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INTRODUCTION |
PYK21 is highly
expressed in osteoclasts, terminally differentiated bone-resorbing
cells of hematopoietic origin, and participates in the signaling
initiated by osteoclast interaction with bone. Adhesion to bone matrix
induces osteoclast differentiation, cytoskeletal reorganization, and
cellular polarization, leading to formation of unique membrane areas
for active bone resorption. These include the following: (i) the
sealing zone, for tight adhesion to bone matrix; (ii) the ruffled
border, for directional secretion of protons and proteases into the
resorption lacuna; and (iii) functional secretory domain toward bone
marrow space, for transcytosis and release of degraded bone matrix
(1-3).
v
3 integrin is highly expressed
in osteoclasts (4) and was suggested to mediate osteoclast adhesion to
various bone matrix proteins and to regulate cytoskeletal organization
required for migration and formation of the sealing zone (5, 6).
Interference with
v
3 integrin function by blocking antibodies or by RGD-containing peptides or proteins leads to
inhibition of osteoclast migration and of bone resorption in
vitro and in vivo (6-12). In addition, targeted
disruption of
3 integrin in mice induces progressive
osteosclerosis without apparent reduction in osteoclast number (13).
These data suggest that
v
3
integrin-mediated adhesion and the signaling initiated by it may play a
key role in the regulating of osteoclastic bone resorption.
The primary adhesion structures in osteoclasts and monocytes are
podosomes, which consist of F-actin core surrounded by a small circle
or rosette of cytoskeletal and signaling proteins as well as
v
3 integrin (14-17). Dynamic regulation
of podosomes and osteoclast spreading has been shown to require the
non-receptor tyrosine kinases c-Src and the proline-rich tyrosine
kinase 2 (PYK2; also known as CAK
, RAFTK, or CADTK) (18-20).
Src-deficient mice are osteopetrotic due to osteoclast dysfunction,
which can be partially rescued by expression of kinase-defective c-Src
(21, 22). We reported previously (16, 19) that PYK2 localizes to
podosomes as well as to sealing zone in bone-resorbing osteoclasts, and
cells treated with antisense PYK2 are not able to resorb bone in
vitro. Furthermore, engagement of
v
3
integrin by ligands or antibodies in osteoclasts leads to PYK2 tyrosine
phosphorylation in a c-Src-dependent manner (16). The role
of PYK2 phosphorylation and the activity to carry these functions have
not been fully defined.
Similar to the focal adhesion kinase (FAK), PYK2 lacks SH2 and SH3
domains but contains other functional domains, including two
proline-rich regions in its C terminus and several phosphorylated tyrosine residues, which can mediate specific protein-protein interactions (23). Tyrosine 402 in PYK2, analogous to tyrosine 397 in
FAK, has been suggested as the primary autophosphorylation site that
provides a docking site for the SH2 domain of c-Src (23-25).
Association of PYK2 with c-Src leads to further phosphorylation of PYK2
at tyrosines 579 and 580 in the kinase domain activation loop, assumed
to be required for its maximal catalytic activity, and at tyrosine 881 at the C terminus, which mediates the association of PYK2 with Grb2
(25). In addition, proline-rich regions in the PYK2 C terminus mediate
interactions with yet other adaptor molecules, p130Cas and
Graf (17, 23).
The objective of this study was to examine if PYK2 kinase activity and
its ability to associate with c-Src are essential for mediating
osteoclastic cytoskeletal organization. We examined the effects of
overexpression of PYK2 kinase-dead (K457A) mutant and of an
autophosphorylation site (Y402F) mutant on the
adhesion-mediated signaling, cell spreading, and migration. We found
that Tyr-402 phosphorylation, but not its kinase activity, is important
for osteoclast spreading and adhesion-induced association of PYK2 with
c-Src. Furthermore, expression of PYK2(Y402F) in osteoclasts prevented normal bone resorption. Whereas both PYK2(Y402F)
and PYK2(K457A) translocate to podosomes,
PYK2(Y402F) acts in a dominant negative fashion to inhibit
adhesion-dependent signaling in osteoclasts by blocking
binding to c-Src and phosphorylation of additional tyrosines in the
regulatory and C-terminal domains of PYK2. On the other hand, we
observe normal phosphorylation of the kinase-defective PYK2(K457A) at Tyr-402, followed by Src recruitment. We thus
suggest here that PYK2 functions as an adaptor for c-Src which allows full activation of PYK2 and other signaling molecules in the adhesion complexes during osteoclastic cytoskeletal organization.
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MATERIALS AND METHODS |
Reagents--
Tissue culture media and cell dissociation buffer
were purchased from Invitrogen; fetal bovine serum (FBS) was from JRH
Bioscience (Lenexa, KS); collagenase was from Wako Chemicals (Richmond,
VA); dispase was from Roche Molecular Biochemicals; and macrophage colony-stimulating factor (M-CSF) was from R & D Systems Inc. (Minneapolis, MN). 1
,25-Dihydroxyvitamin D3
(1
,25(OH)2D3) was a gift of Dr. Milan
R. Uskokovic (Hoffmann-La Roche). Phosphorylation site-specific
polyclonal antibodies for PYK2 were from BIOSOURCE International (Camarillo, CA). Anti-PYK2 polyclonal antibodies were
as described previously (16). Anti-pp60c-Src (mAb GD11) and
anti-phosphotyrosine (mAb 4G10) were from Upstate Biotechnology, Inc.
(Lake Placid, NY). Antibodies to paxillin were from BD Transduction
Laboratories (San Diego, CA), and anti-HSV-Tag was from Novagen
(Madison, WI). Polyclonal antibodies raised against human
3 integrins were a gift from Dr. B. Bednar (Merck).
