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Src Homology 2 (SH2)-Containing 5'-Inositol Phosphatase Localizes to Podosomes, and the SH2 Domain Is Implicated in the Attenuation of Bone Resorption in Osteoclasts

Graduate School of Biological Sciences (K.Y., M.M., K.M., H.T., N.I.-K., T.T.), Nara Institute of Science and Technology, Ikoma, Nara 630-0192, Japan; Division of Microbiology (T.S.), Department of Pathology and Immunology, Akita University School of Medicine, Akita 010-8543, Japan

Address all correspondence and requests for reprints to: Tatsuo Takeya, 8916-5 Takayama, Ikoma, Nara 630-0192, Japan. E-mail: ttakeya{at}bs.naist.jp.

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
c-Src plays an important role in bone resorption by osteoclasts. Here, we show using wild-type and ship^–/– osteoclasts that Src homology 2 (SH2)-containing 5'-inositol phosphatase (SHIP) appeared to negatively regulate bone resorption activated by c-Src. SHIP was found to localize to podosomes under the influence of c-Src, and the presence of either the amino-terminal region comprising the SH2 domain or the carboxyl-terminal region was sufficient for its localization. Although SHIP lacking a functional SH2 domain was still found in podosomes, it could not rescue the hyper-bone resorbing activity and hypersensitivity to receptor activator of nuclear factor-B ligand in ship^–/– osteoclasts, suggesting that the localization of SHIP to podosomes per se was not sufficient and the SH2 domain was indispensable for its function. Cas and c-Cbl, known to function in podosomes of osteoclasts, were identified as novel proteins binding to the SHIP SH2 domain by mass spectrometric analysis, and this interaction appeared to be dependent on the Src kinase activity. These results demonstrate that c-Src enhances the translocation of SHIP to podosomes and regulates its function there through the SH2 domain, leading to an attenuation of bone resorption.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
THE NONRECEPTOR TYROSINE kinase c-Src plays an essential role in organizing the cytoskeleton, adhesion, and migration (1). Targeted disruption of c-src in mice results in osteopetrosis caused by a functional defect of osteoclasts (2, 3). Osteoclasts are multinucleated giant cells and known to differentiate from monocyte/macrophage-lineage cells on stimulation with receptor activator of nuclear factor-B ligand (RANKL). Osteoclasts isolated from c-src knockout mice show an abnormal cytoskeletal structure, retarded cell migration, and reduced bone-resorbing activity (4, 5). It has been reported that stimulation of integrin-_vß₃ with osteopontin or vitronectin could activate c-Src, and subsequently many adaptor proteins or cytoskeleton-related tyrosine kinases such as c-Cbl, Pyk2, and Crk-associated substrate (Cas) (p130Cas) become phosphorylated in osteoclasts (6, 7, 8, 9). c-Src and these substrates form complexes and localize to podosomes, which are specialized adhesion structures closely related to focal adhesions, regulating cell migration and bone resorption (5, 7, 10, 11, 12, 13). Phosphatidylinositol 3'-kinase (PI3K) is one of the major downstream targets of c-Src and also localizes to podosomes in osteoclasts through interaction with gelsolin and c-Src upon the activation of integrin-_vß₃ (14, 15). Inhibition of PI3K by wortmannin was reported to impair the attachment to bone and cause a disappearance of podosomes (16). Furthermore, Miyazaki et al. (11) reported that c-Src phosphorylates residue Y731 of c-Cbl, which is known as a binding site for PI3K, and overexpression of the mutant c-Cbl Y731F decreased bone-resorbing activity in osteoclasts. Together, these results indicated that the Src/PI3K pathway is required for podosome assembly, motility, and bone resorption in osteoclasts.

