Phosphatidylinositol 3,4,5-Trisphosphate Directs Association of Src Homology 2-containing Signaling Proteins with Gelsolin

Abstract

Podosomes are adhesion structures in osteoclasts and are structurally related to focal adhesions mediating cell motility during bone resorption. Here we show that gelsolin coprecipitates some of the focal adhesion-associated proteins such as c-Src, phosphoinositide 3-kinase (PI3K), p130Cas , focal adhesion kinase, integrin ␣ v ␤ 3 , vinculin, talin, and paxillin. These proteins were inducibly tyrosine-phosphorylated in response to integrin activation by osteopontin. Previous studies have defined unique biochemical properties of gelsolin related to phosphatidylinositol 3,4,5-trisphosphate in osteoclast podosomes, and here we demonstrate phosphatidylinositol 3,4,5-trisphosphate/gelsolin function in mediating organization of the podosome signaling complex. Overlay and GST pull-down assays demonstrated strong phosphatidylinositol 3,4,5-trisphosphate-PI3K interactions based on the Src homology 2 domains of PI3K. Furthermore, lipid extraction of lysates from activated osteoclasts eliminated interaction between gelsolin, cSrc, PI3K, and focal adhesion kinase despite equal amounts of gelsolin in both the lipid-extracted and unextracted experiment. The cytoplasmic protein tyrosine phosphatase (PTP)-proline-glutamic acid-serine-threonine amino acid sequences (PEST) was also found to be associated with gelsolin in osteoclast podosomes and with stimulation of ␣ v ␤ 3 -regulated phosphorylation of PTP-PEST. We conclude that gelsolin plays a key role in recruitment of signaling proteins to the plasma membrane through phospholipid-protein interactions and by regulation of their phosphorylation status through its association with PTP-PEST. Because both gelsolin deficiency and PI3K inhibition impair bone resorption, we conclude that phosphatidylinositol 3,4,5-trisphosphate-based protein interactions are critical for osteoclast function.Osteoclasts are multinucleated giant cells with bone-resorbing activity. As osteoclasts crawl over bone surfaces, they require rapid attachment and release from the extracellular matrix. Adhesion structures called podosomes present in highly motile cells are also found in osteoclasts. Osteoclasts are unique because they use the speed of podosome assembly and disassembly to generate high rates of motility. Podosome formation stabilizes the bone matrix-cell interface and forms an isolated compartment between the ruffled border and the bone surface (1, 2). Consistent with their function as adhesion sites, podosomes contain many of the same proteins found in focal adhesions, such as F-actin, vinculin, talin, gelsolin, fimbrin, and ␣-actinin (3-7).We have shown previously that osteopontin (OPN) 1 binding to integrin ␣ v ␤ 3 in osteoclasts stimulates gelsolin-associated PI3K. This leads to increased levels of gelsolin-associated polyphosphoinositides, such as phosphatidylinositol 4,5-bisphosphate (PtdIns 4,5-P 2 ), phosphatidylinositol 3,4-bisphosphate, and phosphatidylinositol 3,4,5-trisphosphate, uncapping of actin barbed ends, and actin filament formation (8). Moreover, OPN stimulates gelsolin-associ...

Sodium fluoride induces podosome formation in endothelial cells

“…Double staining for F-actin and vinculin revealed the characteristic podosomal arrangement of these two proteins in NaF-treated cells, with vinculin forming a ring around each F-actin dot ( Figure 2A). P85α, the regulatory subunit of phosphoinositide 3-kinase and p190RhoGAP-A, a negative regulator for RhoA, both known to localize to podosome actin cores (Burgstaller and Gimona, 2004;Chellaiah et al, 2001;Guegan et al, 2008) co-localized with F-actin in NaF-treated PAE cells ( Figure 2A). Likewise, N-WASP and WIP (WASPinteracting protein), two proteins involved in the actin polymerization machinery, were also recruited to these structures (see Supplementary Figure S2 at http://www.biolcell.org/boc/102/boc1020489add.…”

Phosphorylation of a Wiscott-Aldrich Syndrome Protein-associated Signal Complex Is Critical in Osteoclast Bone Resorption

“…The beads were pelleted and washed three times for 5 min each with ice-cold PBS. Pulldown with GST-fused WASP proteins coupled to glutathione-Sepharose was also performed to determine that the proteins had the capacity to bind FL-WASP and WASP-GP region as described previously (42). The immune complexes were then eluted in electrophoresis sample buffer and subjected to SDS-PAGE.…”

“…The cells were washed and incubated with primary antibodies of interest (WASP, PTP-PEST, and PST-PIP; 1:100 dilution) in the blocking solution for 2 h at 4°C. The primary antibodies were detected with either Cy2-or Cy3-conjugated secondary antibodies as described previously (42,44). Negative controls were performed with nonimmune mouse and goat sera for the double stainings.…”

“…Proteins were expressed in Escherichia coli as glutathione S-transferase (GST) fusions as described previously (42). SDS-PAGE and Coomassie Blue staining tested the purity of the purified proteins.…”

Podosomes at a glance

“…Downstream, a variety of pathways are initiated, most notably PI3K signaling (Chellaiah et al, 1998), which leads to the formation of the phosphatidylinositols PtdIns(3,4)P 2 and PtdIns(3,4,5)P 3 , and activation of focal adhesion kinase (FAK) or its haematopoietic relative Pyk2. 3Ј phosphoinositide signaling is also influenced by the RhoGTPases Rho and (probably) Rac (Chellaiah et al, 2001;Sechi and Wehland, 2000). CDC42, another RhoGTPase, is crucial for the local initiation of actin filament formation, which gives rise to the formation of the actin-rich core structure.…”