Fluorescein isothiocyanate- and TRITC-conjugated IgGs were from The
Jackson Laboratories (West Grove, PA); horseradish
peroxidase-conjugated IgG was from Amersham Biosciences, and Oregon
Green 488 phalloidin was from Molecular Probes (Eugene, OR).
Cell Cultures--
Pre-fusion osteoclasts (pOCs) were prepared
as described previously (26). Briefly, pre-fusion osteoclasts were
obtained from co-cultures of osteoblastic MB1.8 cells and murine bone
marrow cells in
-MEM containing 10% FBS and 10 nM
1
,25(OH)2D3. After 5 days in co-culture,
pOCs were released from dishes with enzyme-free cell dissociation
buffer after removing MB1.8 cells with 0.1% (w/v) collagenase and
dispase in PBS.
Construction of Recombinant Adenoviruses--
The recombinant,
replication-deficient adenovirus vectors were constructed using
p
E1sp1 plasmid consisting of human cytomegalovirus promoter and the
bovine growth hormone polyadenylation site as described previously
(27). Before transfer into adenovirus vector, full-length murine PYK2
cDNA was cloned into pCDNA3 (Invitrogen) as described
previously (16), and Lys-457
Ala and Tyr-402
Phe
mutations were done using U.S.E. mutagenesis kit from Amersham Biosciences. All the constructs had HSV-Tag at their C terminus. Recombinant viruses were produced in HEK293 cells, purified, and titrated according to standard methods (28).
Infection of Cells--
Expression, kinase activity, and
tyrosine phosphorylation of PYK2(wt) and its mutants were
first tested by infecting HEK293 cells. The cells were infected with
recombinant viruses at multiplicity of infection (m.o.i.) of 1-100.
After 24 h, cells were washed once with PBS, lysed into modified
RIPA buffer (10 mM Tris, pH 7.4, 150 mM NaCl,
1% Nonidet P-40, 0.2% sodium deoxycholate, 1 mM EDTA, 5 mM sodium fluoride, 1 mM sodium orthovanadate,
0.5 mM phenylmethylsulfonyl fluoride and a mixture of
protease inhibitors), and used for biochemical analyses.
To produce osteoclast-like cells (OCL) expressing PYK2(wt)
and its mutants, two different strategies of viral infection were used.
First, isolated pOCs were allowed to adhere for 1 h prior to
infection with different m.o.i.s (plaque-forming unit per pOC) of
recombinant viruses for 1 h in
-MEM, 0.1% bovine serum
albumin. After infection,
1
,25(OH)2D3-treated MB1.8 cells were added
to support pOC survival and fusion. After 3 days in 10% FBS and
-MEM in the presence of 10 nM
1
,25(OH)2D3, MB1.8 cells were removed by
collagenase-dispase treatment, and OCLs were lysed and used for
biochemical analyses. In the second strategy, the co-cultures of MB1.8
and murine bone marrow cells were infected with recombinant viruses at
days 2 and 4 in culture. Viral stocks were diluted with
-MEM and
added directly to the co-cultures. After isolation of pOCs, 300,000 cells were used for testing the expression levels of exogenous PYK2 by
immunoprecipitation, using anti-PYK2 antibodies, followed by blotting
with anti-HSV-Tag mAb. Infected pOCs were also used for analyzing cell
adhesion and spreading on vitronectin, cell migration, bone resorption,
and for immunofluorescent stainings.
Immunoblotting and Immunoprecipitation--
HEK293 cells, pOCs
or OCLs expressing PYK2 and its mutants, or uninfected control cells
were lysed into modified RIPA buffer on ice as described previously
(16). Lysates at equal protein concentrations were immunoprecipitated
using anti-PYK2 antibodies for 2-10 h at 4 °C, followed by
G-protein-Sepharose for 1 h at 4 °C, and washed with RIPA
buffer (4 times). Total cell lysates or immunoprecipitated proteins
were separated on 4-12% gradient SDS-PAGE (Invitrogen), transferred
onto Immobilon-P membrane (Millipore, Bedford, MA), which was incubated
subsequently with primary antibodies, followed by horseradish
peroxidase-conjugated secondary antibodies, and developed with enhanced
chemiluminescence system (ECL, Amersham Biosciences). For
co-immunoprecipitation, cells were lysed into Nonidet P-40 lysis buffer
(10 mM Tris, pH 7.4, 150 mM NaCl, 1% Nonidet
P-40, 10% glycerol, 1 mM EDTA, 5 mM sodium
fluoride, 1 mM sodium orthovanadate, 0.5 mM
phenylmethylsulfonyl fluoride, and a mixture of protease inhibitors) on
ice. Lysates at equal protein concentrations were incubated for 2 h with anti-c-Src antibody, followed by G-protein-Sepharose for 1 h at 4 °C, and finally washed with 1/2 times Nonidet P-40
lysis buffer (4 times). Immunoprecipitated proteins were separated on
8% SDS-PAGE, blotted, and stained as described above. The levels of
tyrosine phosphorylation and PYK2 were quantitated using an imaging
densitometer (model GS-700; Bio-Rad) and expressed as fold of
phosphorylated PYK2 in uninfected cells. Each was normalized to total
levels of PYK2 in respective sample.
Kinase Assay--
HEK293 cells or OCLs expressing PYK2 mutants
or uninfected control cells were lysed into modified RIPA buffer and
precipitated with anti-PYK2 or anti-HSV antibodies for 1 h at
4 °C, followed by G-protein-Sepharose for 1 h at 4 °C, and
washed with RIPA buffer (3 times). Half of the immunoprecipitates were
subjected to blotting with anti-PYK2 or anti-HSV antibodies, and the
other half was washed once with low salt buffer (100 mM
NaCl, 10 mM Tris, pH 7.4, 5 mM
MnCl2) and incubated with kinase reaction mixture (10 mM Tris, pH 7.4, 5 mM MnCl2, 1 µM cold ATP, 5 µCi of [
-32P]ATP) for
15 min at 30 °C. The samples were subjected to SDS-PAGE and autoradiography.
