From Research Bone Metabolism, Novartis Pharma AG, CH-4002 Basel, Switzerland
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ABSTRACT |
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Fluoride is known to increase bone mass in
vivo, probably through stimulation of osteoblast proliferation;
however, the mechanisms of fluoroaluminate action in osteoblasts have
not yet been elucidated. We have previously shown that in osteoblastic
MC3T3-E1 cells, fluoroaluminate stimulates G protein-mediated protein
tyrosine phosphorylation (u
a, M., Standke, G. J. R., Jeschke, M., and Rohner, D. (1997) Biochem. Biophys. Res.
Commun. 235, 680-684). Although the Ser/Thr kinases Erk1, Erk2,
and p70S6K were activated in response to fluoroaluminate,
the identity of fluoroaluminate-activated tyrosine kinase(s) remained
elusive. In this study, we show that in MC3T3-E1 cells, fluoroaluminate induces a 110-kDa tyrosine-phosphorylated protein that we identify as Pyk2, a cytoplasmic tyrosine kinase related to Fak
(focal adhesion kinase). The
tyrosine phosphorylation of Pyk2 increased in a dose- and
time-dependent manner. The autophosphorylation activity of Pyk2 increased 3-fold and reached its maximum within 10 min of fluoroaluminate treatment. Fluoroaluminate also induced activation of
Src and the association of Pyk2 with Src. The phosphorylation of
Src-associated Pyk2 increased >20-fold in in vitro kinase
assays, suggesting that Pyk2 is phosphorylated by Src. Although
MC3T3-E1 cells express much more Fak than Pyk2, Src preferentially
associated with Pyk2. In vitro, Pyk2 bound to the Src SH2
domain, suggesting that this interaction mediates the Src-Pyk2
association in cells. These data indicate that osteoblastic cells
express Pyk2, which is tyrosine-phosphorylated and activated in
response to G protein activation by fluoroaluminate, and that the
mechanism of Pyk2 activation most likely involves Src. Thus, Src and
Pyk2 are tyrosine kinases involved in G protein-mediated tyrosine
phosphorylation in osteoblastic cells and may be important for the
osteogenic action of fluoroaluminate.
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INTRODUCTION |
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Fluoride has been known as a bone-forming agent for more than 100 years. Together, the occurrence of osteosclerosis in miners exposed to a mineral consisting of sodium aluminum fluoride (Na3AlF6), the effect of fluoridation of drinking water on bone mass in humans, as well as recent animal studies suggest that fluoride in complex with aluminum is capable of increasing bone formation (reviewed in Refs. 1 and 2). However, a narrow therapeutic window and the questionable quality of newly formed bone prevented the wide use of fluoride for the treatment of osteoporotic patients. A promising new treatment using fluoride has recently been offered in the form of slow-release sodium fluoride (reviewed in Ref. 3). Despite difficulties in its therapeutic use, fluoride remains an interesting bone anabolic agent. Understanding the molecular actions of fluoride on bone is expected to enable the development of drugs that would mimic its anabolic action and could overcome problems of the narrow therapeutic window and side effects.
Little is known about the molecular mechanism of fluoride action in
bone at the cellular level. Fluoride in a complex with aluminum
(fluoroaluminate, most likely AlF4)
(4) binds to
-subunits of heterotrimeric G proteins in
vitro and activates G protein-mediated intracellular signaling
pathways (5-8). Numerous reports indicate that in cultures of isolated primary osteoblastic cells as well as osteoblastic cell lines, fluoride
and fluoroaluminate can induce proliferation (2, 9-11), although this
effect seems to be restricted to a certain population of osteoblastic
cells (12). Activation of several enzymes involved in intracellular
signal transduction has been implicated in mediating the mitogenic
signal of fluoride (including phospholipase C, diglyceride kinase,
phospholipase D, tyrosine kinases, Erk1, Erk2, and p70S6K)
as well as the inhibition of tyrosine phosphatases and adenylate cyclase (2, 13-17).