Src homology 2 (SH2)-containing inositol 5'-phosphatase (SHIP) is an enzyme that catalyzes the dephosphorylation of phosphatidylinositol (3,4,5) triphosphates [PtdIns(3,4,5)P₃] and generates phosphatidylinositol (3, 4) diphosphates [PtdIns(3,4)P₂] (17, 18). Because various kinds of growth factors are known to increase the amount of cellular PtdIns(3,4,5)P₃ through the activation of PI3K, it is widely accepted that SHIP antagonizes the PI3K signaling pathway. In fact, several lines of evidence clearly demonstrated that SHIP negatively regulates macrophage colony-stimulating factor (M-CSF) (18), B cell receptor (19), steel factor (20), IL-3 (21), and integrin signaling (22) in many cell types. SHIP expression is restricted to hematopoietic cells, and targeted deletion of the ship gene in mice results in a myeloproliferative disorder with extensive infiltration of myeloid cells in the lung (21, 23). More recently, Takeshita et al. (24) found that ship-null mice were severely osteoporotic because of increased numbers of osteoclasts. Bone marrow macrophages (BMM) from ship-null mice were hypersensitive to M-CSF and RANKL; consequently, osteoclastogenesis was accelerated, and the survival of osteoclasts was prolonged (24). Because the 5'-phosphatase activity measured in vitro exhibited no significant increase on stimulation with growth factors (17) and the expression of membrane-targeted SHIP decreased levels of PtdIns(3,4,5)P₃ in COS-7 cells (25), it is believed that the translocation of SHIP to the membrane where its substrate seems to be accumulated is central to its activity. Supporting this, coligation of the B-cell antigen receptor with FcRIIB resulted in the recruitment of SHIP to immunoreceptor tyrosine-based inhibitory motif (ITIM) of FcRIIB, leading to an attenuation of the B-cell antigen receptor signaling (26, 27). Likewise, in mast cells, SHIP binds FcRIIB or mast cell function-associated antigen and negatively regulates FcR1-induced degranulation (28, 29). In contrast to the immunoreceptor signaling, little is known about how SHIP activity is regulated and/or what molecule regulates the localization of SHIP in osteoclasts.

We previously searched for c-Src-binding proteins in osteoclast precursors using a combination of pull-down assays and mass spectrometric analysis (9). As a result, we identified 10 species of proteins, and SHIP was one. In this study, we investigated the functional role of c-Src in phosphatidylinositol signaling in osteoclasts, particularly focusing on SHIP in wild-type as well as ship^–/– osteoclasts. SHIP was found mainly in podosomes under the influence of c-Src. Although direct interaction with c-Src was not required for the localization of SHIP, the SH2 domain of SHIP appeared to be indispensable for the function of SHIP in podosomes. The significance of this novel function of c-Src as a modulator of SHIP is discussed.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Cells and reagents
RAW264 cells were cultured in Eagle’s MEM (Nissui, Tokyo, Japan) supplemented with nonessential amino acids (Invitrogen, Carlsbad, CA), L-glutamine, and 10% fetal calf serum (FCS). HeLa, human embryonic kidney (HEK) 293, and NIH3T3 cells were cultured in DMEM (Invitrogen) supplemented with 10% FCS, penicillin, and streptomycin. Glutathione-S-transferase (GST)-RANKL was prepared as described previously. Anti-SHIP monoclonal antibody (sc-8425), anti-C-terminal Src kinase (anti-CSK) antibody (sc-286), and normal mouse IgG were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-Src monoclonal antibody (OP-07) was obtained from Oncogene Research Products (San Diego, CA). Anti-green fluorescent protein (anti-GFP) monoclonal antibody (JL-8), anti-Cbl monoclonal antibody, and anti-Cas monoclonal antibody were purchased from BD Biosciences (San Diego, CA). Anti-ß-actin monoclonal antibody (AC-74) and antivinculin monoclonal antibody were from Sigma Chemical Co. (St. Louis, MO). Anti-Src (pY⁴¹⁸) was from Biosource International (Camarillo, CA), and antiphosphotyrosine antibody (4G10) was from Upstate Biotechnology (Lake Placid, NY). Horseradish peroxidase-conjugated sheep antimouse IgG and horseradish peroxidase-conjugated protein-A were purchased from Amersham-Pharmacia Biotech (Piscataway, NJ). LY294002 and PP1 were obtained from Calbiochem (La Jolla, CA) and BIOMOL (Plymouth Meeting, PA), respectively.

Isolation of bone marrow cells and culture of BMM
Conditioned medium of NIH3T3 cells stably expressing a human M-CSF vector (RIKEN DNA Bank, RDB no. 1524) (30) was used as a source of M-CSF. This conditioned medium contains approximately 3.3 µg/ml M-CSF, as judged by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay (M-CSF-dependent growth) using BMM. In addition, FACS analysis showed that 94.8% of cells were Mac-1 positive when bone marrow cells were cultured with 3% conditioned medium. BMM were prepared as described previously (31) with some modification. In brief, bone marrow cells were collected by flashing the femurs and tibias of 4- to 8-wk-old wild-type or ship knockout mice (20) with -MEM (Invitrogen), and red blood cells were removed by treatment with ammonium chloride solution. After a wash, the cells were cultured in -MEM supplemented with 10% FCS. After 12–16 h, nonadherent cells were collected and cultured an additional 1–3 d in -MEM supplemented with 10% FCS and 100 ng/ml M-CSF. These BMM were used for osteoclastogenesis and retroviral gene transfer as described below.

