal adhesion-associated protein
paxillin in vitro (12). PYK2 is thus suggested to
participate in the transfer of signals from the cell surface to the cytoskeleton.
p130Cas was first identified as a major
tyrosine-phosphorylated protein in v-src and
v-crk transformed cells (13, 14). p130Cas
contains an N-terminal SH3 domain, a substrate domain, a proline-rich region, and a C-terminal domain with multiple tyrosine residues (14).
This unique structure suggested that p130Cas may serve as a
docking protein for multiple SH2 and SH3 domain-containing molecules.
In addition, p130Cas is tyrosine-phosphorylated during
integrin-mediated adhesion and localizes to focal adhesions in
fibroblasts (15-17). p130Cas was shown to bind to FAK both
in vitro and in vivo (15, 18). Integrin-dependent p130Cas phosphorylation was
absent in c-Src deficient fibroblasts (19). Recently, PYK2 was shown to
be tyrosine phosphorylated and associated with p130Cas upon
B cell adhesion to fibronectin and stimulation of the antigen receptor
(10).
Osteoclasts are highly differentiated bone resorbing cells. Osteoclast
activation is initiated by adhesion to bone matrix and formation of the
sealing zone, a specialized adhesion structure responsible for the
tight attachment of osteoclasts to mineralized bone matrix (20, 21). We
found that PYK2 is highly expressed in osteoclasts and is
tyrosine-phosphorylated upon integrin-mediated adhesion and is
activated by osteoblastic MB1.8 cells, which are essential for
osteoclast function in vitro (22, 23). Furthermore, PYK2
localizes to podosomes, the primary adhesion structures in osteoclasts
which become the sealing zone in actively resorbing osteoclasts (22).
We also showed that tyrosine phosphorylation of p130Cas is
involved in the organization of the podosome-rich ring structures in
osteoclasts (24). Tyrosine phosphorylation of p130Cas was
markedly reduced in osteoclasts derived from Src(
/
) mice, in which
osteoclast activity is severely compromised (24).
In this study, we demonstrate that p130Cas participates in
adhesion-mediated signaling in osteoclasts. p130Cas is
tyrosine-phosphorylated upon
3-integrin engagement by
ligand binding or antibody-induced clustering. In osteoclasts,
p130Cas is stably associated with PYK2, via the SH3 domain
of p130Cas and the C-terminal domain of PYK2, independent
of tyrosine phosphorylation. Furthermore, p130Cas
co-localizes with PYK2 in the sealing zone of resorbing osteoclasts on
bone. These findings suggest that the engagement of
3-integrin initiates the activation of the
p130Cas·PYK2 complex which plays a role in osteoclast activation.
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EXPERIMENTAL PROCEDURES |
Reagents--
Fibronectin was from NY Blood Center (New York,
NY); vitronectin and laminin were from Life Technologies, Inc.; and
collagen was from Collaborative Biomedical Products (Bedford, MA).
Anti-PYK2 and anti-p130Cas polyclonal antibodies were
developed as described (15, 22). Anti-N-domain of PYK2 antibodies was
from Santa Cruz Biotechnology (Santa Cruz, CA). Antibodies to
p130Cas (mAb 21), paxillin (mAb 349), and PYK2 (mAb 11)
were from Transduction Labs (Lexington, KY). Anti-vinculin (mAb
VIN-11-5) was from Sigma. Anti-
2 (mAb 18/2) was from
ATCC (Manassas, VA), and anti-
1 (mAb 9EG7) and
anti-
3 (mAb 2C9.G2) integrins were from PharMingen (San
Diego, CA). Horseradish peroxidase-conjugated anti-phosphotyrosine antibodies were from Transduction Labs and Upstate Biotechnology. Other
conjugated secondary antibodies were from Jackson Labs (West Grove,
PA), Amersham Pharmacia Biotech, Organon Teknika (Durham, NC), and
Sigma. Collagenase was from Wako Chemicals U.S.A., Inc. (Dallas, TX),
and dispase was from Boehringer-Mannheim. 1
,25-Dihydroxy-vitamin D3 (1
,25(OH)2D3) was a gift from
Dr. M. Uskokovic, Hoffmann-LaRoche (Nutley, NJ). GST fusion proteins of
Fyn, Lyn, and PI3-kinase were from PharMingen and Santa Cruz
Biotechnology. GST-fusion proteins of c-Src, PYK2, and
p130Cas were generated as described (15, 22).
