Src-dependent repression of ARF6 is required to maintain podosome-rich sealing zones in bone-digesting osteoclasts

Abstract

Bone digestion occurs when osteoclasts adhere onto bone surfaces and polarize to form acidic, hydrolase-rich resorption lacunae. For this process, they condense their actin-rich podosomes in tight belts to establish sealing zones, which segregate their basal membranes from those facing resorption lacunae. This polarization process remains poorly understood. Here, we combined quantitative proteomics and gene silencing to identify new substrates of the Src tyrosine kinase, a key regulator of osteoclast function. We now report that a depletion of the ARF GTPase-activating protein GIT2, which localizes to sealing zones upon Src phosphorylation, or a lack of GTP hydrolysis on ARF6 impairs sealing zone formation and polarized membrane traffic. Surprisingly, the Rho guanine nucleotide exchange factors ␣ and ␤ PIX, which usually coordinate ARF and Rho signaling, were found to be dispensable. We conclude that the Src-dependent localization of GIT2 is essential for downregulating ARF6 activity at sealing zones, and thus for maintaining osteoclast polarity.polarity ͉ GIT2 ͉ podosomes ͉ quantitative proteomics

Tissue‐ and development‐dependent expression of the small GTPase Arf6 in mice

“…Osteoclasts expressed a low level of Arf6 mRNA (open arrows in Fig. 15H), as Arf6 has been reported to play a role in sealing zone formation in osteoclasts (Heckel et al, 2009). Importantly, osteocytes did not show the signal of Arf6 mRNA expression (white arrows in Fig.…”

Bone remodelling at a glance

“…This leads to activation of signalling pathways that involve phosphoinositide 3-kinase (PI3K) and phospholipase C (PLC) (which are also activated by c-fms and complement v3 signalling), and activation of the small GTPases Rac and Cdc42 by guanine nucleotide-exchange factors (GEFs) such as Vav3 (Novack and Faccio, 2009), combined with deactivation of ADP-ribosylation factor 6 (Arf6) through its GTPase-activating protein (GAP) GIT2 (G-protein-coupled receptor kinase-activator 2) (Heckel et al, 2009). Together with changes in the activity of Rho and downstream effects on microtubule acetylation and stabilisation, the combined action of these pathways promotes actin and microtubule reorganisation, thereby leading to the formation of the SZ and subsequently the RB.…”

NSP-CAS Protein Complexes: Emerging Signaling Modules in Cancer

“…For example, little is known about the role of NSP phosphorylation, which represents an additional putative regulatory aspect for the NSP family. Multiple phosphorylation sites have been identified, mostly in the amino-terminal half of all 3 NSP proteins, in normal and cancer cells 80,81,86,[88][89][90][91] (phosposite.org) (Fig. 1).…”