title: An osteoclastic protein-tyrosine phosphatase regulates the β3-integrin, syk, and shp1 signaling through respective src-dependent phosphorylation in osteoclasts.
abstract: This study utilized the glutathione transferase (GST) pull-down assay to identify novel substrates of an osteoclastic protein-tyrosine phosphatase, PTP-oc. Consistent with the previous findings that the phosphorylated tyr-527 (pY527) of Src is a substrate of PTP-oc, the major protein pulled down with the phosphatase-deficient (PD)-PTP-oc-GST trapping mutant in RAW264.7 cells was Src. The GST-PD-PTP-oc also pulled down pY-Syk and pY-β(3)-integrin, but not after PP2 pretreatment. However, PTP-oc transgenic osteoclasts or PTP-oc-overexpressing RAW264.7 cells had elevated, and not reduced, levels of pY525/526-Syk and pY759-β(3) integrin, and the PTP-oc siRNA treatment drastically reduced levels of pY525/526 Syk and pY759-β(3)-integrin in RAW264.7 cells. These findings are incompatible with the premise that they are substrates of PTP-oc. The PTP-oc-dependent increases in pY525/526-Syk and pY759-β(3)-integrin levels were completely blocked by PP2, indicating that these effects are secondary to PTP-oc-mediated activation of the Src protein-tyrosine kinase (PTK). Overexpression of PTP-oc increased, and siRNA-mediated suppression of PTP-oc reduced, pY160-Vav1, pY173-Vav3, and pY783-PLCγ levels, and Rac1 activation, which are downstream mediators of the ITAM/Syk signaling. Overexpression of PTP-oc also increased, and PTP-oc siRNA treatment decreased, the pY-Shp1 levels, which were blocked by PP2. Since Shp1 is a negative regulator of osteoclast activity and is a key mediator of the ITIM signaling, these findings suggest that PTP-oc is an upstream suppressor of the ITIM/Shp1 signaling through PTP-oc-induced Src-dependent Shp1 phosphorylation. In summary, PTP-oc plays a central regulatory role in the concerted regulation of the β(3)-integrin, the ITAM/Syk, and the ITIM/Shp1 signaling indirectly through activation of Src PTK.
authors.affiliation: Musculoskeletal Disease Center, Loma Linda, CA 92357, USA. William.Lau@med.va.gov
authors.forename: K-H William
authors.initials: KH
authors.lastname: Lau
authors.forename: Virginia
authors.initials: V
authors.lastname: Stiffel
authors.forename: Mehran
authors.initials: M
authors.lastname: Amoui
mesh_terms: D000818:Animals; D001862:Bone Resorption; D002460:Cell Line; D039661:Integrin beta3 / Q000235:genetics / Q000378:metabolism*; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D009479:Neuropeptides / Q000096:biosynthesis / Q000378:metabolism; D010010:Osteoclasts / Q000378:metabolism* / Q000502:physiology; D051966:Phospholipase C gamma / Q000096:biosynthesis; D010766:Phosphorylation; D053824:Protein Tyrosine Phosphatase, Non-Receptor Type 6 / Q000378:metabolism*; D017027:Protein Tyrosine Phosphatases / Q000235:genetics / Q000378:metabolism*; D011505:Protein-Tyrosine Kinases / Q000235:genetics / Q000378:metabolism*; D051567:Proto-Oncogene Proteins c-vav / Q000096:biosynthesis; D034622:RNA Interference; D034741:RNA, Small Interfering; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D015398:Signal Transduction; D000072377:Syk Kinase; D020744:rac GTP-Binding Proteins / Q000096:biosynthesis / Q000378:metabolism; D020830:rac1 GTP-Binding Protein; D019061:src-Family Kinases / Q000378:metabolism*
chemical_list: D039661:Integrin beta3; D047908:Intracellular Signaling Peptides and Proteins; D009479:Neuropeptides; D051567:Proto-Oncogene Proteins c-vav; D034741:RNA, Small Interfering; C484602:Rac1 protein, mouse; D011993:Recombinant Fusion Proteins; C496619:Vav1 protein, mouse; C496622:Vav3 protein, mouse; D011505:Protein-Tyrosine Kinases; D000072377:Syk Kinase; C000606897:Syk protein, mouse; D019061:src-Family Kinases; D053824:Protein Tyrosine Phosphatase, Non-Receptor Type 6; D017027:Protein Tyrosine Phosphatases; D051966:Phospholipase C gamma; D020744:rac GTP-Binding Proteins; D020830:rac1 GTP-Binding Protein