Dynamin Forms a Src Kinase–sensitive Complex with Cbl and Regulates Podosomes and Osteoclast Activity

Abstract

Podosomes are highly dynamic actin-containing adhesion structures found in osteoclasts, macrophages, and Rous sarcoma virus (RSV)-transformed fibroblasts. After integrin engagement, Pyk2 recruits Src and the adaptor protein Cbl, forming a molecular signaling complex that is critical for cell migration, and deletion of any molecule in this complex disrupts podosome ring formation and/or decreases osteoclast migration. Dynamin, a GTPase essential for endocytosis, is also involved in actin cytoskeleton remodeling and is localized to podosomes where it has a role in actin turnover. We found that dynamin colocalizes with Cbl in the actin-rich podosome belt of osteoclasts and that dynamin forms a complex with Cbl in osteoclasts and when overexpressed in 293VnR or SYF cells. The association of dynamin with Cbl in osteoclasts was decreased by Src tyrosine kinase activity and we found that destabilization of the dynamin-Cbl complex involves the recruitment of Src through the proline-rich domain of Cbl. Overexpression of dynamin increased osteoclast bone resorbing activity and migration, whereas overexpression of dynK44A decreased osteoclast resorption and migration. These studies suggest that dynamin, Cbl, and Src coordinately participate in signaling complexes that are important in the assembly and remodeling of the actin cytoskeleton, leading to changes in osteoclast adhesion, migration, and resorption. INTRODUCTIONOsteoclasts are multinucleated bone-resorbing cells that are responsible for the degradation of bone. After the engagement of integrins with extracellular matrix proteins, osteoclasts undergo cytoskeletal reorganization and cellular polarization, resulting in the establishment of distinct apical and basolateral membrane domains (Baron et al., 1985;Vaananen et al., 2000). The domain facing the bone surface is comprised of a highly convoluted ruffled border membrane, circumscribed by a ring-like structure called the sealing zone. The sealing zone contains highly dynamic actin-containing adhesion structures known as podosomes, which mediate cell attachment and motility and which are also observed in leukocytes, macrophages, and v-Src or Rous sarcoma virus (RSV)-transformed cells (Marchisio et al., 1987;Ochoa et al., 2000). Within the core of individual podosomes, actin adopts a columnar structure perpendicular to the plasma membrane and undergoes continuous cycles of polymerization and depolymerization (Ochoa et al., 2000;Pfaff and Jurdic, 2001;Destaing et al., 2003). F-actin, cortactin, gelsolin, and the actin-regulatory proteins N-WASP and Arp2/3 are present within the podosome core, whereas integrins and the integrin-associated proteins paxillin, talin, and vinculin are localized around the actin core (Linder and Aepfelbacher, 2003). Although podosomes and focal adhesions have many of these proteins in common, podosome turnover occurs within minutes (Stickel and Wang, 1987; Chen, 1989;Destaing et al., 2003), in contrast to the relatively stable structure of focal adhesions which serve to anchor cells to the...

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