Constructs, transfection, and retroviral gene transfer
Mouse SHIP cDNA was kindly provided by Dr. Ravichandran (University of Virginia, Charlottesville, VA) and subcloned into the GFP expression vector (a gift from Dr. Umezono). Various mutants of SHIP were generated by PCR-based mutagenesis, all constructs were sequenced, and the presence of appropriate mutations was confirmed. Mouse c-Src and CSK were cloned by PCR, and mutations were introduced similarly. c-Src wild-type, Y527F, and K295M were subcloned into pRc/CMV (Invitrogen). The c-Src SH3 domain (amino acids 1–149) and SH3-SH2 domain (amino acids 1–262) were subcloned into pGEX-3X (Amersham Biosciences, Arlington Heights, IL). GFP-SHIP, c-Src K295M, and CSK wild-type and CSK kinase dead (CSK-KD) (K222R) cDNA were subcloned into the pCX4puro retroviral vector (a gift from Dr. Akagi, Osaka Bioscience Institute, Osaka, Japan). c-Cbl expression vector was kindly provided by Dr. Shishido, Nara Institute of Science and Technology (NAIST), Nara, Japan. Transfections were performed using the FuGene6 transfection reagent (Roche, Indianapolis, IN) according to the manufacturer’s recommendations. To prepare the retrovirus, the plasmid was introduced into HEK293 cells with the pE-eco vector and pGP vector (TaKaRa, Tokyo, Japan) and cultured for 48 h. The supernatant was collected and filtered through a 0.22-µm pore filter. For infection, BMM prepared as described above were cultured with the retrovirus for 2 d in -MEM supplemented with 10% FCS and 100 ng/ml M-CSF and additionally cultured for 3 d in fresh medium in the presence of 3 µg/ml puromycin for selection.

Osteoclastogenesis in vitro
BMM were harvested and seeded on either plastic or calcium-phosphate-coated plates (BD Biosciences) at 2 x 10⁵ cells/ml on the day before stimulation with M-CSF and RANKL. M-CSF and RANKL were added to final concentrations of 100 and 50 ng/ml, respectively. After 72 h, the medium was changed to fresh medium containing M-CSF and RANKL, and incubation was continued for another 24–48 h. Osteoclastogenesis using the mouse monocyte/macrophage cell line RAW264 was performed as reported (32). The tartrate-resistant acid phosphatase (TRAP) assay and bone resorption assay were performed as described previously (32, 33).

Immunocytochemistry
Cells grown on coverslips were fixed with 3.7% paraformaldehyde, permeabilized with 0.2% Triton X-100/PBS, and blocked with 5% skim milk/PBS for 1 h. Then cells were incubated with appropriate antibodies for 1 h and visualized with secondary antibody conjugated to Alexa fluor 488 or Texas-red (Molecular Probes, Eugene, OR). Samples were viewed with a LSM410 laser scanning microscope (Zeiss, Oberkochen, Germany).

Pull-down assay, immunoprecipitation, and Western blotting
Cells were lysed with a 1% Triton X-100-containing buffer [50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 2 mM EDTA, 2 mM phenylmethylsulfonyl fluoride, 2 mM Na₃VO₄, 20 mM NaF, 100 kIU/ml aprotinin, and 1% Triton X-100). The lysates were clarified by centrifugation for 20 min. For the pull-down assay, the lysates were incubated for 4 h with 10 µg GST or GST fusion protein immobilized on glutathione-Sepharose beads (Amersham). After the beads had been washed four to five times by centrifugation, they were suspended in sample buffer. For immunoprecipitation, the lysates were incubated with 1 µg anti-SHIP, anti-Cbl, anti-GFP, or anti-Src antibody at 4 C for 12–16 h and then with 20 µl protein G-Sepharose beads (Amersham) for 1 h. The immunoprecipitates were washed four to five times with lysis buffer and suspended with sample buffer. The proteins were separated by SDS-PAGE, transferred to polyvinylidene difluoride membrane, and detected by Western blotting.

Mass spectrometry
Osteoclasts differentiated from RAW264 cells were stimulated with 500 µM pervanadate for 15 min. The lysates were collected and incubated with the GST-SHIP-SH2 domain (amino acids 1–169) immobilized to glutathione beads for 4 h at 4 C. The beads were washed with lysis buffer four to five times by gentle centrifugation, and proteins binding SHIP-SH2 were eluted with a 0.5 M NaCl-containing lysis buffer. The proteins were separated with SDS-PAGE and visualized with Sypro-Ruby stain (Molecular Probes). The mass spectrometric analysis of these proteins was performed as previously described (34).

