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Serine 785 phosphorylation of the ß1 cytoplasmic domain modulates ß1A-integrin-dependent functions

Department of Biology, Wesleyan University, Hall-Atwater Labs, Room 257, Lawn Ave, Middletown, CT 06459, USA

View larger version (32K): [in a new window]   Fig. 1. Expression of chicken ß1 in GD25 cells. Three constructs expressing chicken ß1 with site directed substitutions at S785 of the cytoplasmic domain (A) were transfected into GD25 cells. Biotin surface labeled cells from clones expressing chicken S785S, S785M or S785D ß1 were immunoprecipitated using anti-chicken-ß1 antibody (W1B10) and the precipitates analyzed by SDS-PAGE followed by western blotting probed with streptavidin (B). Biotin surface labeled cells from clones expressing chicken S785S, S785M or S785D ß1 were immunoprecipitated using anti-mouse-6 antibody (GOH3) and the precipitates analyzed by SDS-PAGE followed by western blotting probed with streptavidin (C). As a loading control, supernatant fractions (see Methods) were analyzed for spectrin content by western analysis (D).

View larger version (32K): [in a new window]   Fig. 2. Role of S785 in cell attachment. Attachment to laminin (LAM) was examined for F9 and GD25 cell lines expressing chicken ß1. Untransfected F9 cells attachment is inhibited by increasing concentrations of CD29 HA2/5, a mouse-specific function-blocking ß1 antibody. Both the F9-S785S and F9-S785D cell lines continue to attach in the presence of the antibody, whereas the F9-S785M cell line does not (A). Untransfected and transfected GD25 cells attach equally well to serum coated substrates (B). By contrast, only GD25ß1A, GD25-S785S4 and GD25-S785D2 attach well to laminin (LM) (B). Error bars represent ±s.e. of quadruplicate experiments.

View larger version (104K): [in a new window]   Fig. 3. Role of S785 in cell spreading. F9 and GD25 cell lines were assayed for their ability to spread on laminin coated substrates. All F9 cell lines are able to spread on laminin in the absence of the ß1 specific antibody CD29 HA2/5 (A). In the presence of the antibody, untransfected F9 and F9-S785D fail to spread. By contrast, F9-S785S and F9-S785M cells are able to spread (A). Spreading of F9-S785S cells is inhibited if cells are incubated with both anti-chicken-ß1 (W1B10) and anti-mouse-ß1 antibodies (CD29 HA2/5) (B). Untransfected GD25 cells and GD25-S785D2 cells are unable to spread on laminin. Cells expressing either wild-type mouse ß1 or S785S, S785A or S785M spread on laminin (C).

View larger version (54K): [in a new window]   Fig. 4. Role of S785 in directed cell migration. F9 outgrowth and GD25 cell migration was examined on laminin coated substrates. Parietal endoderm outgrowth assays were used to measure the role of S785 in F9 cell migration (A). All cell lines produce extensive outgrowth when plated on laminin. F9-S785S and F9-S785M produce moderate to extensive outgrowth in the presence of the antibody CD29 HA2/5. Untransfected and F9-S785D cell outgrowth is inhibited. (B) Extent of outgrowth, for cells treated (AB) or untreated (CTL) with the ß1 function-blocking antibody CD29 HA2/5, was quantified by measuring the distance from the edge of the embryoid body to the outermost edge of outgrowth at ten points for ten embryoid bodies. Error bars represent ±s.e. of triplicate experiments. (C) Boyden chamber assays were used to measure migration of GD25 cell lines. Error bars represent ±s.e. of quadruplicate experiments. An intermediate level of migration is seen with the GD25-S785M2 and significantly reduced migration with GD25-S785D2.

View larger version (82K): [in a new window]   Fig. 5. Localization of ß1 in GD25 cells expressing site directed substitutions at S785. Cells were plated on fibronectin and double labeled with antibodies directed against vinculin and chicken ß1. There is no ß1 in untransfected GD25 cells. ß1 does not localize to focal adhesion sites in GD25-S785D2 cells. By contrast, ß1 in both GD25-S785S4 and GD25-S785M2 cells colocalizes with vinculin to these structures.