Cell Attachment and Spreading--
pOCs expressing PYK2 and its
mutants were allowed to attach to vitronectin-coated dishes (20 µg/ml, Invitrogen) at indicated times in the absence of serum. After
gently washing with PBS, attached cells were fixed and stained for
TRAP, and the cell area was counted using an image-analysis system
(Empire Image System, Milford, NJ).
Bone Resorption--
Isolated pOCs were cultured together with
10 nM 1
,25(OH)2D3-pretreated (48 h) MB1.8 cells on dentine slices in the presence of 10 nM
1
,25(OH)2D3 for 24 h. Cells were then
removed by ultrasonication in NH4OH, and resorption pits on
dentine slices were examined by scanning electron microscopy
(ElectroScan model 2010).
Cell Migration--
Cell migration was assayed using a Boyden
chamber type apparatus (Neuroprobe, Cabin John, MD) as described before
(29). Briefly, 5 nM M-CSF was placed in the bottom chamber
and isolated pOCs at a density of 20,000 cells/well in the upper
chamber. Cells were allowed to migrate through a polycarbonate filter
for 8 h in a humidified incubator at 37 °C. Then the cells that
migrated to the bottom of the filter were stained for TRAP and counted from 10 sequentially selected fields using an Olympus IX70 microscope with a ×10 objective.
Immunofluorescence--
pOCs expressing PYK2 and its mutants
were allowed to attach onto vitronectin-coated glass coverslips for
1-2 h, fixed with 4% paraformaldehyde, and permeabilized with 0.2%
Triton X-100 in PBS. Cells were stained using anti-
3 or
anti-PYK2 polyclonal antibodies, and anti-HSV, anti-phosphotyrosine,
and anti-paxillin mAbs, followed with the appropriate secondary
antibodies, or with Oregon-Green 488 phalloidin. Stainings were viewed
with a Leica TCS SP Spectral confocal laser scanning microscope
equipped with Argon-Krypton laser (Leica Microsystems Heidelberg GmbH,
Heidelberg, Germany).
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RESULTS |
Kinase Activity and Tyrosine Phosphorylation of PYK2 and Its
Mutants in HEK293 Cells--
Kinase activity and tyrosine
phosphorylation levels of exogenous murine PYK2(wt), PYK2
(Y402F), and PYK2(K457A) were first characterized
in HEK293 cells, which do not express endogenous PYK2. Cells were
infected with recombinant adenovirus constructs expressing HSV-tagged
PYK2(wt) or the mutants at m.o.i. of 1-10 for 24 h and
lysed for biochemical analyses. In vitro kinase assay of
immunoprecipitated PYK2 shows autophosphorylation activity of wild type
PYK2 and of PYK2(Y402F). This suggests that additional tyrosines in PYK2, beside the known major autophosphorylation site
Tyr-402, can be phosphorylated in vitro. In contrast, the ATP-binding site defective PYK2(K457) mutant shows no kinase
activity under the same condition (Fig.
1A). Western blots for HSV-Tag and PYK2 reveal expression levels of exogenous PYK2 and the mutants in
HEK293 cells (Fig. 1A, lower panels).

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Fig. 1.
Kinase activity and tyrosine phosphorylation
of PYK2 mutants expressed in HEK293 cells. A,
uninfected HEK293 cells (Control) and HEK293 cells infected
with the indicated m.o.i./cell of adenovirus expressing wild type PYK2
(wt PYK2), PYK2(K457A), or PYK2(Y402F) for
24 h were lysed and subjected for PYK2 immunoprecipitation
(IP) and in vitro kinase assay as described under
"Materials and Methods." Half the immunoprecipitates were used for
blotting for HSV-Tag, stripped and re-blotted for PYK2, to normalize
for the amounts of expressed PYK2 forms. B, uninfected
HEK293 cells (Control) and HEK293 cells infected with
adenovirus expressing wild type PYK2 (wt PYK2), PYK2(K457A),
or PYK2(Y402F) for 24 h were lysed and subjected for
PYK2 immunoprecipitation followed by blotting with site-specific
anti-phosphotyrosine PYK2 antibodies, pY402,
pY579/580, and pY881. Then membranes were
stripped and re-blotted for HSV-Tag and PYK2 to normalize for the
amounts of expressed protein.
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Phosphorylation of various tyrosines in PYK2(wt) and its
mutants was investigated using phosphorylation site-specific antibodies for PYK2. Fig. 1B shows that in HEK293 cells, when expressed
at the similar concentrations, PYK2(wt) was highly
phosphorylated at tyrosines 402, 579/580, and 881, whereas only weak
phosphorylation in PYK2(K457A) was detected at these
tyrosines. Undetectable phosphorylation at Tyr-402, Tyr-579/Tyr-580,
and Tyr-881 was observed in PYK2(Y402F). This suggests that
phosphorylation of PYK2 at these tyrosines was initiated by the
autophosphorylation at Tyr-402, which was previously suggested to
recruit an Src kinase for further phosphorylation at the C-terminal
tyrosines. Interestingly, the kinase-defective PYK2 appears to interact
with another tyrosine kinase in HEK293 cells, which weakly
phosphorylates Tyr-402. Our results also suggest that tyrosines other
than 402, 579/589, or 881 is a substrate for in vitro kinase
activity of PYK2(Y402F) as observed in Fig. 1A.
