Escherichia coli MutY protein cleaves A/G- or a/7,8-dihydro-8-oxo-guanine (A/GO)-containing DNA on the A-strand by N-glycosylase and apurinic/apyrimidinic endonuclease or lyase activities. In this paper, we show that MutY can be trapped in a stable covalent enzyme-DNA intermediate in the presence of sodium borohydride, a new finding that supports the grouping of MutY in that class of DNA glycosylases that possess concomitant apurinic/apyrimidinic lyase activity. To potentially help determine the substrate recognition site of MutY, mutant proteins were constructed. MutY proteins with a Gly116 --> Ala (G116A) or Asp (G116D) mutation had reduced binding affinities for both A/G- and A/GO-containing DNA substrates. The catalytic parameters, however, were differentially affected. While A/G- and A/GO-containing DNA were cleaved by MutY with specificity constants (kcat/Km) of 10 and 3.3 min-1 microM-1, respectively, MutY(G116D) cleaved these DNAs 2, 300- and 9-fold less efficiently. The catalytic activities of MutY(G116A) with A/G- and A/GO-containing DNA were about the same as that of wild-type MutY. Both MutY(G116A) and MutY(G116D) could be trapped in covalent intermediates with A/GO-containing DNA, but with lower efficiencies than the wild-type enzyme in the presence of sodium borohydride. MutY(G116A) also formed a covalent intermediate with A/G-containing DNA, but MutY(G116D) did not. Since Gly116 of MutY lies in a region that is highly conserved among several DNA glycosylases, it is likely this conserved region is in the proximity of the substrate binding and/or catalytic sites.

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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...

An immortalized cell line representing the primitive erythroid (EryP) lineage was established from in vitro–differentiated progeny (embryoid bodies [EBs]) of embryonic stem (ES) cells using a retroviral insertional mutation, and has been termed EB-PE for embryoid body–derived primitive erythroid. Even though EB-PE cells are immortalized, they show characteristics of normal EryP cells, such as gene expression and growth factor dependency. In addition, EB-PE cells can differentiate further in culture. Investigation of growth factor requirements of EB-PE cells showed that basic fibroblast growth factor (bFGF) and erythropoietin (Epo) play unique roles in EB-PE proliferation and differentiation. While bFGF was a strong mitogen, Epo was required for both proliferation and differentiation. The unique proliferative response to bFGF coincided with upregulation of its receptor, fibroblast growth factor receptor (fgfr-1), and downregulation of erythropoietin receptor (EpoR) gene expression. Studies of primary EryP cells derived from early EBs, when tested in a colony-formation assay, also provided evidence for the mitogenic role of bFGF in concert with Epo.