Src kinase activity is essential for osteoclast function.
Deletion of the c-src gene impairs osteoclast bone resorbing activity, causing osteopetrosis. Although it has been concluded that restoring only the Src adaptor function at least partly rescues the cell attachment and skeletal phenotypes, the contribution of Src kinase activity remains controversial. Src forms a complex with Pyk2 and Cbl after adhesion-induced stimulation of alpha(V)beta(3) integrin. To demonstrate the importance of the Pyk2-Src association in osteoclasts and to distinguish the contributions of the Src adaptor and kinase activities in cytoskeletal organization and osteoclast function, we expressed mutants of Src and Pyk2 in osteoclasts using adenovirus vectors. Eliminating the Src-binding site on Pyk2 (Pyk2(Y402F)) markedly inhibited bone resorption by osteoclast-like cells, whereas kinase-dead Pyk2 had little effect. Kinase-dead Src, unlike kinase-dead Pyk2, markedly inhibited the bone-resorbing activity of wild type osteoclasts and failed to significantly restore bone-resorbing activity to Src(-/-) osteoclast-like cells. Activation of Src kinase by overexpressing kinase-dead Csk failed to reverse the inhibitory effect of Pyk2(Y402F), suggesting that osteoclastic bone resorption requires both c-Src kinase activity and the targeting of Src kinase by Pyk2. Src-catalyzed phosphorylation of Cbl on Tyr-731 is reported to induce the activation and recruitment of phosphatidylinositol 3-kinase to the cell membrane in a signaling pathway that is critical for osteoclast function. Expressing the Cbl(Y731F) mutant in osteoclasts markedly reduced their bone resorbing activity, suggesting that phosphorylation of Cbl(Y731) and the subsequent recruitment and activation of phosphatidylinositol 3-kinase may be critical signaling events downstream of Src in osteoclasts.
Miyazaki
Tsuyoshi
T
Department of Cell Biology, Yale University School of Medicine, New Haven, Connecticut 06510, USA.
Sanjay
Archana
A
Neff
Lynn
L
Tanaka
Sakae
S
Horne
William C
WC
Baron
Roland
R
D000256:Adenoviridae / Q000235:genetics; D000818:Animals; D001665:Binding Sites; D001862:Bone Resorption; D002384:Catalysis; D002460:Cell Line; D002470:Cell Survival; D002478:Cells, Cultured; D018920:Coculture Techniques; D003599:Cytoskeleton / Q000378:metabolism; D004789:Enzyme Activation; D051418:Focal Adhesion Kinase 2; D017353:Gene Deletion; D005822:Genetic Vectors; D051379:Mice; D008856:Microscopy, Fluorescence; D009154:Mutation; D010010:Osteoclasts / Q000378:metabolism*; D010641:Phenotype; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D010766:Phosphorylation; D011233:Precipitin Tests; D011485:Protein Binding; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D015398:Signal Transduction; D013997:Time Factors; D014443:Tyrosine / Q000737:chemistry; D019061:src-Family Kinases / Q000502:physiology*
D014443:Tyrosine; D019869:Phosphatidylinositol 3-Kinases; D011505:Protein-Tyrosine Kinases; D051418:Focal Adhesion Kinase 2; C496053:Ptk2b protein, mouse; D019061:src-Family Kinases