Expression and Kinase Activity of Exogenous PYK2 and Its Mutants in
Osteoclast-like Cells--
Expression of PYK2 and its mutants in OCLs
was characterized by infecting isolated pOCs at increased m.o.i. of
adenovirus constructs and then culturing pOCs with osteoblastic MB1.8
cells to support osteoclast survival and fusion. After 3 days, MB1.8 cells were removed with collagenase-dispase, and OCLs were lysed for
biochemical analysis. Immunoprecipitation analysis shows
dose-dependent increase in the expression of all exogenous
HSV-tagged PYK2 forms (Fig. 2).
Concentrations of adenovirus resulting in ~3-4-fold expression of
exogenous over endogenous PYK2 were used in all following experiments.
No defects in OCL growth or survival were observed at the highest
expression levels of exogenous PYK2.

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Fig. 2.
Expression levels of PYK2 mutants in
osteoclast-like cells. Isolated pOCs were left untreated
(C) or infected with various m.o.i./pOC of adenovirus
expressing wt PYK2, PYK2(K457A), or PYK2(Y402F)
for 1 h and cultured with the osteoclast-inducing MB1.8 cells for
3 days. Then MB1.8 cells were removed with collagenase-dispase as
described under "Materials and Methods," and osteoclast-like cells
were lysed and subjected to PYK2 immunoprecipitation (IP)
followed by blotting for HSV-Tag to illustrate the expression of
exogenous PYK2 and then for PYK2 to illustrate both exogenous and
endogenous PYK2. PYK2 fold expression was calculated as the ratio of
the PYK2 amount in lanes 1-6 to PYK2 amount in controls
(C). 1-6 in the graphs correspond to
lanes 1-6 in the blots.
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Next kinase activities of PYK2(wt) and its mutants
overexpressed in osteoclast-like cells were characterized in in
vitro kinase assay. Fig. 3
demonstrates high autophosphorylation activity of exogenously expressed
PYK2(wt) in immunoprecipitates using anti-HSV antibodies.
PYK2(Y402F) clearly displayed lower autophosphorylation activity as compared with the wild type, and no activity was found to
associate with PYK2(K457A). Western blots for HSV and PYK2 demonstrate similar expression levels of exogenous PYK2 forms recovered
in HSV immunoprecipitates (Fig. 3, left lanes). These results confirm the functional expression of PYK2 and its mutants in
osteoclast-like cells, similar to that observed in HEK293 cells. In
PYK2 immunoprecipitates (Fig. 3, right lanes), expression
levels of exogenous PYK2 forms were similar (Fig. 3, blot:
HSV) and 3-4 times that of endogenous PYK2 (Fig. 3, blot:
PYK2, as comparing control to the last three right
lanes in IP: PYK2). Autophosphorylation in PYK2
immunoprecipitates from osteoclast-like cells overexpressing PYK2(wt), PYK2(K457A), or PYK2(Y402F)
results from both endogenously and exogenously expressed pools of PYK2.
Phosphorylation levels of PYK2 in PYK2(K457A)-expressing
cells were similar to that of endogenous PYK2 (comparing lanes
5 and 7 in in vitro kinase assay) in the
uninfected cells, suggesting that endogenous PYK2 failed to
cross-phosphorylate the exogenously introduced mutant.

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Fig. 3.
Kinase activity of PYK2 mutants overexpressed
in osteoclast-like cells. Isolated pOCs were left untreated
(Control) or infected with adenovirus expressing wt PYK2,
PYK2(K457A), or PYK2(Y402F) for 1 h and
cultured with MB1.8 cells for 3 days. Osteoclast-like cells were lysed,
and half of the lysates were subjected to HSV-Tag immunoprecipitation
(IP) and half to PYK2 immunoprecipitation. Half of each
immunoprecipitation was used for in vitro kinase assay, and
the other half was subjected to blotting first for HSV-Tag to visualize
the expression of exogenous PYK2 and then for PYK2 to visualize both
exogenous and endogenous PYK2.
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Tyrosine Phosphorylation of PYK2 and Its Mutants in Osteoclast-like
Cells--
Because PYK2 tyrosine phosphorylation correlates with its
kinase activity in osteoclasts, we characterized phosphorylation pattern of PYK2 and its mutants in osteoclasts using phosphorylation site-specific antibodies. Similar to HEK293 cells, PYK2(wt)
in OCLs was highly phosphorylated at tyrosines 402, 579/580, and 881 as
found in immunoprecipitates using anti-HSV-Tag antibodies (Fig.
4, A-C). Furthermore,
PYK2(wt) was tyrosine-phosphorylated to the same extent as
endogenous PYK2 in control cells, as determined by the
ratio of Tyr(P) to PYK2 (Fig. 4, A-C),
suggesting that exogenous PYK2 was fully activated in OCLs. In contrast
to HEK293 cells, kinase-defective PYK2(K457A) in OCLs showed
even higher tyrosine phosphorylation levels at tyrosines 402, 579/580,
and 881 when compared with wild type PYK2. This suggests that PYK2 kinase activity is not necessary for phosphorylation of tyrosines 402, 579/580, or 881 of PYK2 in osteoclasts and implicates that another
kinase(s) can efficiently phosphorylate these tyrosines in osteoclasts.
However, PYK2(Y402F) showed no phosphorylation at Tyr-402
(Fig. 4A) and only very low phosphorylation at Tyr-579/580 and Tyr-881 (Fig. 4, B and C), suggesting that
phosphorylation at Tyr-402 is important for additional phosphorylation
at the regulatory (Tyr-579/580) and C-terminal (Tyr-881) domains of
PYK2 in osteoclasts. Tyrosine phosphorylation in PYK2
immunoprecipitates (Fig. 4, A-C, right lanes)
results from both endogenous and exogenously expressed pools of
PYK2.

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Fig. 4.