Animals--
Heterozygote Src(+/
) mice were obtained from
Jackson Laboratory (Bar Harbor, ME), and Src(
/
) mice were
phenotypically distinguished from their Src(+/?) siblings by lack of
tooth eruption. All animals were cared for according to Institutional
Animal Care and Use Committee (IACUC) Guide.
Cell Cultures--
Prefusion osteoclasts (pOCs) were prepared as
described previously (22, 23). Briefly, mouse bone marrow cells were
co-cultured with osteoblastic MB1.8 cells for 5-6 days in the presence
of 10 nM 1
,25(OH)2D3. pOCs were
released from dishes with 10 mM EDTA after removing MB1.8
cells with collagenase-dispase. Alternatively, the co-cultures were
kept for 7-8 days to achieve multinucleated osteoclast-like cells
(OCLs) and purified as described (22). Src(
/
) and Src(+/?) OCLs
were obtained from co-cultures of MB1.8 cells and spleen cells isolated
from 2-3 wk old Src(
/
) or their normal littermates as described above.
Cell Adhesion--
After isolation, pOCs (105
cells/plate) were washed twice with serum-free
-minimal essential
medium containing 0.1% bovine serum albumin (Sigma) and kept in
suspension or allowed to attach to polystyrene dishes, coated with ECM
proteins (fetal bovine serum, fibronectin (25 µg/ml), vitronectin (10 µg/ml), osteopontin (50 µg/ml), laminin (25 µg/ml), type I or
type IV collagen (25 µg/ml)). After 5-60 min at 37 °C, an equal
volume of 2× TNE lysis buffer (20 mM Tris, pH 7.8, 300 mM NaCl, 2 mM EDTA, 2% Nonidet P-40, 2 mM NaVO3, 20 mM NaF, 20 µg/ml
leupeptin, 1 trypsin inhibitory units/ml aprotinin and 2 mM
phenylmethylsulfonyl fluoride) was added to the plates. Clarified
lysates were subjected to immunoprecipitation and -blotting.
Alternatively, pOCs were allowed to attach to vitronectin-coated plates
in serum-free medium for the indicated times and then fixed and stained
for TRAP as described (24). Numbers and area of attached pOCs were
measured using the Empire Imaging Analyzing Systems (Milford, NJ).
Results are expressed as the means (± S.E.) of four fields for the
number of attached cells and of more than 300 cells for the area of pOCs.
Immunoblotting and Immunoprecipitation--
Lysates were
separated on a 4-20% gradient or 8% SDS-PAGE (Novex, San Diego, CA),
and electrotransferred to Immobilon-P membrane (Millipore, Bedford, MA)
overnight. After blocking with 100 mM NaCl, 10 mM Tris, 0.1% Tween 20, 1% bovine serum albumin, the membrane was incubated with primary antibodies, followed by horseradish peroxidase-conjugated secondary antibodies, and detected with the ECL
chemiluminescence system (Amersham Pharmacia Biotech). For
immunoprecipitation, the lysates were precleared with Sepharose-4B and
precipitated with anti-PYK2 polyclonal antibodies or
anti-p130Cas monoclonal antibody for 1 h at 4 °C
followed by protein G-Sepharose for 1 h at 4 °C, respectively.
Immunoprecipitated proteins were washed with lysis buffer (5×),
followed by SDS-PAGE, blotted, and stained as described above.