We (17) and others (2, 18) have recently reported that fluoroaluminate and fluoride increase protein tyrosine phosphorylation in osteoblast-like cells. In our system, fluoroaluminate induced prominent tyrosine phosphorylation of several proteins with apparent molecular masses of ~70, 120, and 130 kDa (17). However, the identity of tyrosine kinases mediating fluoroaluminate-induced tyrosine phosphorylation has remained elusive. In this study, we describe the tyrosine phosphorylation of a previously undetected 110-kDa protein in response to treatment of MC3T3-E1 cells with fluoroaluminate. The phosphorylation of this protein was weaker than the tyrosine phosphorylation of major proteins that were previously described, and its resolution from the major 120-kDa protein was critical for its detection. Furthermore, we demonstrate that this 110-kDa protein is immunologically indistinguishable from Pyk2 (proline-rich tyrosine kinase 2), a recently described tyrosine kinase shown to be regulated by G protein-coupled receptors in neuronal cells (19, 20). This finding prompted us to investigate the mechanism of Pyk2 activation by fluoroaluminate in osteoblastic MC3T3-E1 cells.
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EXPERIMENTAL PROCEDURES |
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Materials-- NaF was purchased from Fluka, and AlCl3, bradykinin, protein A-Sepharose CL-4B, and protein G-Sepharose 4B Fast Flow were from Sigma. Fluoroaluminate solution was prepared immediately before the experiment by mixing stock solutions (500 mM NaF and 10 mM AlCl3) to give a final concentration of 10 mM NaF and 10 µM AlCl3, which has been recommended for formation of fluoroaluminate complexes (4). [32P]ATP (specific activity of 3000 Ci/mmol) was acquired from Amersham Pharmacia Biotech. The SH2 domains immobilized on agarose beads were from Oncogene Science Inc. (PhosphoBindTM SH2 reagents). Antibodies were obtained from the following sources: anti-phosphotyrosine monoclonal antibody 4G10 (Upstate Biotechnology, Inc.), anti-Fak monoclonal antibody (mAb)1 2A7 (Upstate Biotechnology, Inc. and Transduction Laboratories), goat anti-Pyk2 polyclonal antibody (raised against amino acids 990-1009 of human Pyk2; Santa Cruz Biotechnology), horseradish peroxidase-conjugated secondary antibodies (Cappel), anti-Src monoclonal antibodies LA-074 (Quality Technology Inc.) and 05-184 (Upstate Biotechnology, Inc.), and anti-Src polyclonal antibody SRC2 (Santa Cruz Biotechnology).
Cell Culture--
The mouse osteoblastic cell line MC3T3-E1 was
provided by Dr. J. Caverzasio (University of Geneva, Geneva,
Switzerland). Cells were cultured in -minimum Eagle's medium
supplemented with 10% fetal calf serum (Life Technologies, Inc.),
penicillin/streptomycin, and L-glutamine. For experiments,
cells were grown in 10- or 15-cm tissue culture dishes for 3 days to
~90% confluency. Prior to stimulation, cell cultures were generally
deprived of growth factors for 24 h in the absence of serum. PC12
cells and NIH3T3 fibroblasts were obtained from the American Type
Culture Collection (ATCC 12705 and CRL 1658, respectively). Human
osteoblastic cells were derived from explants of trabecular bone as
described (21); primary calvarial osteoblasts were isolated from
3-7-day-old MA01 mice as described (22).
Cell Extraction-- Agonist-treated MC3T3-E1 cells were washed with ice-cold phosphate-buffered saline and lysed in ice-cold Nonidet P-40 lysis buffer (25 mM Tris (pH 7.4), 10% (v/v) glycerol, 1% (v/v) Nonidet P-40, 50 mM NaF, 1 mM sodium vanadate, 1 mM phenylmethylsulfonyl fluoride, and 10 µg/ml each aprotinin, leupeptin, and pepstatin A) on ice for ~15 min. Cleared cell lysates were obtained by subsequent centrifugation in a microcentrifuge (14,000 rpm at 4 °C for 5 min). Protein concentrations were determined using a Bradford micromethod (Bio-Rad).
