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

Abstract

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-kappaB 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.

A pleckstrin homology‐related domain in SHIP1 mediates membrane localization during Fcγ receptor‐induced phagocytosis

“…Regions above or below the threshold of 0.5 are considered disordered or ordered, respectively. C ) Secondary structure predictions (helices, red cylinders; strands, blue arrows) for residues 292–401 were obtained using the webserver algorithms HNN (46), GOR4 (47), PSIPRED (48,49), SOPMA (50), Jpred3 (51), PORTER (52), PROF (53), Sspro (54), and JUFO (55,56). D ) A consensus score was generated for each residue, with predicted helices (red) valued as +1, strands (blue) as −1, and coils as 0.…”

Siglec-15 Protein Regulates Formation of Functional Osteoclasts in Concert with DNAX-activating Protein of 12 kDa (DAP12)

“…In brief, mouse bone marrow cells were cultured in tissue culture dishes with ␣-minimal essential medium containing 10% heatinactivated fetal bovine serum (FBS) and 0.5% macrophage colony-stimulating factor (M-CSF)-conditioned medium (obtained from NIH3T3/pCAhMCSF cells) (20). After an overnight incubation, nonadherent cells were harvested and cultured in plastic dishes with 5% M-CSF conditioned medium for 3 days.…”

The role of the inositol polyphosphate 5-phosphatases in cellular function and human disease

“…SHIP1 contains an N-terminal SH2 domain, a central 5-ptase domain which hydrolyses PtdIns(3,4,5)P 3 and Ins(1,3,4,5)P 4 and a Cterminal PRD containing two NPXY motifs [223]. The N-terminal SH2 domain binds tyrosine-phosphorylated proteins such as Shc [223], the Gabs (Grb2-associated binders) [224,225], the Doks (downstream of tyrosine kinases) [226][227][228], SHP-2 (SH2domain-containing protein tyrosine phosphatase 2) [229,230], c-Cbl [231] and LAT (linker for activation of T-cells) [232]. SHIP1 is recruited to the cytoplasmic tail of ITIM (immunoreceptor tyrosine-based inhibitory motif)-containing receptors Fcγ RBIIb and Cd22 following their co-ligation with the BCR via its interaction with Shc (reviewed in [1]).…”