Integrin Clustering--
Antibody-induced clustering in pOCs was
performed as reported (25). Cell suspensions (1 × 106
cells/ml) were incubated with various anti-
subunit antibodies at 25 µg/ml, 4 °C for 30 min, washed twice with ice-cold serum-free medium containing 100 µM Na3VO4,
and incubated with 50 µg/ml goat F(ab')2 anti-rat IgG or
anti-hamster IgG at 37 °C for 1 h. Cells were lysed in modified
RIPA buffer (50 mM Tris-HCl, pH 7.5, 150 mM
NaCl, 1% Nonidet P-40, 0.2% sodium deoxycholate, 1 mM
EDTA, 50 mM NaF, 1 mM
Na3VO4, and proteinase inhibitors) and
subjected to immunoprecipitation and blotting as described above.
In Vitro Protein Association Assays--
These experiments were
performed with GST-fusion proteins containing the SH3 domains of Fyn,
Lyn, Src, PI3-kinase, and p130Cas or the kinase and N- and
C-terminal domains of PYK2. OCLs lysates (1 mg/ml) were incubated with
GST-fusion protein coupled with glutathione-Sepharose beads for 2 h at 4 °C. The beads were washed (3×) with lysis buffer and with
phosphate-buffered saline (1×), and precipitated proteins were
separated by SDS-PAGE and subjected to immunoblot analysis using
anti-PYK2 or anti-p130Cas antibody.
Immunofluorescence--
pOCs were seeded on glass coverslips or
on bone slices together with
1
,25(OH)2D3-treated MB1.8 cells. At the
indicated times, cells were fixed with 4% paraformaldehyde and
permeabilized with 0.5% Triton X-100 in phosphate-buffered saline.
Cells were stained using polyclonal anti-PYK2, poly- or monoclonal
anti-p130Cas, anti-vinculin and anti-paxillin antibodies,
followed with the appropriate secondary antibodies, or with FITC- or
rhodamine-conjugated phalloidin. Immunofluorescent stainings were
viewed with a fluorescence microscope or with a confocal laser scanning
microscope (Leica, Heidelberg, Germany), equipped with a multiline
Omnichrome argon-crypton laser (Chino, California) (26).
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RESULTS |
Adhesion-dependent Tyrosine Phosphorylation of
p130Cas--
Expression of p130Cas and PYK2 in
osteoclasts was examined using Western blot analysis. Both pOCs and
multinucleated OCLs express significant levels of p130Cas
and PYK2 similar to that of IC-21 macrophages (Fig.
1A). We have previously found
that the osteoblastic MB1.8 cells do not express detectable levels of
PYK2 (22).

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Fig. 1.
Expression of p130Cas and PYK2 in
osteoclasts and tyrosine phosphorylation of p130Cas in pOCs
on various matrices. A, Western blot analysis for
p130Cas (top panel) and PYK2 (bottom
panel) in cell lysates (6 µg) from IC21 macrophages, isolated
prefusion osteoclasts (pOC), and multinucleated
osteoclast-like cells (OCL) was carried out as described
under "Experimental Procedures." B, pOCs were allowed to
attach to plates coated with serum, vitronectin (VN),
osteopontin (OPN), fibronectin (FN), type I
collagen (Col I), type IV collagen (Col IV), or
laminin (LN) for 1 h. Cell lysates were subjected to
p130Cas immunoprecipitation, followed by blotting for
phosphotyrosine (PY) and p130Cas, as described
under "Experimental Procedures."
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The effect of adhesion on p130Cas tyrosine phosphorylation
was examined in isolated pOCs, which can be reseeded on various ECM proteins in the absence of serum. Attachment to vitronectin,
osteopontin, fibronectin, and serum stimulated p130Cas
tyrosine phosphorylation, whereas lower levels of tyrosine
phosphorylation were detected in pOCs adhering to type I or type IV
collagen, or to laminin (Fig. 1B). Relative to pOCs
maintained in suspension, p130Cas was
tyrosine-phosphorylated in a time-dependent manner upon
attachment to vitronectin. An increase in p130Cas tyrosine
phosphorylation was detected within 5 min of seeding and reached the
maximum around 30 min (Fig.