Western Blotting-- Proteins were resolved on 12% SDS-polyacrylamide gels with an acrylamide/bisacrylamide ratio of 150:1 at 25 mA/gel using a Bio-Rad Minigel apparatus. Electroblotting onto polyvinylidene difluoride membrane (Millipore Corp.) was performed overnight at 30 V and 4 °C (Bio-Rad Mini Trans-Blot unit). Blots were placed in blocking buffer (phosphate-buffered saline and 3% gelatin) for 1 h to prevent nonspecific binding, followed by incubation with a specific antibody for 1 h. Detection of immunoreactive bands was performed after incubation with horseradish peroxidase-conjugated secondary antibodies with an enhanced chemiluminescence kit (ECL, Amersham Pharmacia Biotech) according to the manufacturer's instructions.
Immunodepletion-- The anti-Pyk2 polyclonal antibody was bound to 100 µl of a 1:1 mixture of protein A and protein G-Sepharose (protein A/G-Sepharose) in 500 µl of Nonidet P-40 lysis buffer by incubation at room temperature on a shaker for 1 h. Subsequently, the protein A/G-Sepharose beads were washed three times with 1 ml of lysis buffer to remove unbound antibodies and resuspended in lysis buffer. An aliquot of cleared cell lysate prepared from fluoroaluminate-stimulated MC3T3-E1 cells (200 µl, 150 µg of total protein) was incubated on ice with 5 µg of Pyk2-specific antibody prebound to protein A/G-Sepharose beads for 1 h. The beads were removed by centrifugation, and the supernatant was incubated with the immobilized antibody again. Equal amounts of protein of the original lysate and of the supernatants after immunodepletion of Pyk2 were analyzed by Western blotting as described above.
Immunoprecipitation-- Cleared cell lysates were diluted with Nonidet P-40 lysis buffer to a final protein concentration of 1 mg/ml and incubated with 2 µg of the appropriate antibody for 2-4 h on ice on an orbital shaker. The immune complexes were subsequently captured with 10 µl of protein A- or protein G-Sepharose for 30 min. The beads were washed twice with 1 ml of 50 mM Tris-HCl (pH 8.0), 500 mM NaCl, 2 mM EDTA, and 0.5% Nonidet P-40; twice with 1 ml of 50 mM Tris-HCl (pH 8.0), 500 mM NaCl, and 2 mM EDTA; and once with 30 mM Tris (pH 7.5). For co-immunoprecipitation experiments, the NaCl concentration was 140 mM (nonstringent conditions). The proteins in immune complexes were analyzed either in kinase assays or by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting. For reprecipitation of denatured proteins from immune complex kinase assays, the immune complexes on protein A-Sepharose were boiled in SDS loading buffer to release immunoprecipitated proteins. Sepharose beads were removed by centrifugation, and the supernatant was diluted with 800 µl of Nonidet P-40 lysis buffer and used for immunoprecipitation as described above.
Immune Complex Kinase Assay-- Pyk2, Src, or Fak immune complexes were washed as described above. An additional wash was performed in kinase buffer (30 mM Tris (pH 7.5), 2 mM MgCl2, and 5 mM MnCl2). The protein kinase assays were performed in 30 µl of kinase buffer and 10 µCi of [32P]ATP (3000 Ci/mmol) per reaction at 37 °C for 10 min. Immune complexes were washed three times with 1 ml of ice-cold NET (50 mM Tris-HCl, pH 8.0, 500 mM NaCl, and 2 mM EDTA) to remove free [32P]ATP. The antigens were released from the beads by boiling them in 50 µl SDS of loading buffer and subjected to 12% SDS-PAGE. Src immune complexes were separated on longer gels (8 × 9 cm, Mighty Minigel, Amersham Pharmacia Biotech) at 20 mA/gel to achieve a better separation. Gels were dried and exposed on a PhosphorImager screen (Molecular Dynamics, Inc.) for 4-40 h. The bands of interest were quantified with the ImageQuant program (Molecular Dynamics, Inc.) and corrected for background signal.