Defective tyrosine phosphorylation of
autophosphorylation site-mutated PYK2(Y402F) but not
of kinase-dead PYK2(K457A) in osteoclast-like
cells. Isolated pOCs were left untreated (Control) or
infected with adenovirus expressing wt PYK2, PYK2(K457A), or
PYK2(Y402F) for 1 h and cultured with MB1.8 cells for 3 days. Osteoclast-like cells were lysed, and half of the lysates were
subjected to HSV-Tag immunoprecipitation (IP) and half to
PYK2 immunoprecipitation. Immunoprecipitates were then subjected for
blotting for site-specific anti-phosphotyrosine PYK2 antibodies,
pY402 (A), pYpY579/580 (B),
or pY881 (C). Membranes were then stripped and
re-blotted for HSV-Tag and then for PYK2. HSV immunoprecipitates
visualize tyrosine phosphorylation of exogenously expressed PYK2 forms
(left lanes), whereas PYK2-immunoprecipitates visualize
tyrosine phosphorylation of both exogenously and endogenously expressed
PYK2 (right lanes). Tyr(P)/PYK2 ratio demonstrates relative
amounts of phosphorylation at each tyrosine in the samples.
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Expression of PYK2(Y402F) Inhibits Osteoclast-like Cell Spreading
and Bone Resorption--
Because we demonstrated previously (19) the
role of PYK2 antisense in inhibiting cytoskeletal reorganization in
osteoclasts during cell adhesion and spreading, we thus examined the
effects of PYK2 mutants on spreading of pOCs on vitronectin under
serum-free conditions. From the time course of pOC spreading as shown
in Fig. 5A, there were no
statistically significant differences between the rate of cell
spreading in control pOCs expressing only endogenous PYK2 and pOCs
overexpressing wild type or kinase-defective PYK2. In contrast,
spreading of pOCs overexpressing PYK2(Y402F) was severely
impaired (Fig. 5, A and B, p < 0.001 at 30- and 60-min time points). These results suggest that the
poorly tyrosine-phosphorylated PYK2(Y402F) functions as
dominant negative PYK2 preventing cytoskeletal reorganization during
osteoclast spreading. On the other hand, the kinase activity of PYK2
appears to be unnecessary for supporting osteoclast spreading.

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Fig. 5.
Autophosphorylation site-mutated
PYK2(Y402F) prevents pOCs spreading on vitronectin and
bone resorption. pOCs were isolated from control co-cultures
(Control) or from co-cultures infected with adenovirus
expressing wt PYK2, PYK2(K457A), or PYK2(Y402F)
as described under "Materials and Methods." A, pOCs were
then allowed to adhere and spread on vitronectin-coated surfaces for
indicated times up to 60 min, fixed, stained for TRAP and quantitated
for cell area as described under "Materials and Methods."
Statistically significant differences were tested using t
test, ***, p < 0.001, and ns = no
significant difference when compared with the control of the same time
point. B, examples of typically spread pOCs in each
treatment. C, pOCs were cultured with MB1.8 cells on bone
slices for 24 h, and cells were then removed, and the formed
resorption pits were demonstrated using S.E.M. Note very shallow pits
formed by pOCs expressing PYK2(Y402F) (panels C
and D) compared with the control (panels A and
B).
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Next we investigated if reduced cell spreading, caused by
overexpressing PYK2(Y402F), affects bone resorption. Control
and PYK2(Y402F)-expressing pOCs were cultured on dentine
slices together with MB1.8 cells for 24 h. The cells were removed,
and resorption pits were examined by S.E.M. Only very shallow
resorption pits were made by PYK2(Y402F)-expressing
osteoclasts (Fig. 5C, panels C and D),
when compared with uninfected cells (Fig. 5C, panels A and B). This indicates that expression of
PYK2(Y402F) prevents both cell spreading and bone
resorption, similar to cells expressing PYK2 antisense or to
c-Src-deficient osteoclasts.
c-Src Tyrosine Phosphorylation and Its Association with PYK2
Mutants in Osteoclast-like Cells--
Since the rate of cell spreading
of osteoclasts overexpressing PYK2(Y402F) was inhibited in a
similar manner as found in the Src-deficient osteoclasts and
phosphorylated Tyr-402 has been suggested to mediate the recruitment of
c-Src to PYK2 via its association to Src-SH2 domain, we investigated
the ability of PYK2 mutants binding to endogenous c-Src in osteoclasts
by co-immunoprecipitation methods. Fig. 6
demonstrates the association of PYK2(wt) and
PYK2(K457A), but not PYK2(Y402F) with c-Src (Fig.
6A), suggesting that phosphorylated tyrosine 402 of PYK2 is
the major docking site for c-Src in this cell type. In addition, we
could not detect any differences in the levels of tyrosine
phosphorylation (Fig. 6B) or kinase activity (data not
shown) of c-Src in osteoclast-like cells overexpressing various PYK2
mutants, confirming our previous results that c-Src is not the
substrate of PYK2 in osteoclasts.

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Fig. 6.
c-Src tyrosine phosphorylation and its
association with PYK2 mutants in osteoclast-like cells. Isolated
pOCs were left untreated (Control) or infected with
adenovirus expressing wt PYK2, PYK2(K457A), or
PYK2(Y402F) for 1 h and cultured with MB1.8 cells for 3 days. Then MB1.8 cells were removed with collagenase-dispase as
described under "Materials and Methods," and osteoclast-like cells
were lysed. A, lysates were subjected to c-Src
immunoprecipitation (IP) and blotting for HSV-Tag. Membranes
were then stripped and re-probed first for PYK2 and then for c-Src.
Sample of total cell lysate of each treatment was used for Western
blotting (TCL, right lanes). IgG is
immunoglobulin heavy chain used for immunoprecipitation. B,
similar lysates were subjected for c-Src immunoprecipitation and
blotting for phosphotyrosine (pY) and c-Src.