2A). Interestingly, pOC
attachment to vitronectin-coated plates appears to precede the peak
phosphorylation of p130Cas (Fig. 2B), suggesting
that tyrosine phosphorylation of p130Cas may play a role in
the cytoskeletal organization of osteoclast precursors upon adhesion.
Furthermore, tyrosine phosphorylation parallels to the time course of
osteoclast spreading (Fig. 2C).

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Fig. 2.
Tyrosine phosphorylation of
p130Cas correlates with pOC adhesion and spreading.
A, pOCs were kept in suspension or allowed to attach to
vitronectin-coated plates in the absence of serum at 37 °C for
indicated times, 5-60 min. Cell lysates were subjected to
p130Cas immunoprecipitation, followed by blotting for
phosphotyrosine (PY) and p130Cas, as described
under "Experimental Procedures." B and C, in
the parallel cultures, pOCs were fixed and stained for TRAP at the
indicated times, and the number and area of attached TRAP(+) cells were
quantitated as described under "Experimental Procedures." Data are
expressed as the means ± S.E. of four fields in panel
B and as the means as ± S.E. of more than 300 pOCs in
panel C.
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3-Integrin Clustering Induces Tyrosine
Phosphorylation of p130Cas--
Matrix preferences for
inducing p130Cas phosphorylation suggested a selective
integrin-mediated phenomenon. Osteoclasts express
v
3-,
v
1-,
and
2
1-integrins (26-29). Immature
osteoclast precursors were shown to express
2- and
5-integrins (30, 31). Integrin involvement and
specificity for p130Cas tyrosine phosphorylation was
examined by antibody-mediated clustering of integrins in pOCs in
suspension. Clustering with anti-
3-antibody strongly
induced p130Cas tyrosine phosphorylation, whereas
clustering with anti-
1- or anti-
2-antibody induced low levels of
p130Cas phosphorylation in pOCs (Fig.
3), indicating that the vitronectin receptor
v
3 is the major integrin
responsible for this effect. Clustering of
5-integrin
was not examined in this study because antibodies that recognize the
extracellular domain of the murine
5-integrin are not
available.

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Fig. 3.
Clustering of
3-integrins induces tyrosine
phosphorylation of p130Cas in pOCs. pOCs were
incubated with antibodies against 1-, 2-,
or 3-integrin subunits (1 × 106
cells/ml) and subsequently with secondary antibodies to induce integrin
clustering. The control cells (Susp.) were incubated with
the secondary antibodies only. Cell lysates were subjected to
p130Cas immunoprecipitation and blotting for
phosphotyrosine (PY) and p130Cas.
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Localization of p130Cas in Podosomes and in the Sealing
Zone of Osteoclasts and Co-localization with PYK2--
Because
3-mediated adhesion is thought to be involved in
osteoclast activation, the subcellular localization of
p130Cas was examined in OCLs plated on glass coverslips and
on bone. On glass coverslips, p130Cas was localized in
podosomes (Fig. 4) and, along with
F-actin, organized in a typical podosomal-rich adhesion structure at
the periphery of OCLs (24). Double staining of p130Cas and
PYK2 in OCLs on glass showed that p130Cas also co-localized
with PYK2 (Fig. 4). On mineralized bone matrix, osteoclasts polarize
and form the sealing zone that circumscribes the resorptive ruffled
border membrane, where protons and proteases are secreted. Sealing zone
formation can be followed using vinculin immunostaining to visualize
the transition from small rings in podosomes to a double circle
structure in the mature sealing zone (21). Similarly, on bone,
p130Cas localized in OCLs to podosomes and to the nascent
sealing zone, as well as to the double circle structure in the mature
sealing zone (Fig. 5A).
p130Cas also co-localized with the cytoskeletal proteins
F-actin, vinculin, and paxillin (Fig. 6).