SH2 Binding Assay--
Association of Pyk2 with SH2 domains of
phospholipase C, PI 3-kinase (C-terminal SH2 domain), and Src was
measured using PhosphoBindTM SH2 reagents according to the protocol
provided by the manufacturer (Oncogene Science Inc.). Briefly, Pyk2 was
immunoprecipitated from 500 µg of cellular protein and allowed to
autophosphorylate in the presence of [32P]ATP in
vitro as described above. The 32P-labeled Pyk2 was
recovered from the beads by boiling in SDS loading buffer. The
supernatant was harvested by centrifugation and diluted in SH2 binding
buffer (phosphate-buffered saline, 1% Triton X-100, and 5 mM dithiothreitol). Equal volumes of diluted 32P-labeled Pyk2 were added to 15 µl of each prewashed
SH2-agarose conjugate or protein A-Sepharose and incubated for 2 h
on ice. In parallel, immunoprecipitation of Pyk2 was done with 1 µg
of anti-Pyk2 antibody. The beads were washed four times with 1 ml of
SH2 binding buffer and analyzed by SDS-PAGE.
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RESULTS |
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Fluoroaluminate Induces Tyrosine Phosphorylation of a 110-kDa Protein in Osteoblastic MC3T3-E1 Cells-- Previously, we had shown that fluoroaluminate induced the tyrosine phosphorylation of several proteins in osteoblastic MC3T3-E1 cells, which reached a maximum between 30 and 60 min of stimulation (17). Here, we have stimulated serum-depleted cultures with 10 mM NaF and 10 µM AlCl3 for 20 min. Control cells were left untreated or were treated with 10 µM AlCl3. In addition to previously observed tyrosine-phosphorylated proteins of 120, 130, and 65-70 kDa, more precise analysis of the Western blots revealed enhanced tyrosine phosphorylation of another 110-kDa protein (Fig. 1A) that is herein referred to as p110. NaF alone induced a qualitatively identical but weaker phosphotyrosine increase (data not shown). Stimulation of MC3T3-E1 cells with 10 µM AlCl3 did not result in an alteration of tyrosine-phosphorylated proteins (Fig. 1A).
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Identification of p110 as Pyk2 Tyrosine Kinase-- Pyk2, a cytoplasmic tyrosine kinase of the Fak family, has recently been shown to participate in signal transduction involving G protein-coupled receptors (19, 20). Pyk2 has an apparent molecular mass of 112 kDa (19) and could therefore be a candidate for the 110-kDa tyrosine-phosphorylated protein detected in fluoroaluminate-treated osteoblasts. Pyk2 has been reported to have a restricted tissue distribution (24-26), but no data are available about the presence of the kinase in bone cells. To evaluate whether or not Pyk2 is expressed in osteoblastic cells, Pyk2 levels were analyzed by Western blot analysis of extracts from different cell lines and from primary human osteoblast-like cells using an anti-Pyk2 polyclonal antibody that does not cross-react with Fak (see "Experimental Procedures") (Fig. 2A). An immunoreactive band of the expected molecular mass was detected in MC3T3-E1 cells (Fig. 2A). The signal was stronger in rat pheochromocytoma (PC12) cells, which have been reported to abundantly express Pyk2 (19, 27). Consistent with earlier observations (25), Pyk2 was not detected in NIH3T3 fibroblasts. Low amounts of Pyk2 were present in primary human osteoblasts (Fig. 2A) and primary mouse osteoblasts (data not shown), suggesting that Pyk2 is expressed in osteoblasts in situ.
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Pyk2 Kinase Is Activated in Primary Murine Calvarial Osteoblast-like Cells-- The data shown above indicated that Pyk2 is expressed in the MC3T3-E1 cell line and in primary human osteoblast-like cells and that fluoroaluminate can stimulate Pyk2 phosphorylation in MC3T3-E1 cells. To investigate whether fluoroaluminate-induced Pyk2 phosphorylation also occurs in primary cells, we measured Pyk2 phosphorylation in primary cultures of murine calvarial osteoblasts. These cultures expressed higher amounts of Pyk2 compared with human osteoblast-like cells (data not shown). Pyk2 was immunoprecipitated, and its phosphorylation was analyzed by anti-phosphotyrosine Western blotting. In these primary cultures, fluoroaluminate-induced tyrosine phosphorylation of Pyk2 increased with time of treatment up to 60 min (Fig. 3A). A Western blot for Pyk2 was done to confirm that equal amounts of Pyk2 were present in Pyk2 precipitates (Fig. 3B). These data indicate that fluoroaluminate induces Pyk2 phosphorylation in primary cultures of osteoblast-like cells and not only in a cell line.