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Enhanced mineral-hydrolyzing activity by c-Src in ship-null osteoclasts
To examine the functional relationship between c-Src and SHIP in osteoclasts, we first examined the effect of c-Src kinase activity on bone resorption in wild-type and ship-null mouse osteoclasts. To this end, dominant negative CSK (CSK-KD, K222R) was expressed in BMM by retroviral gene transfer to enhance c-Src kinase activity (Fig. 1A) and cells were cultured on synthetic calcium phosphate thin film-coated plates in the presence of RANKL and M-CSF. Consistent with a previous report (24), the hydrolyzing activity of ship-null osteoclasts was stronger than that of mock-transfected wild-type cells (Fig. 1B), indicating that SHIP plays a negative role in bone-resorbing activity. The expression of CSK-KD increased the hydrolysis in both wild-type and ship-null osteoclasts, whereas the fold increase in the hydrolyzing activity caused by CSK-KD expression was much greater in ship-null osteoclasts than in wild-type cells (Fig. 1B). Regarding this, the expression level of CSK-KD and the level of phosphorylation at the autophosphorylation site of c-Src (Y418) were comparable between both cells (Fig. 1A) and the differentiation into TRAP-positive multinucleated cells was not essentially affected by the expression of CSK-KD (Fig. 1C), suggesting that a deficiency of SHIP and c-Src activity enhanced the bone-resorbing reactions synergistically and that some functional association between c-Src and SHIP existed. Incidentally, the difference in the number of TRAP-positive multinucleated cells between ship wild-type and ship-null osteoclasts on either plastic or calcium-phosphate-coated plates (Fig. 1C and supplemental Fig. 1A, published as supplemental data on The Endocrine Society’s Journals Online web site at http://endo.endojournals.org.) was not significant in our system, in contrast to the previous work (24). Instead, we observed hypersensitivity to RANKL, enlarged cell shape, and an increased number of nuclei in ship-null osteoclasts (see supplemental Fig. 1B and Fig. 6).

View larger version (50K): [in this window] [in a new window]   FIG. 1. Src kinase activation markedly increased bone resorption in ship–/– osteoclasts. A, Expression of CSK-KD and phosphorylation at the autophosphorylation site of c-Src (Y418) were monitored by Western blotting using anti-CSK antibody and anti-pY418 Src antibody, respectively. Anti-ß-actin was used as a loading control. WCL, Whole-cell lysate. B, Empty vector or CSK-KD was expressed in BMM from ship+/+ or ship–/–, and osteoclastogenesis was induced by RANKL (50 ng/ml) and M-CSF (100 ng/ml) on synthetic calcium phosphate thin film-coated plates for 5 d. The mineral resorption of the plates was assessed morphometrically by the von Kossa method (left). The area of resorption in 10 fields was measured, and data representative of two independent experiments are shown (n = 2) (right). **, P < 0.01. The area of resorption observed in mock-transfected ship+/+ cells was taken as 1.0, and all values were calculated and shown as fold differences. C, BMM were prepared as in A and stimulated with RANKL on plastic culture plates for 4 d. Osteoclastogenesis was monitored by TRAP staining. Bar, 200 µm (left). Right, Quantification of TRAP-positive multinucleated cells (MNCs) of three independent experiments (n = 3). The number of TRAP-positive MNCs observed in mock-transfected ship+/+ cells was taken as 1.0, and all values were calculated and shown as fold differences.

View larger version (50K): [in this window] [in a new window]   FIG. 6. SHIP SH2 mutant could not rescue the hyper-bone-resorbing activity and hypersensitivity to RANKL in ship–/– osteoclasts. A, Expression of GFP, GFP-SHIP wild-type (wt), and GFP-SHIP R34L in BMM. The preparation of BMM and expression of SHIP using retroviral vectors are described in Materials and Methods. Expression of SHIP was detected by Western blotting using anti-SHIP antibody. Actin was used to monitor the amount of protein applied. B, Bone-resorbing activity of the SH2 mutant. The expression vectors indicated were introduced into BMM from ship+/+ or ship–/–, and osteoclasts were generated with RANKL and M-CSF. The mineral resorption on the plate was assessed as in Fig. 1 (left). Quantification of resorbing activity (right) was done as in Fig. 1. *, P < 0.05. C, The expression vectors indicated were introduced into BMM from ship+/+ or ship–/–, and the cells were stimulated with various concentrations of RANKL (12.5–50 ng/ml). After 4 d, osteoclastogenesis was monitored by TRAP staining. Bar, 200 µm (left). The relative numbers of TRAP-positive multinucleate cells (MNCs) formed in the presence of 12.5 ng/ml RANKL were counted in two independent experiments performed in triplicate (right). **, P < 0.01. D, Double staining of SHIP wild-type or R34L and F-actin in ship–/– osteoclasts. Cells were prepared as in C, and staining was performed as in Fig. 2. Bar, 25 µm.