The distribution of paxillin (Fig. 6h), shown here for the
first time in osteoclasts on bone, follows the staining pattern of
vinculin. However, in resorbing OCLs, p130Cas also
co-localized with PYK2 in the nascent sealing zone, as well as in the
mature sealing zone (Fig. 5B). This morphological evidence for the co-localization of p130Cas and PYK2 in osteoclasts
in vivo supports a role for p130Cas in the
morphological changes associated with osteoclast activity.

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Fig. 4.
Localization of p130Cas in
podosomes and co-localization with PYK2 in OCLs on glass. pOCs
were cultured with MB1.8 cells on glass coverslips for 48 h in the
presence of 1,25(OH)2D3, leading to fusion and
formation of multinucleated OCLs. After formation of OCLs, MB1.8 cells
were removed by collagenase/dispase, and OCLs were double-stained with
monoclonal anti-p130Cas (a and d) and
polyclonal anti-PYK2 antibody (b and e).
Co-localization is seen as yellow in overlaid images
(c and f). Note the staining in the peripheral
areas of two osteoclasts side by side (d-f).
Bars, 25 µm (a-c), 10 µm
(d-f).
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Fig. 5.
Characteristic patterns of
p130Cas localization in OCLs on bone, and its
co-localization with PYK2. pOCs were cultured with MB1.8 cells on
bone in the presence of 1,25(OH)2D3 to obtain
resorbing OCLs. A, cells stained with monoclonal
anti-p130Cas antibody, showing the characteristic patterns
of p130Cas localization in OCLs in confocal microscopic
optical sections during different stages of bone resorption
(a-c). Note the highest concentration of
p130Cas at the edges of the mature sealing zone as a double
circle structure (c). B, pseudocolored confocal
images of double staining of p130Cas (green,
a and d) with PYK2 (red, b
and e) in resorbing OCLs on bone. Co-localization is seen as
yellow in overlaid images (c and f). Localization
of p130Cas and PYK2 in the forming sealing zone
(a-c) and in the mature sealing zone
(d-f). Images merged from optical sections from
1.0 (a-c) and 1.5 (d-f)
µm thickness close to the bone surface. Bars, 10 µm.
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Fig. 6.
Co-localization of p130Cas with
cytoskeletal proteins in OCLs on bone. Pseudocolored confocal
images of double stainings of p130Cas (red,
a, d, and g) with F-actin
(b), vinculin (e), and paxillin (h)
(green) in resorbing OCLs on bone. Colocalization is seen as
yellow in overlaid images (c, f, and
i). Note two osteoclasts in different phases of resorption
cycle in p130Cas and vinculin double staining (d
and e), both showing co-localization (f). Images
merged from optical sections from 2.6 (a-c), 4.0 (d-f), and 1.8 (g-i) µm
thickness close to the bone surface. Bars, 10 µm.
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Association of p130Cas with PYK2 in
Osteoclasts--
Because p130Cas colocalizes with PYK2 in
the sealing zone of active osteoclasts, we investigated the interaction
of the two proteins in these cells. Immunoprecipitation and
immunoblotting experiments show association of p130Cas with
PYK2 in pOCs in situ (Fig. 7).
Furthermore, this association was present both in suspended and
attached cells (Fig. 7), suggesting that it is independent of tyrosine
phosphorylation.

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Fig. 7.
Stable association of p130Cas
with PYK2 in pOCs. pOCs were kept in suspension (Susp.)
or allowed to adhere to vitronectin for 1 h (Attached),
followed by solubilization in TNE lysis buffer, as described under
"Experimental Procedures." Cell extracts (1 mg/ml) were
immunoprecipitated with anti-N-terminal domain of PYK2 antibodies (2 µg/ml), separated on SDS-PAGE (4-20%), and subjected to
immunoblotting with anti-phosphotyrosine (PY, left
panel) and then with anti-p130Cas and anti-PYK2
(right panel).