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Pyk2 Kinase Activity in MC3T3-E1 Cells Is Increased by Fluoroaluminate in a Dose- and Time-dependent Manner-- As shown in Fig. 1, p110/Pyk2 becomes tyrosine-phosphorylated in response to fluoroaluminate. Increased tyrosine phosphorylation of Pyk2 in response to G protein-coupled receptor agonists has been reported by several groups (19, 20, 29). However, the autocatalytic activity of Pyk2 was either unchanged (30) or not documented in most studies. In this study, we consistently observed a maximal 2-3-fold increase in Pyk2 kinase activity upon fluoroaluminate treatment of MC3T3-E1 cells (Fig. 4). The dose dependence of this effect was studied by treating MC3T3-E1 cells for 30 min with different concentrations of fluoroaluminate, and Pyk2 activation was analyzed in Pyk2 immune complex kinase assays (Fig. 4A). Maximal autocatalytic activity of Pyk2 was detected with 10 mM NaF (Fig. 4A). The kinetics of the autophosphorylation of Pyk2 were studied by analyzing extracts of cells that had been treated for increasing periods of time with fluoroaluminate (1-90 min). Stimulation of osteoblastic MC3T3-E1 cells with fluoroaluminate induced a rapid elevation of Pyk2 kinase activity (Fig. 4B). The peak of Pyk2 activity was reached after 10-15 min of treatment and declined slowly over 1.5-2 h to basal activity. Elevated Pyk2 kinase activity was also measured with enolase as an exogenous substrate (data not shown). Thus, the data from the immune complex kinase assays suggest that activation of heterotrimeric G proteins by fluoroaluminate induces a moderate, but reproducible increase in Pyk2 kinase activity in MC3T3-E1 cells.
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Src Kinase Mediates the Tyrosine Phosphorylation of Pyk2 in MC3T3-E1 Cells-- The observation that the increase in Pyk2 tyrosine phosphorylation is much more pronounced than the increase in autophosphorylation activity (compare Figs. 1 and 4) suggested that an associated kinase contributed to Pyk2 tyrosine phosphorylation. Src kinase has been demonstrated to associate with Pyk2 (20, 28) and is required for high activation of Erk by Pyk2 in PC12 cells (20). Thus, we studied the association of Pyk2 and Src in MC3T3-E1 cells by Src immune complex kinase assays in fluoroaluminate-treated cells. To avoid possible interference of antibody with Src-Pyk2 complex formation, we used a mAb recognizing N-terminal amino acids of Src (LA-074), a region unlikely to be important in Src-Pyk2 interactions. The immune complexes were washed under nonstringent conditions, subjected to kinase assay, and analyzed by SDS-PAGE and autoradiography.
Autophosphorylation of Src was induced within 1 min, showed a peak of activity 5 min after addition of fluoroaluminate and slowly decreased with time of treatment (band at ~60 kDa) (Fig. 5, A and B). The maximal activation of Src was ~3-fold after 5 min. This increase in Src activity is in agreement with other studies (20, 31). A control blot of the Src immunoprecipitates with anti-Src antibodies showed that a similar amount of enzyme was present in all samples (Fig. 5C). Several other proteins became phosphorylated in Src immune complex kinase assays. These Src-associated proteins are likely to represent either Src substrates or kinases capable of autophosphorylation. The strongest incorporation of 32P into specifically bound proteins was observed in a unknown protein of ~140 kDa (Fig. 5A). Weaker bands were detected at ~70 and 50 kDa; the latter one most likely represents nonspecific phosphorylation of the the antibody heavy chain. Interestingly, a protein of ~110 kDa (p110) was detected in Src immune complex assays in fluoroaluminate-treated MC3T3-E1 cell lysates, but not in untreated controls (Fig. 5A). The intensity of radioactivity incorporated into this protein increased with time (Fig. 5, A and B).