View larger version (30K): [in this window] [in a new window]   FIG. 2. SHIP localizes to podosomes in osteoclasts. A, Osteoclasts of ship+/+ or ship–/– were differentiated in vitro, and the localization of SHIP was monitored immunocytochemically using anti-SHIP antibody. Bar, 25 µm. B, Double staining of SHIP and F-actin in osteoclasts using a confocal microscope equipped with a x100 oil immersion lens. The arrows indicate the podosomes doubly stained. Bar, 10 µm. C, Osteoclasts were differentiated from BMM expressing empty vector, c-Src K295M, or CSK, and the localization of SHIP was monitored. F-actin was stained as in B. Bar, 25 µm (left). Expressions of c-Src, c-Src K295M, and CSK were detected by Western blotting using anti-c-Src and anti-CSK antibody. Phosphorylation of Y418 of c-Src was monitored as in Fig. 1A (right).

View larger version (27K): [in this window] [in a new window]   FIG. 3. c-Src induces translocation of SHIP to focal adhesions in HeLa cells. A, GFP-SHIP was expressed in HeLa cells together with empty vector, c-Src wild-type (WT), Y527F, or K295M, and the localization of SHIP and Src was monitored at 24–48 h after transfection. Note that SHIP localized to the tips of membranes only when c-Src Y527F was coexpressed (arrowheads). Bar, 25 µm. B, GFP-SHIP and c-Src Y527F were expressed in HeLa cells, and the localization of SHIP, vinculin, and actin was monitored immunocytochemically. Bar, 25 µm. C, HeLa cells expressing GFP-SHIP and c-Src Y527F were treated with 10 µM LY294002 or solvent (dimethylsulfoxide) for 3 h, followed by immunocytochemical staining with SHIP and c-Src antibody. Bar, 25 µm.

Identification of the domain required for the localization of SHIP to focal adhesions
To understand the mechanism involved in the Src-induced translocation of SHIP, we tried to identify the region in SHIP required for its localization to focal adhesions in HeLa cells. GFP-SHIP variants constructed for this purpose are illustrated in Fig. 4A. Regarding this, SHIP has two NPXY motifs, Y917 and Y1020, which are presumed to be potential sites of phosphorylation by tyrosine kinases. c-Src actually could phosphorylate SHIP, and the level of phosphorylation was increased when Src Y527F was expressed, whereas no phosphorylation was detected in cells expressing kinase dead c-Src (K295M) (Fig. 4B). Furthermore, the phosphorylation level of SHIP 2YF in which both Y917 and Y1020 were substituted with phenylalanine was decreased to less than 20% of that of SHIP wild-type, suggesting that Y917 and Y1020 are major sites of phosphorylation by c-Src.

View larger version (35K): [in this window] [in a new window]   FIG. 4. Identification of domain required for the localization of SHIP to focal adhesions. A, Schema of GFP-SHIP variants used. SHIP consists of a SH2 domain, a linker domain, a 5'-phosphatase domain, two NPXY motifs, and a C-terminal domain. The two Ys in the C-terminal domain indicate Y917 and Y1020 of NPXY motifs. All constructs were tagged with GFP at the N terminus. The activity for translocation to focal adhesions in each construct is summarized on the right. B, GFP-SHIP wild-type and GFP-SHIP Y917/1020F were expressed in HEK293 cells with c-Src wild-type, Y527F, or K295M. Immunoprecipitated SHIP was detected with antiphosphotyrosine antibody (4G10) and anti-GFP antibody (top). The relative phosphorylation level was determined by densitometric analysis (bottom). C, GFP-SHIP wild-type and variants were expressed in HeLa cells with (a'–f') or without (a–f) c-Src Y527F, and the localization of SHIP was detected as in Fig. 3. The arrowheads indicate the SHIP in focal adhesions. Bar, 25 µm. D, Localization of the SHIP variants and vinculin was monitored as in Fig. 3B. Bar, 25 µm. WT, Wild type.