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c-Src Is Not Required for the Association of p130Cas
and PYK2--
This was confirmed in Src(
/
) osteoclasts. Src has
been implicated in p130Cas and PYK2 tyrosine
phosphorylation, and osteoclast function is severely compromised in
Src(
/
) mice (9, 32). As observed previously, the level of tyrosine
phosphorylation of PYK2 immunoprecipitated from Src(
/
) OCLs is
markedly reduced, although the same level of PYK2 is expressed in
Src(
/
) and Src(+/?) OCLs (Fig. 8)
(22). Interestingly, a comparable level of p130Cas was
immunoprecipitated with PYK2 from both Src(
/
) and Src(+/?) OCLs
(Fig. 8, middle panel). We previously reported the
expression of both p130Cas isoforms (Cas A and B) in
Src(
/
) OCLs, as compared with the predominant expression of Cas B
in Src(+/?) OCLs. Both isoforms of p130Cas appeared to
associate with PYK2 in Src(
/
) OCL lysates (Fig. 8, middle
panel). Furthermore, treatment of Src(+/?) OCLs with cytochalasin
D for 20 min caused dephosphorylation of PYK2 and dissociation of the
Src and PYK2 complex (Fig. 8, bottom panel) but had no
significant effect on the association of p130Cas with PYK2
(Fig. 8, middle panel). These results indicate that the
association of p130Cas and PYK2 is not dependent on an
intact cytoskeleton or on c-Src function in osteoclasts.

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Fig. 8.
Association of p130cas
and PYK2 in Src( / ) OCLs. Src( / ) and Src(+/?) OCLs
were obtained from co-cultures and purified as described under
"Experimental Procedures." Total cell lysates from Src( / ) and
Src(+/?) OCLs with or without 5 µM cytochalasin D
(CD) for 20 min were immunoprecipitated with anti-PYK2
antibody, separated on SDS-PAGE (8%), and subjected for immunoblotting
with anti-phosphotyrosine (PY, upper left panel)
and then with anti-p130Cas (upper middle panel),
anti-PYK2 (upper right panel), and anti-Src (lower
left panel) antibodies. Two isoforms of p130Cas were
shown as Cas A and Cas B.
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Association of p130Cas and PYK2 Is Mediated by SH3
Domains of p130Cas and the C-terminal Domain of
PYK2--
To characterize the domains that mediate the association of
p130Cas with PYK2, GST-fusion proteins encoding for SH3
domains from various signaling molecules, as well as the kinase and N-
and C-terminal domains of PYK2 were incubated with lysates prepared from OCLs. GST-fusion protein encoding the SH3 domain of
p130Cas binds to PYK2 from OCL lysates, whereas the
GST-fusion proteins encoding the SH3 domains of Src, Lyn, Fyn, or
PI3-kinase or GST alone do not bind to PYK2 in OCL lysates (Fig.
9A). On the other hand, the
GST-fusion protein encoding the C-terminal domain of PYK2, but not the
kinase or N-terminal domains or GST alone, interacts with
p130Cas (Fig. 9B). Lysates from attached OCLs
were used for this experiment, and the p130Cas that
associated with the C-terminal domain of PYK2 was
tyrosine-phosphorylated, suggesting that tyrosine-phosphorylated
p130Cas can associate with PYK2.

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Fig. 9.
Interaction between p130Cas and
PYK2 is mediated by the SH3 domain of p130Cas and the
C-terminal domain of PYK2. A, OCL lysates (1 mg/ml)
were incubated with GST alone or with GST-fusion proteins (10 µg)
containing the SH3 domains of Src, Lyn, Fyn, PI3-kinase, or
p130Cas. B, lysates were incubated with GST or
with GST-fusion proteins (10 µg) containing the N-terminal
(GST-PYK2(N)), the kinase
(GST-PYK2(K)), or the C-terminal
domain (GST-PYK2(C)) of PYK2, followed
by incubation with gluthathione-Sepharose beads for 2 h at
4 °C. After washings, precipitated proteins were separated on
SDS-PAGE and subjected to immunoblotting for PYK2 (A) or for
phosphotyrosine (PY) and p130Cas
(B).
