Characterization of unique functionalities in c-Src domains required for osteoclast podosome belt formation.

Abstract Deletion of c-Src, a ubiquitously expressed tyrosine kinase, results in osteoclast dysfunction and osteopetrosis, in which bones harden into “stone”. In contrast, deletion of the genes encoding other members of the Src family kinase (SFK) fails to produce an osteopetrotic phonotype. This suggests that c-Src performs a unique function in the osteoclast that cannot be compensated for by other Src family kinases. We aimed to identify the molecular basis of this unique role in osteoclasts and bone resorption. We found that c-Src, Lyn and Fyn were the most highly expressed SFKs in wildtype osteoclasts, whereas Hck, Lck, Blk and Fgr displayed low levels of expression. Formation of the podosome belt, clusters of unique actin assemblies, was disrupted in src-/- osteoclasts; introduction of constitutively activated SFKs revealed that only c-Src and Fyn could restore this process. To identify the key structural domains responsible, we constructed chimeric Src-Hck and Src-Lyn constructs in which the Unique, SH3, SH2 or catalytic domains had been swapped. We found that the Src Unique, SH3 and kinase domains were each crucial to establish Src functionality. The SH2 domain could however be substituted with Lyn or Hck SH2 domains. Furthermore, we demonstrate that c-Src’s functionality is, in part, derived from an SH3-PPR interaction with c-Cbl, leading to phosphorylation of c-Cbl Tyr700. These data help to clarify Src’s unique functionality in the organization of the cytoskeleton in osteoclasts, required for efficient bone resorption and explain why c-Src cannot be replaced, in osteoclasts, by other SFKs.