Dynamin Reduces Pyk2 Y402 Phosphorylation and Src Binding in Osteoclasts

Abstract

Signaling via the Pyk2-Src-Cbl complex downstream of integrins contributes to the assembly, organization, and dynamics of podosomes, which are the transient adhesion complexes of highly motile cells such as osteoclasts and dendritic cells. We previously demonstrated that the GTPase dynamin is associated with podosomes, regulates actin flux in podosomes, and promotes bone resorption by osteoclasts. We report here that dynamin associates with Pyk2, independent of dynamin's GTPase activity, and reduces Pyk2 Y402 phosphorylation in a GTPase-dependent manner, leading to decreased Src binding to Pyk2. Overexpressing dynamin decreased the macrophage colony-stimulating factor-and adhesion-induced phosphorylation of Pyk2 in osteoclastlike cells, suggesting that dynamin is likely to regulate Src-Pyk2 binding downstream of integrins and growth factor receptors with important cellular consequences. Furthermore, catalytically active Src promotes dynamin-Pyk2 association, and mutating specific Src-phosphorylated tyrosine residues in dynamin blunts the dynamin-induced decrease in Pyk2 phosphorylation. Thus, since Src binds to Pyk2 through its interaction with phospho-Y402, our results suggest that Src activates a negative-feedback loop downstream of integrin engagement and other stimuli by promoting both the binding of dynamin to Pyk2-containing complexes and the dynamin-dependent decrease in Pyk2 Y402 phosphorylation, ultimately leading to the dissociation of Src from Pyk2.Podosomes are specialized transient actin-containing adhesion structures (11,14,37,60) that are found in highly motile cells, such as osteoclasts, macrophages, dendritic cells, transformed metastatic cells, and v-src-transformed cells (37, 43), where they are thought to play important roles in cellular migration and invasion (34). In resorbing osteoclasts on bone, podosomes are concentrated within the sealing zone, a beltlike actin-rich structure that is important for adhesion and which delineates the resorptive region of the cell known as the ruffled border. Unlike focal adhesions, which are relatively stable structures (11, 60), the assembly and disassembly of podosomes occurs within minutes (t 1/2 ϭ 2 to 4 min) and involves the recruitment and activation of integrins, signaling proteins and scaffolding proteins (11,14,35,47,60). However, the mechanisms of action of key signaling proteins involved in podosome assembly and disassembly are only partially understood.The focal adhesion kinase Pyk2 has been linked to the proliferation, migration, and activity of a variety of mesenchymal, epithelial, and hematopoietic cell types. Several groups, including our own, have reported the importance of Pyk2 in podosome belt organization, cell spreading, and bone-resorbing activity in osteoclasts (18,26,31,40,65,66). Pyk2 is recruited to activated ␤ 2 and ␤ 3 integrins (9, 20) at adhesion sites and is autophosphorylated at Y402 (17, 47, 50) via an intermolecular trans-acting mechanism (46). Although Pyk2 is partially activated by integrin-induced Ca 2ϩ signaling (20, 5...

Microtubule Dynamic Instability Controls Podosome Patterning in Osteoclasts through EB1, Cortactin, and Src

“…Deacetylation of these same lysines by HDAC6 reverses this process (58). Using HEK293VnR cells, a model system we have used extensively to dissect molecular signaling events linked to OC adhesion (15,21,23,55,95), we observed (i) that phosphorylation and acetylation of cortactin are inversely and dynamically related and (ii) that acetylated (and thereby dephosphorylated) cortactin exhibits enhanced association with EB1. In OCs, both increased acetylation and decreased phosphorylation of cortactin are associated with stabilization of the podosome belt.…”

“…Since defects in podosome patterning or signaling often lead to a decrease in OC activity (54,55), we next determined whether CTTN Ϫ/Ϫ OCs were impaired in their ability to resorb bone in vitro. The average resorbed area produced on dentin slices per individual OC was reduced in the absence of cortactin, and the average pit depth showed a similar pattern (Fig.…”

Oncogenic Src requires a wild-type counterpart to regulate invadopodia maturation

“…These data in podosomes support our findings in invadopodia, but since this report did not address the functionality of these cells to degrade ECM, it is not known whether WT Src is needed for maturation in this system. These results are particularly interesting because Src has been suggested to act as a 'molecular switch' to regulate dynamin-Cbl signaling complexes (Bruzzaniti et al, 2005) and as a protein with an important (kinase independent) adaptor function (Bruzzaniti et al, 2009) in osteoclast podosomes. Manipulation of Src activity in WT osteoclasts also suggest a dual function for Src in the regulation of actin dynamics through cortactin phosphorylation in podosome assembly and subsequent maturation into more highly organized structures known as sealing zones (Luxenburg et al, 2006).…”

Podosome organization drives osteoclast-mediated bone resorption

“…In turn, dynamin GTPase activity reduces Pyk2 Y402 phosphorylation, which induces Src dissociation from the complex, providing a possible negative feedback of the Src signaling cascade and suggesting a coordinated role of Src, Cbl, and dynamin in regulating actin remodeling. 103,104 Moreover, Src can activate another tyrosine kinase Syk, which is involved in podosomal rearrangement by facilitating Rac GTPase activation. 105 Indeed, mice lacking Src, Syk, or Vav3 expression suffer from increased bone mass due to defective OC-resorption.…”