Binding to c-Src was not essential for translocation of SHIP
It has been reported that a constitutively active form of Src localized to focal adhesions and that SHIP could associate with c-Src in several cell systems (38). We examined whether the interaction with c-Src was required for SHIP to localize to focal adhesions. To this end, we first tried to identify the domain(s) responsible for the binding in each protein. Using pull-down assays, we found that the Src SH3 domain was sufficient for this interaction (Fig. 5A). Because SHIP has several PXXP motifs that are known as putative SH3 domain-binding sites in both the N-terminal (1–457) and C-terminal (739–1190) regions, we examined which domain could bind to the Src SH3 domain. GFP-SHIP wild-type (full length), GFP-SHIP 1–457, or GFP-SHIP 739-1190 was expressed in HEK293 cells, and a pull-down assay was performed using the GST-c-Src SH3 domain. It was found that the Src SH3 domain could bind to GFP-SHIP wild-type and the 1–457 variant but not to 739-1190 (Fig. 5B). Because GFP-SHIP 1–457 was shown to contain three typical proline-rich motifs, P126–129P, P152–155P, and P251–254P, we substituted alanine for proline in each motif and tested the effect on the interaction with c-Src. As shown in Fig. 5C, the P126/129A variant (SHIP PA) lost the binding activity, whereas the other mutants showed no change. These results indicated that the proline-rich region in the N-terminal domain of SHIP was responsible for the binding to the c-Src SH3 domain when examined by pull-down assay.

View larger version (53K): [in this window] [in a new window]   FIG. 5. Interaction of c-Src and SHIP. A, Pull-down assay using GST-tagged c-Src SH3-SH2 domain or SH3 domain. The ship wild-type (WT) or ship-null (KO) mouse osteoclast lysates and GST-Src fusion proteins were mixed, and the bound proteins were separated by SDS-PAGE, followed by Western blotting using anti-SHIP antibody. B, GFP, GFP-SHIP wild-type (SHIP-wt), GFP-SHIP 1–457, and GFP-SHIP 739-1190 were expressed in HEK293 cells, and a pull-down assay was performed using the GST-Src SH3 domain. The whole-cell lysate (WCL) and Src-bound SHIP were separated by SDS-PAGE, and the GFP signal was detected by Western blotting using anti-GFP antibody. C, GFP-SHIP wild-type, P126/129A, P152/155A, and P251/254P were expressed in HEK293 cells. A pull-down assay was performed as in B. D, GFP-SHIP wild-type, GFP-SHIP P126/129A, GFP-SHIP Y917/1020F (2YF), GFP-SHIP 1–167, GFP-SHIP 1–457, and GFP-SHIP 1–457 R34L were expressed in HEK293 cells with c-Src Y527F. Immunoprecipitates (IP) with anti-Src antibody were detected with anti-GFP and anti-Src antibodies. E, GFP-SHIP wild-type and 1–167 were expressed with c-Src Y527F in HeLa cells, and the localization of SHIP and vinculin was observed as in Fig. 3.

SHIP-SH2 domain is essential for the regulation of bone resorption
We then investigated the significance of the SHIP SH2 domain to bone resorption and osteoclastogenesis in vitro. To this end, BMM expressing GFP (control), GFP-SHIP wild-type, or GFP-SHIP R34L were prepared and induced to become osteoclasts by RANKL. The expression of exogenously expressed GFP-SHIP was comparable to that of endogenous SHIP, and no significant difference was found between the wild-type and R34L mutant (Fig. 6A). We confirmed the strong resorbing activity of ship-null osteoclasts and found that GFP-SHIP R34L could not rescue at all the hyper-bone-resorbing activity of ship-null osteoclasts, in contrast to GFP-SHIP wild-type (Fig. 6B). Next, we performed a rescue experiment for osteoclastogenesis. BMM were prepared as above and induced to rescue osteoclastogenesis in the presence of various concentrations of RANKL. As reported previously (24), ship-knockout BMM could differentiate into osteoclasts even with the lower concentration of RANKL. GFP-SHIP could rescue this phenotype, whereas the R34L variant could not (Fig. 6C). Because the R34L variant still could be found in podosomes like the wild-type (Fig. 6D), these results suggested that the recruitment to podosomes was not sufficient for the function of SHIP and that the SH2 domain was necessary for SHIP to negatively regulate bone resorption and the RANKL signaling pathway.

SHIP SH2 domain interacts with Cas and c-Cbl in a Src kinase activity-dependent manner
To understand the functional mechanism of the SHIP SH2 domain in podosomes, we searched for protein(s) binding this domain in osteoclasts. Mass spectroscopic analysis revealed that Cas and c-Cbl bound to the domain (Fig. 7A). Although Src was not included in the list of bound proteins, the interaction of Src with the SHIP SH2 domain was confirmed by pull-down assay (supplemental Fig. 2). Cas and c-Cbl are known to act downstream of Src, to interact with PI3K, and to be involved in organizing the actin cytoskeleton (6, 7, 8, 9). Moreover, the localization as well as function of these proteins in osteoclast podosomes was reported (5, 6, 8, 10, 11). As shown in Fig. 7B, we detected the direct binding of the SHIP SH2 domain to Cas and c-Cbl using pull-down assays and found that the interaction was diminished when PP1, a Src family kinase inhibitor, was administered to osteoclasts. We then examined the endogenous interaction of these proteins with SHIP by coimmunoprecipitation with anti-SHIP antibody. The interaction of these proteins with SHIP was detected in RAW264-derived osteoclasts (Fig. 7C). The interaction was relatively weak at the basal level in osteoclasts prepared from BMM, but overexpression of dominant negative CSK (CSK-KD) enhanced the binding of Cas and c-Cbl to SHIP (Fig. 7D). The involvement of the SH2 domain in binding was further confirmed using GFP-SHIP R34L (Fig. 7E). These results, therefore, suggested that SHIP binds to Cas and c-Cbl through the SH2 domain in a Src kinase activity-dependent manner.