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Localization of Exogenous PYK2 and Its Mutants to Podosomes in
Osteoclast-like Cells--
Since cell spreading was impaired in pOCs
expressing PYK2(Y402F), we next characterized the
localization of PYK2(wt) and its mutants in pOCs. Double
stainings of HSV, showing the localization of exogenous PYK2, and
F-actin showed that both PYK2(wt) and PYK2(K457A) can be readily localized to podosomes in pOCs adhered to vitronectin (Fig. 7, B,
C, F, and G). Although the spreading of
PYK2(Y402F) expressing pOCs were greatly reduced, the cells
adhere to vitronectin, and a few of them started to form podosomes
containing lamellipodia, and PYK2(Y402F) could be found to
translocate into the podosomes in these cells as seen with anti-HSV
antibodies (Fig. 7, D and H; Fig.
8, C and D). Double
stainings of HSV and PYK2 demonstrated that in addition to peripheral
podosomes substantial amounts of all exogenously expressed PYK2 forms
were localized in the cytoplasm (Fig. 8). In addition,
3
integrin and paxillin co-localized with HSV and surrounded F-actin
stained podosomes, respectively, in pOCs expressing PYK2 forms (Fig.
9). This demonstrates that pOCs expressing kinase-dead PYK2(K457A) or PYK2(Y402F)
are able to form normal podosomes. In addition, the results suggest
that PYK2 might be constitutively recruited to the podosomes upon cell
adhesion, and phosphorylation of Tyr-402, Tyr-579/580, or Tyr-881 or
association with c-Src might not be necessary for podosomal targeting
of PYK2.

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Fig. 7.
Localization of PYK2 mutants to
podosomes in pOCs. pOCs were isolated from control co-cultures
(Control, A and E) or from co-cultures
infected with adenovirus expressing wt PYK2 (B and
F), PYK2(K457A) (C and G),
or PYK2(Y402F) (D and H) as described
under "Materials and Methods." pOCs were then allowed to adhere and
spread on vitronectin-coated surfaces and subjected for staining for
F-actin (A-D) and HSV-Tag (E-H). Extended
confocal images from the adhesion structures close to the glass surface
show punctate podosomal staining of F-actin surrounded by HSV tag
staining for PYK2 forms in adenovirus transfected pOCs
(arrows). Note that also the less spread pOC expressing
PYK2(Y402F) contains podosomes with HSV staining
(D and H) at the cell periphery
(arrows). Bar, 20 µm.
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Fig. 8.
High expression of PYK2 mutants also in the
cytoplasm of pOCs. pOCs were isolated from co-cultures infected
with adenovirus expressing wt PYK2 (A and D),
PYK2(K457A) (B and E), or
PYK2(Y402F) (C and F) as described
under "Materials and Methods." pOCs were then allowed to adhere and
spread on vitronectin-coated surfaces and subjected for double staining
for PYK2 (A-C) and HSV-Tag (D-F).
Extended confocal images through the cells show in addition to
peripheral podosomal staining (arrows) also prominent
cytoplasmic staining for all PYK2 forms. Note the higher magnification
in C and F that was used because of reduced
spreading of PYK2(Y402F) expressing pOC, showing also clear
podosomes at cell periphery (arrows). Bars, 20 (A, B, D, and E) and 10 µm (C and F).
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Fig. 9.
Localization of
3 integrin (A') and
paxillin (B') in podosomes in PYK2 mutants expressing
pOCs. A', pOCs were isolated from control co-cultures
(Control, A and E) or from co-cultures
infected with adenovirus expressing wt PYK2 (B and
F), PYK2(K457A) (C and G),
or PYK2(Y402F) (D and H) as described
under "Materials and Methods." pOCs were then allowed to adhere and
spread on vitronectin-coated surfaces and subjected for double staining
for 3 integrin (A-D) and HSV-Tag
(E-H). B', pOCs were isolated from co-cultures
infected with adenovirus expressing PYK2(K457A)
(A and C) or PYK2(Y402F) (B
and D) as described under "Materials and Methods,"
allowed to adhere and spread on vitronectin-coated surfaces, and
subjected for double staining for F-actin (A and
B) and paxillin (C and D). Extended
confocal images from the adhesion structures close to the glass surface
show podosomal staining of 3 integrin and HSV-tag
(A', arrows) and paxillin surrounding punctate
F-actin staining (B', arrows) in
adenovirus-transfected pOCs. Bars (A') 20 (A, C-E, G, and H), 10 (B and F), (B') and 10 µm.
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Overexpressing of PYK2 and Its Mutants in Osteoclast-like Cells Did
Not Affect Cell Migration toward M-CSF--
We demonstrated previously
(30) that although cell spreading of Src-deficient osteoclasts is
severely impaired, their migration toward M-CSF is normal. Similar to
Src-deficient osteoclasts, we showed here that the migration of
osteoclasts overexpressing PYK2(Y402F) toward M-CSF is
comparable with that of uninfected pOCs or of cells infected with
adenovirus expressing PYK2(wt) or PYK2(K457A)
(Fig. 10). However, we observed a
consistent increase in the number of migrated pOCs that overexpress
exogenous PYK2(wt) or kinase-defective PYK2 as compared with control
uninfected cells, suggesting that an increase in PYK2 expression over
the endogenous levels might accelerate the rate of osteoclast migration
toward M-CSF. It should also be noted that pOCs expressing
PYK2(Y402F) when cultured together with MB1.8 cells in the
presence of serum for 3 days were normally spread (data not shown).
This suggested that M-CSF secreted by MB1.8 cells can also induce
normal spreading of PYK2(Y402F) expressing osteoclasts.

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Fig. 10.
Migration of PYK2 mutants expressing pOCs
toward M-CSF. pOCs were isolated from control co-cultures
(Control) or from co-cultures infected with adenovirus
expressing wt PYK2, PYK2(K457A) or PYK2(Y402F) as
described under "Materials and Methods." pOCs were then allowed to
migrate in a Boyden chamber for 8 h, and cells migrated on lower
surface of the membrane were stained for TRAP and counted.