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|
 |
DISCUSSION |
Bone resorbing osteoclasts are highly differentiated cells
specialized in the digestion of mineralized matrix. Osteoclast activation is initiated by recognition of and adhesion to the bone
surface, followed by cellular polarization and formation of the sealing
zone, a specialized membrane structure mediating tight adhesion between
the osteoclast cell membrane and the bone surface. It is well
documented from pharmacological studies that
v
3, the major integrin in osteoclasts, is
important for osteoclast function (27, 33, 34).
v
3 mediates osteoclast adhesion to
several RGD matrix proteins including vitronectin, osteopontin, bone
sialoprotein, and fibronectin (35). Although
v
3 localizes to podosomes, the initial
adhesion structure in osteoclasts (26, 36), the presence of this
integrin in the mature sealing zone in resorbing osteoclasts is
controversial (26, 34, 37, 38). The signaling mechanisms regulating the
adhesion-dependent activation of osteoclasts are not well
understood. We have recently found that PYK2 is the major adhesion
kinase in osteoclasts, which undergoes tyrosine phosphorylation upon
3-integrin engagement and that localizes to the sealing
zone (22).
Similarly, p130Cas tyrosine phosphorylation correlates with
the formation of the podosomal-rich adhesion structures in osteoclasts plated on glass or on plastic culture dishes (24). These findings suggest that both PYK2 and p130Cas may be involved in the
adhesion-dependent signaling that mediates cytoskeletal
organization in osteoclasts. This is further supported by the present
study which shows that both p130Cas and PYK2 are
tyrosine-phosphorylated upon pOC adhesion to the same ECM proteins and
with a similar time course. In addition, p130Cas, similar
to PYK2, is highly tyrosine-phosphorylated in pOCs upon
3-integrin clustering by either ligand engagement or
antibody cross-linking. However, whereas PYK2 appears to be selectively tyrosine-phosphorylated upon clustering of the
3-integrin (22), p130Cas tyrosine
phosphorylation is also induced, albeit to a lower level by other
osteoclast integrins including
1 and
2-integrins.
The adhesion-dependent tyrosine phosphorylation of
p130Cas in osteoclasts is consistent with findings in other
cell types although the kinetics are slightly different. In fibroblasts
(17) and primary chicken embryo cells (15), maximal phosphorylation was seen at 15-20 min, whereas in osteoclasts, optimal phosphorylation is
not reached until after 30 min, paralleling the time course of
osteoclast spreading. The preferences for ECM proteins is also different, osteopontin, vitronectin, and fibronectin in osteoclasts rather than primarily fibronectin in fibroblasts.
3
seems to be the major integrin that mediates these effects in osteoclasts.
We further examined the localization of p130Cas in
osteoclasts and found it to be present in podosomes, where it
co-localizes with PYK2 and cytoskeletal proteins. More importantly, it
colocalizes with PYK2 in the sealing zone. During bone resorption,
osteoclast adhesion to bone matrix leads to reorganization of
cytoskeletal structures and formation of the sealing zone, a tight
attachment between the osteoclast plasma membrane and bone matrix, with
specific organization of F-actin and associated proteins, such as
vinculin and talin (21). Initial formation and accumulation of
podosomes precedes these changes (21). In this study, we localized
p130Cas by confocal microscopy in resorbing osteoclasts on
bone and found it is present in the newly forming sealing zone and
mainly at the edges (the double circle structure) of mature sealing
zones. In these structures, p130Cas colocalizes with
vinculin and paxillin and as mentioned with PYK2. Together with our
recent results on PYK2 (22), these findings strongly suggest that
p130Cas and the p130Cas·PYK2 complex play a
role in the cytoskeletal reorganization and formation of the sealing
zone during osteoclast activation.