View larger version (51K): [in this window] [in a new window]   FIG. 7. Cas and c-Cbl are SHIP-binding partners in osteoclasts. A, Mass spectrometric analysis. The GST-SHIP SH2 domain and osteoclast lysate were mixed, and the SHIP-bound proteins were separated by SDS-PAGE and visualized with Sypro Ruby staining, followed by liquid chromatography-tandem mass spectrometry analysis. The proteins identified (GenBank accession number) were as follows: tight junction protein 2 (NP_035727), Cas (AK079554), c-Cbl (B43817), mKIAA0384 (BAC41421), SEC23A (AAF08301), SEC23B (NP_033173), BTK (P35991), ARF-GEF6 (AK053163), Pstpip1 (NP_035323), phosphoprotein (AAA21731), Dok-like protein (NP_038767), dynactin 2 (NP_081427), integrin-linked protein kinase 2 (P57043), leupaxin (AK077085), Arp1 homolog (XP_238177), -parvin (XP_235771), and Ras suppressor protein 1 (Q01730). The band indicated by the asterisk was a nonspecific GST-bound protein. B, Pull-down assay using GST-SHIP-SH2. Osteoclasts differentiated from RAW264 cells were treated with or without 10 µM PP1 for 3 h, and the proteins bound to GST or GST-SHIP-SH2 were detected with anti-Cas antibody or anti-c-Cbl antibody. C, Immunoprecipitates (IP) with normal mouse IgG or anti-SHIP antibody from RAW264-derived osteoclasts were detected with anti-Cas antibody, anti-c-Cbl antibody, and anti-SHIP antibody. D, Immunoprecipitates with anti-SHIP antibody from BMM-derived osteoclasts expressing empty vector or CSK-KD were detected with anti-Cas or anti-c-Cbl antibody. The level of phosphorylation at the autophosphorylation site of c-Src (Y418) was detected by immunoblotting using pY418-specific antibody. E, GFP-SHIP wild-type (WT) or R34L mutant was expressed in HEK293 cells together with c-Src Y527F and c-Cbl. Immunoprecipitates with anti-GFP antibody were detected with anti-c-Cbl antibody or anti-GFP antibody. WCL, Whole-cell lysate.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

The intracellular distribution of SHIP has not been fully investigated even in other cell systems. Using an immunocytochemical technique, we found that SHIP mainly localized around the actin ring in osteoclasts, in particular with F-actin in podosomes. Podosomes are specialized cytoskeletal structures consisting of integrin, adaptor proteins, kinases, actin, and actin regulators (37, 39, 40) and are implicated in attachment to the extracellular matrix, migration, and bone resorption in osteoclasts. PI3K also localizes to podosomes upon stimulation with integrin (13), and PtdIns(3,4,5)P₃ induced actin polymerization through the activation of guanine-nucleotide exchange factors for Rho family small GTPases such as Vav (41), SWAP-70 (42), and P-Rex1 (43). Recently, Vav3-deficient osteoclasts were reported to be defective in actin cytoskeletal reorganization, spreading, and bone-resorbing activity (44). Therefore, SHIP seems to regulate actin polymerization negatively in podosomes by dephosphorylating PtdIns(3,4,5)P₃.