Statistically significant differences were tested using t
test, *, p < 0.05 when compared with the control,
ns = no significant difference when compared with the
control, PYK2(K457A), or PYK2(Y402F).
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DISCUSSION |
PYK2 has been shown to be one of the major adhesion-induced
tyrosine kinase in osteoclasts, necessary for osteoclastic bone resorption (16, 19). The present data characterize the importance of
various PYK2 domains for its function in osteoclasts. First, we
verified adenovirus expression of wild type PYK2 and its mutants in
HEK293 cells, which do not express endogenous PYK2. In HEK293 cells,
both kinase-defective PYK2(K457A) and autophosphorylation site-mutated PYK2(Y402F) were very poorly phosphorylated at
tyrosines 402, 579/580, and 881. This suggests that autophosphorylation of tyrosine 402 is necessary for further tyrosine phosphorylation of
PYK2 in HEK293 cells. Similar observations using transfection of HEK293
cells (20, 31) and endothelial cells (32) have been described recently.
Interestingly, Tyr-402 is not the only autophosphorylation site of
PYK2, because we consistently observe weak phosphorylation of tyrosines
other than Tyr-402, Tyr-579/580, and Tyr-881 in PYK2(Y402F)
in the in vitro kinase assay and by blotting with antibodies
that recognize any phosphotyrosine residues (data not shown). The
identity and potential role of the additional autophosphorylation sites
of PYK2 in the adhesion-dependent signaling pathway in
osteoclasts or other cells are subjects of future investigations.
We and others (33, 34) have demonstrated previously the use of
replication-defective recombinant adenovirus as an effective method to
highly introduce exogenous genes into osteoclasts and that adenovirus
expressing PYK2 antisense inhibits osteoclast spreading and function
(19). By using the same method, this study describes
dose-dependent expression of wild type PYK2 and two mutants
PYK2(K457A) and PYK2(Y402F) in osteoclast-like
cells. Although endogenous PYK2 is an abundantly expressed kinase in osteoclasts, 3-4-fold higher expression of exogenous PYK2 over the
endogenous level could readily be achieved without affecting osteoclast survival.
In contrast to HEK293 cells, overexpression of the kinase-defective
PYK2(K457A) mutant in osteoclasts showed no reduction in
phosphorylation at the tyrosines 402, 579/580, or 881 and did not
prevent cell spreading on vitronectin, suggesting that its kinase
activity is not necessary for the integrin
v
3-dependent signaling in
osteoclasts mediated by PYK2. Interestingly, phosphorylation of
Tyr-402, which leads to its association with c-Src, is normal in
PYK2(K457A). Given PYK2 homodimerization, it is highly
unlikely that phosphorylation of the overexpressed mutant was due to
the endogenous wild type PYK2, suggesting that Tyr-402 was
phosphorylated by another tyrosine kinase(s) expressed in osteoclasts.
Src family of kinases as well as the tyrosine receptor kinases,
platelet-derived growth factor or vascular endothelial growth factor
receptors, have been implicated in the activation of FAK independently
of FAK kinase activity (35, 36). Because unlike many cell types osteoclasts highly express c-Src, c-Src might be responsible for phosphorylation of Tyr-402 in PYK2(K457A), to promote
additional Src docking to this site, leading to maximal activation of
PYK2 via phosphorylation of Tyr-579/580 in the regulatory domain and Tyr-881 in its C-terminal domain. Phosphorylation of these tyrosines is
thus suggested to regulate downstream signaling and osteoclast spreading. This notion is in agreement with recent findings (37) demonstrating that adhesion-induced FAK auto-phosphorylation at tyrosine 397 as well as phosphorylation at tyrosine 577 in the kinase
domain require Src family kinase function. On the other hand, Sanjay
and co-workers (20) suggested that integrin-induced PYK2 tyrosine
phosphorylation is c-Src-independent but is mediated by an increase in
cytoplasmic Ca2+ concentration. In addition, PYK2 kinase
activity was found to be necessary for cell spreading and migration and
for angiogenesis in pulmonary vascular endothelial cells (32).
Pulmonary vascular endothelial cells express both PYK2 and FAK, and FAK
expression as well as p130Cas were decreased following
transfection with the kinase-defective PYK2. These findings suggested
that the kinase activity-dependent role of PYK2 on focal
adhesion formation and cytoskeletal reorganization in vascular
pulmonary endothelial cells was mediated through FAK and
p130Cas (32).
An important finding in the present paper is the dominant negative
function of the major autophosphorylation site mutant
PYK2(Y402F) in osteoclast spreading. PYK2(Y402F)
displayed in vitro kinase activity, albeit lower than wild
type or endogenous PYK2. This is in agreement with results observed in
HEK293 cells (31) (Fig. 1) and in pulmonary vascular endothelial cells
(32). The lower kinase activity of PYK2(Y402F) could be due
to poor phosphorylation of kinase domain tyrosines 579/580, suggested
to be required for full activity of PYK2 (23). PYK2(Y402F)
was unable to associate with c-Src, which may be the reason of reduced
phosphorylation of tyrosines 579/580 as well as tyrosine 881 at the C
terminus in PYK2(Y402F). Reduced tyrosine phosphorylation
may prevent association with and activation of downstream signaling
molecules, such as association of Grb2 to phosphorylated Tyr-881,
required for cytoskeletal organization and osteoclast spreading. More
importantly, reduced cell spreading also reflected to the impaired bone
resorption because only very shallow resorption pits were formed by
osteoclasts expressing PYK2(Y402F). Further studies are
needed to identify important downstream factors, whose association to
signaling complex and/or activation is prevented by
PYK2(Y402F) in osteoclasts. In other systems, PYK2
phosphorylation at tyrosine 402 and association with c-Src leads to
activation of mitogen-activated protein kinase cascades via distinct
mediators (31). Phosphorylation of Shc and Grb2 and their association
with PYK2 and Sos mediate activation of extracellular signal-regulated
kinase, whereas tyrosine phosphorylation of p130Cas and its
association to PYK2 and Crk and possible Crk effectors lead to
activation of other mitogen-activated protein kinase cascades, e.g. the c-Jun N-terminal kinase cascade (31). In
osteoclasts,
v
3-mediated adhesion
induces c-Src-dependent phosphorylation of extracellular
signal-regulated kinases (30), which points to Grb2-Sos complex as one
possible mediator.