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Association of in Vitro Phosphorylated as Well as Native Pyk2 with
the Src SH2 Domain--
Recruitment of Src to Fak and Pyk2 was
reported to be mediated by the Src SH2 domain (20, 28, 32). To test
whether phosphorylated Pyk2 associated with the SH2 domain of Src,
in vitro phosphorylated, immunopurified, denatured, and
renatured Pyk2 was incubated with immobilized SH2 domains of Src,
phospholipase C, and the p85 subunit of phosphatidylinositol
3-kinase (the p85 PI 3-kinase C-terminal SH2 domain). The binding of
32P-labeled Pyk2 to individual SH2 domains was analyzed by
SDS-PAGE and quantified with a PhosphorImager (Fig.
6A). As a control, Pyk2 was
immunoprecipitated under the same conditions. Of three SH2 domains
tested, only the Src SH2 domain efficiently bound Pyk2 (Fig.
6A). Thus, purified renatured Pyk2 directly associates with
the Src SH2 domain.
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DISCUSSION |
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Tyrosine phosphorylation is important for G protein-coupled
receptor signaling (for a recent review, see Ref. 33). Tyrosine kinases
activated by G protein-coupled receptors include the cytoplasmic enzymes Fak and Src family kinases (31, 34, 35), whose substrates are
likely to participate in cytoskeletal rearrangement
(e.g. paxillin, p130Cas, and vinculin). The
importance of tyrosine phosphorylation for the mitogenic effect of
fluoride on osteoblasts was suggested based on the observation that
cell proliferation was markedly reduced by tyrosine kinase inhibitors
(2, 16). Very limited information is available about the tyrosine
kinases participating in fluoride-induced osteoblast signal
transduction. Previously, we reported that tyrosine phosphorylation of
several intracellular proteins increases when osteoblastic MC3T3-E1
cells are treated with the G protein activator fluoroaluminate; major
bands were observed at 70, 120, and 130 kDa (17). In this study, we
described a 110-kDa protein whose tyrosine phosphorylation increased in response to fluoroaluminate. Subsequently, we identified this protein
as the tyrosine kinase Pyk2. In addition to fluoroaluminate, the
phosphorylation of Pyk2 in MC3T3-E1 cells was also increased upon
stimulation of cells with ligands of G protein-coupled receptors, such
as bradykinin, thrombin, and endothelin-1, indicating that Pyk2
phosphorylation occurs via a G protein activation mechanism. In
agreement with our findings, Pyk2 kinase was originally reported to be
activated by intracellular Ca2+ generated by G
protein-coupled receptor signaling in neuronal cells (19). This is the
first report of Pyk2 expression in osteoblast-like cells and of its
regulation by G protein signaling in these cells. Presently, the
identity of the G protein regulating Pyk2 activity remains elusive.
Taking into account that a common G protein that is activated by
bradykinin, thrombin, and endothelin-1 is Gq, it is
possible that G
q mediates Pyk2 activation. However, the involvement of G
i cannot be excluded since Pyk2
activation is partially sensitive to pertussis toxin (data not
shown).
Pyk2 is also known as Cak (cell adhesion
kinase-
) (24) and RAFTK (related
adhesion focal tyrosine
kinase) (36), and it belongs to the Fak kinase family.
Similar to Fak, Pyk2 consists of a kinase domain and large amino- and
carboxyl-terminal sequences that contain neither SH2 nor SH3 domains.
Both proteins also seem to share some functional characteristics, such
as the downstream targets paxillin and p130Cas (26, 30, 37,
38) and the ability to associate with Src family kinases (20, 32, 39).
On the other hand, Pyk2 and Fak clearly show different modes of
regulation (29, 40) and a distinct subcellular localization (24) in
certain cell types. Pyk2 is abundantly expressed in neuronal and
hematopoietic cells and is not present in some other cell types,
whereas Fak is expressed in a variety of tissues at similar levels (25,
26, 36). In relation to the possible different roles of Pyk2 and Fak,
it is interesting that preferential association of Src with Pyk2, but
not with Fak, was observed in fluoroaluminate-stimulated cells. Although Fak also becomes tyrosine-phosphorylated upon fluoroaluminate treatment of MC3T3-E1 cells, we failed to detect a significant increase
in Fak autophosphorylation activity under the same conditions (data not
shown). In addition, Fak was not increased in Src immune complexes of
fluoroaluminate-treated cells. This observation is in agreement with
the unaffected Fak kinase activity since autophosphorylation of Fak at
tyrosine 397 is required for Src binding (32). This interesting
differential behavior of Fak and Pyk2 could be due to different modes
of activation of the two related kinases.