c-Src Y527F induced the translocation of SHIP to focal adhesions in HeLa cells, and inhibition of Src kinase activity impaired the localization of SHIP in osteoclasts. These results clearly demonstrated that the activation of Src was essential for the proper localization of SHIP. It has been reported that c-Src kinase activity was essential for podosome assembly, and SHIP was found to interact with c-Src. In addition, although the spatial and temporal regulation of c-Src’s activation in osteoclasts is not clear, Sandilands et al. (45) reported recently that Src becomes activated in endosomes and is translocated to the plasma membrane in fibroblasts. Therefore, a possible mechanism of Src-induced translocation of SHIP could be that c-Src accompanies SHIP to podosomes; otherwise, SHIP may be recruited by other proteins that can be activated by the kinase activity of Src. The critical difference between these two assumptions is whether the interaction between SHIP and c-Src is essential or not for the localization of SHIP to podosomes. In this study, we identified the c-Src-binding domain of SHIP for the first time and found that the SHIP mutant lacking this domain still could localize to focal adhesions. Conversely, although another SHIP variant (1–457 R34L) could bind to c-Src, it appeared not to localize to focal adhesion. Therefore, it is likely that the interaction of SHIP with partner protein(s), not direct interaction with c-Src, plays a more vital role for the localization of SHIP. Regarding this, we found that either the N-terminal nor C-terminal domain of SHIP was sufficient in the localization to Src-induced focal adhesions in HeLa cells, suggesting that the N-terminal and C-terminal regions of SHIP are regulated independently, and probably each domain has respective partner proteins for translocation and/or stabilization in podosomes. The mechanism involved in the localization of SHIP, however, remains to be elucidated.

We subsequently found using a SHIP variant lacking a functional SH2 domain (GFP-SHIP R34L) that the localization to podosomes was not sufficient for the function of SHIP. To understand the functional role of the SH2 domain, we searched for binding proteins and found that Cas and c-Cbl bound SHIP SH2 in a Src kinase activity-dependent manner. The functional importance of c-Cbl in podosome dynamics, osteoclast migration, and bone resorption has been revealed both in vitro (6, 10, 11, 13) and in vivo (46). Although deletion of the Cas gene resulted in embryonic lethality, actin stress fiber formation appeared to be severely impaired in recovered Cas^–/– fibroblasts (47). More importantly, these proteins are known to localize to podosomes of osteoclasts (15) and are well recognized as substrates of c-Src. Therefore, Cas and c-Cbl seem to be strong candidates for partner proteins in podosomes. How the interaction of Cas and/or c-Cbl with SHIP controls the function of SHIP under the control of c-Src in podosomes needs to be clarified. In this regard, 13 other proteins were also detected as novel SHIP-binding proteins including many cytoskeleton-related proteins, e.g. leupaxin, integrin-linked kinase 2, and -parvin. Although these proteins could be binding partners for SHIP or form complexes with Cas/c-Cbl and SHIP in podosomes in osteoclasts, their exact roles in the function and/or localization of SHIP also remain elusive.

Finally, PtdIns(3,4,5)P₃ can activate numerous proteins and regulate cell proliferation, survival, motility, and vesicle transport. To maintain cell homeostasis, the amount of intracellular PtdIns(3,4,5)P₃ must be strictly regulated spatially and temporally. In the present study, we showed that SHIP is recruited to podosomes under the influence of c-Src in osteoclasts. This may contribute to such a spatial and temporal regulation of PtdIns(3,4,5)P₃ during the bone resorption process. In other words, it is feasible to assume that when c-Src activates the signaling machinery required for bone-resorbing reactions, a signaling cascade that antagonizes the activated machinery could be stimulated to regulate the resorbing reactions, and SHIP may be such a modulator. In this regard, it has been reported that Src induces negative regulation under certain conditions; for example, v-src activates both mitogenic and survival signaling pathways, but the oncogenic form of Src induces apoptosis when the functions of Ras and PI3K are inhibited in fibroblasts (48). Our findings provide additional insight into the function of c-Src and phosphoinositides in osteoclasts.

	Acknowledgments

 
We are grateful Josef Penninger (Institute of Molecular and Biotechnology of the Austrian Academy of Sciences) for providing ship-knockout mice. We are also grateful Kazuyuki Ito (Osaka Medical Center for Cancer and Cardiovascular Diseases) for valuable discussions. We thank Masayoshi Kuwano [Nara Institute of Science and Technology (NAIST)] for technical support with the LC-MS/MS analysis.

	Footnotes

 
This work was supported by the NAIST Frontier Bio-COE Project.

Conflict of interest statement: No conflicts are declared.

First Published Online April 6, 2006

Abbreviations: BMM, Bone marrow macrophages; Cas, Crk-associated substrate; CSK, C-terminal Src kinase; CSK-KD, CSK kinase dead; FCS, fetal calf serum; GFP, green fluorescent protein; GST, glutathione-S-transferase; HEK, human embryonic kidney; M-CSF, macrophage colony-stimulating factor; PI3K, phosphatidylinositol 3'-kinase; PtdIns(3,4,5)P₃, phosphatidylinositol (3,4,5) triphosphates; RANKL, receptor activator of nuclear factor-B ligand; SH2, Src homology 2; SHIP, SH2-containing 5'-inositol phosphatase; TRAP, tartrate-resistant acid phosphatase.

Received October 14, 2005.

Accepted for publication March 24, 2006.

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