title: 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), p130(Cas), focal adhesion kinase, integrin alpha(v)beta(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, c-Src, 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 alpha(v)beta(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.
authors.affiliation: Department of Oral and Craniofacial Biological Sciences, University of Maryland, 666 W. Baltimore St., Baltimore, MD 21201, USA. mac001@dental.umaryland.edu
authors.forename: M A
authors.initials: MA
authors.lastname: Chellaiah
authors.forename: R S
authors.initials: RS
authors.lastname: Biswas
authors.forename: D
authors.initials: D
authors.lastname: Yuen
authors.forename: U M
authors.initials: UM
authors.lastname: Alvarez
authors.forename: K A
authors.initials: KA
authors.lastname: Hruska
mesh_terms: D000818:Animals; D001717:Birds; D015153:Blotting, Western; D002448:Cell Adhesion; D016895:Culture Media, Serum-Free / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D018260:Gelsolin / Q000737:chemistry* / Q000378:metabolism; D005982:Glutathione Transferase / Q000378:metabolism; D050356:Lipid Metabolism; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck) / Q000378:metabolism; D051379:Mice; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D008954:Models, Biological; D010010:Osteoclasts / Q000378:metabolism; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D018129:Phosphatidylinositol Phosphates / Q000378:metabolism*; D010743:Phospholipids / Q000378:metabolism; D010766:Phosphorylation; D011233:Precipitin Tests; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000378:metabolism; D016392:Proto-Oncogene Proteins pp60(c-src) / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D015398:Signal Transduction; D018909:src Homology Domains
chemical_list: D016895:Culture Media, Serum-Free; D018260:Gelsolin; D018129:Phosphatidylinositol Phosphates; D010743:Phospholipids; D011506:Proteins; D011993:Recombinant Fusion Proteins; C060974:phosphatidylinositol 3,4,5-triphosphate; D005982:Glutathione Transferase; D019869:Phosphatidylinositol 3-Kinases; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck); D016392:Proto-Oncogene Proteins pp60(c-src)