Both Fak and Pyk2 are potently activated by integrins, receptors that mediate cell adhesion to extracellular matrix proteins (26, 30, 38, 41, 45). In addition, recent reports indicate that stimulation of the platelet-derived growth factor receptor can lead to Fak activation (42, 43). However, there are no reports of Pyk2 phosphorylation mediated by receptor tyrosine kinases, such as the receptors for platelet-derived growth factor or epidermal growth factor. Moreover, we did not observe a significant increase in Pyk2 tyrosine phosphorylation upon treatment of MC3T3-E1 cells with platelet-derived growth factor, epidermal growth factor, or insulin (data not shown). One possible difference between Pyk2 and Fak could be that Pyk2 is more responsive to activation by G protein-coupled receptors, whereas Fak might be more responsive to activation by receptor tyrosine kinases. Consistent with this proposal is the observation that Fak phosphorylation and activity were not changed with increasing intracellular Ca2+ levels, which strongly activated Pyk2 (19), and that Pyk2, but not Fak, associated with paxillin upon stimulation of epithelial cells with angiotensin II (44). Future studies will determine which conditions account for the selective activation of one or the other of the two related kinases.
Our data indicate that fluoroaluminate transduces a signal to a G protein, which, in turn, activates (directly or indirectly) Src and Pyk2 kinases and causes their association. The kinetics of Src activation are slightly faster than those of Pyk2 activation, phosphorylation, and association with Src, suggesting that Src activation occurs first and therefore may be a trigger for Pyk2 activation. Autophosphorylation of tyrosine 402 in Pyk2 has been shown to be a prerequisite for association with Src (20). This is consistent with our data showing that there is a substantial Pyk2 basal activity in unstimulated cells. This basal Pyk2 activity may come from continuous integrin stimulation via adherence of cells in culture to plastic or in vivo to the extracellular matrix. Thus, Pyk2 activation may result from a combination of basal integrin signaling and activated G protein/ Src-mediated signaling.
What is the role of Pyk2 in osteoblast-like cells? Tyrosine phosphorylation has been proposed to be required for osteoblast proliferation in response to fluoroaluminate (2). Among the tyrosine kinases activated in fluoroaluminate-treated MC3T3-E1 osteoblasts, Pyk2 is the first enzyme that shows somewhat restricted tissue distribution. Therefore, Pyk2 activation might contribute to the higher susceptibility of bone-forming osteoblasts to fluoroaluminate as compared with cells from other tissues. Furthermore, recent evidence suggests that adhesion-mediated signaling in osteoblast-like cells may be necessary for bone morphogenic protein-2-induced differentiation (46). Pyk2 has been suggested to mediate Erk (19, 20) and c-Jun N-terminal protein kinase activation (27, 29). Erk activation is generally recognized as a signal in mitogenesis and, in rare cases, in differentiation (47). Presently, there is not much information on the role of c-Jun N-terminal protein kinase in osteoblasts. Generation of Pyk2-deficient mice and analysis of their bones and bone cells in vitro should be a useful approach to address some of these questions.
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ACKNOWLEDGEMENTS |
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We thank Dr. Kurt Ballmer-Hofer for valuable discussions and for providing anti-Src antibodies. Dr. Lee Graves and Prof. H. S. Earp are acknowledged for the kind gift of a Pyk2 polyclonal antibody that was used in initial experiments. We are very grateful to Drs. Patrick B. Dennis and Jean Feyen for critical reading of the manuscript and for fruitful discussions. Ngoc-Hong Luong-Nguyen is acknowledged for technical help in some experiments.
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FOOTNOTES |
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* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Research Bone
Metabolism, Novartis Pharma AG, K-125.9.12, P. O. Box, CH-4002 Basel, Switzerland. Tel.: 4161-696-44-49; Fax: 4161-696-38-49.
1 The abbreviations used are: mAb, monoclonal antibody; PAGE, polyacrylamide gel electrophoresis; PI, phosphatidylinositol 3-kinase.
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REFERENCES |
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