It is well known that in fibroblastic cells, which do not express
endogenous PYK2 but use FAK as the main adhesion-related tyrosine
kinase, overexpression of PYK2 induces apoptosis (38). Apparently,
competition of PYK2 to endogenous FAK in fibroblasts results in
different biological effect compared with what we observed here with
osteoclasts, expressing high level of endogenous PYK2 but little FAK.
Furthermore, although PYK2 expression is elevated in FAK null
fibroblasts, it is not able to fully substitute FAK function or
localize to focal adhesions (39). In FAK null fibroblasts, PYK2 is
targeted to focal adhesions only when it is expressed as a chimera
containing FAK C-terminal domain (40). Thus PYK2 localization to
adhesion contacts is a fairly unique feature. In addition to
osteoclasts, PYK2 has been reported to localize to adhesion contacts
only in monocytes and during nerve growth factor-induced neuronal cell
differentiation (41-43). The present results of the dominant
negative effect of PYK2(Y402F) on cytoskeletal organization,
osteoclast spreading, and bone resorption further support the specific
role of PYK2 function in osteoclasts.
Interestingly, osteoclasts expressing any of the PYK2 forms were able
to form podosomes, and all the PYK2 forms were able to localize to
podosomes in osteoclasts on vitronectin. This suggests that the kinase
activity or tyrosine phosphorylation of PYK2 is not necessary for its
localization to podosomes. Similarly autophosphorylation site- or
kinase domain-mutated FAK localizes to focal adhesions in fibroblasts
(44). The mechanism for podosome-targeting of PYK2 is not well
understood. The presented data are in agreement with recent results
demonstrating direct binding of PYK2 to the
3
cytoplasmic tail in vitro regardless of PYK2 tyrosine
phosphorylation or paxillin association (45). Thus it is unlikely that
PYK2 tyrosine phosphorylation provides a regulatory mechanism for its targeting to podosomes. However, PYK2 tyrosine phosphorylation may
affect its association with and recruitment of other components to
podosomes or activity of downstream targets that regulate podosome dynamics. Evidently this could have crucial effects on cell
spreading. As a matter of fact, targeting of PYK2(Y402F) to
podosomes may be necessary for its dominant negative action on
osteoclast spreading or enhance that effect. The molecular mechanism by
which PYK2(Y402F) prevents osteoclast spreading remains to
be elucidated. In the case of c-Src, it has been suggested that the
reduced spreading and migration of the c-Src-deficient osteoclasts
are caused by defect in podosome disassembly rather that assembly (20).
Further studies are required to characterize cytoskeletal or other
morphological changes preventing bone resorption of
PYK2(Y402F) overexpressing osteoclasts.
Finally, migration of osteoclasts toward M-CSF is not disturbed by
expression of the PYK2 mutants used in this study. Similar observations
have been made recently (37) showing that FAK tyrosine phosphorylation
by G-protein-coupled receptor agonists is not c-Src-dependent. One possibility is that the M-CSF
stimulates osteoclast cytoskeletal organization and migration by
activating the pathway downstream of the c-Src-PYK2 complex, consistent
with our previous finding (30) that migration of c-Src-deficient osteoclasts toward M-CSF is not impaired. Phospholipase C-
was found
to be a common downstream mediator for
v
3
and M-CSF signals in osteoclasts and M-CSF-induced cytoskeletal
reorganization and modulated the
v
3
integrin ligand interaction and recruitment of signaling molecules,
including PYK2, to adhesion structures in the absence of c-Src (30).
The present study suggests that phosphorylation of Tyr-402 in PYK2 is
not required for M-CSF-induced cytoskeletal organization in
osteoclasts. Although the previous study (30) showed that M-CSF did not
highly induce PYK2 tyrosine phosphorylation in c-Src-deficient
osteoclasts, we cannot rule out an effect of M-CSF on the binding of
the C-terminal domain of PYK2 to phospholipase C-
, which depends on
both SH2 and SH3 domains of phospholipase C-
(30).
In summary, our results show that the mutant of major
autophosphorylation site PYK2(Y402F) functions as a dominant
negative PYK2 by preventing its association with c-Src, which has been shown to regulate both PYK2 kinase activity and its ability to recruit
downstream adhesion-dependent signaling molecules (16, 17,
30). Interestingly, although overexpression of PYK2(Y402F) results in inhibition of osteoclast spreading, it can be readily localized to a few forming podosomes, suggesting that c-Src recruitment to the adhesion contacts plays a significant role in modulating cytoskeletal organization in osteoclasts. Furthermore, overexpression of PYK2(Y402F) leads to impaired osteoclastic bone
resorption in vitro. On the other hand, tyrosine
phosphorylation of the kinase-defective PYK2(K457A) is
similar to wild type PYK2 in osteoclasts, has normal interaction with
c-Src, localizes to podosome, and does not affect osteoclast spreading.
Taken together, our data thus suggest that PYK2 may function as an
adaptor for c-Src which allows downstream signaling, required for to
normal osteoclast spreading and bone resorption.