Furthermore, co-immunoprecipitation of p130Cas and PYK2 in
osteoclasts shows stable association of the two proteins. This
resembles the association of p130Cas with FAK (15, 18) and
is consistent with recent findings in B cells (10). Using GST-fusion
proteins, this association was found to be mediated by the SH3 domain
of p130Cas and the C-terminal domain of PYK2, similar to
the association of p130Cas with FAK (15). This is in
agreement with recent findings by Ohba et al. (39), showing
that the first proline-rich sequence in the C-terminal domain of PYK2
can bind SH3-domain of p130Cas. Interestingly, SH3 domains
of Src, Fyn, Lyn, or PI3-kinase did not bind to PYK2, suggesting
preferential interaction of the SH3 domain of p130Cas with
PYK2. These results suggest that tyrosine phosphorylation of
p130Cas and PYK2 are not necessary for the association of
these two molecules.
This was further confirmed using osteoclasts derived from Src(
/
)
mice, in which tyrosine phosphorylation of PYK2 (22) and
p130Cas (24) are markedly reduced, but p130Cas
still associates with PYK2. Interestingly, p130Cas
association with FAK was not readily detected in lysates from Src(
/
) fibroblasts. This association was found to be mediated by
the N-terminal fragment of c-Src which includes its SH3 and SH2 domains
(40), suggesting that c-Src serves as an adapter for the
p130Cas-FAK interaction (40). Our results indicate that in
osteoclasts p130Cas and PYK2 associate in the absence of
c-Src but do not localize to the podosomes and ring-like structures
(22, 24). These observations suggest that c-Src may be necessary in
osteoclasts for the integrin-mediated activation of the
p130Cas·PYK2 complex required for its recruitment to the
adhesion structures. Tyrosine phosphorylation of the
p130Cas·PYK2 complex could regulate and possibly enhance
downstream signaling events initiated by
3-integrins
ligation in osteoclasts.
In osteoclasts, c-Src is highly expressed and is essential for
osteoclast function (32). Src-deficient mice have an osteopetrotic phenotype caused by nonfunctional osteoclasts (32). The majority of
c-Src is localized in the osteoclast ruffled border and in vacuoles
rather than in the sealing zone (41, 42), suggesting that the
interaction of c-Src and the p130Cas·PYK2 complex
precedes formation of the actin ring and sealing zone. However, the
direct involvement of c-Src in this process is clearly indicated by our
recent findings showing adhesion-induced association of PYK2 with c-Src
in osteoclasts (22) and PYK2 phosphorylation by c-Src in
vitro. In addition, both PYK2 and c-Src were shown to translocate
to the cytoskeleton upon osteoclast adhesion (22, 43), pointing to Src
interaction with PYK2 in vivo. Furthermore,
integrin-mediated p130Cas tyrosine phosphorylation is
substantially diminished in both Src-deficient fibroblasts and
osteoclasts (19, 24). In addition, c-Src was shown to bind directly to
p130Cas (44), suggesting that p130Cas could be
a substrate of c-Src in vivo. In human B cells, PYK2 phosphorylates p130Cas following
1-mediated
stimulation (45). Taken together, these observations suggest that
cooperation between PYK2 and Src kinases may be required for the
complete phosphorylation of p130Cas, which is probably
involved in downstream signaling.
In summary, the findings presented here show adhesion- and
3-integrin-mediated, Src-dependent tyrosine
phosphorylation of p130Cas in osteoclasts with similar
kinetics as recently shown for PYK2. Furthermore, p130Cas
and PYK2 are stably associated via the SH3 domain of
p130Cas and the proline-rich C-terminal domain of PYK2,
independent of tyrosine phosphorylation or c-Src. Finally,
p130Cas localizes to podosomes and the sealing zone, the
functionally important adhesion structures in osteoclasts, where it
co-localizes with PYK2. Taken together these findings suggest a role
for p130Cas and the p130Cas·PYK2 complex in
the c-Src and adhesion-dependent signaling that leads to
osteoclast activation, cytoskeletal reorganization, and formation of
the sealing zone.