Abstract
Expression of activated mutants of c-Src in epithelial cells can induce tumorigenicity. In addition to such oncogenic transformation, the cells undergo a dramatic morphological transformation: cell-cell contacts are disrupted, spreading on extracellular matrix proteins is suppressed, actin stress fibers and focal contacts are lost, and podosomes are formed. We have previously shown that integrin alphavbeta3 strongly supports Src-mediated oncogenic transformation through an interaction at the beta3 cytoplasmic tail. Our current findings demonstrate that this interaction does not affect Src-mediated morphological alterations, thus separating oncogenic from morphological transformation. Moreover, beta1 and beta3 integrins differently affect the various aspects of Src-induced morphological transformation. High levels of beta3, but not beta1, integrins can prevent Src-induced cell rounding although stress fiber disassembly and podosome formation still occur. Studies using chimeric integrin subunits demonstrate that this protection requires the beta3 extracellular domain. Finally, like tumor formation, podosome assembly occurs independent of beta3 phosphorylation. Instead, phosphorylation of beta1 is required to suppress Rho-mediated contractility in order to assemble podosomes. Thus, integrins regulate Src-mediated oncogenic transformation and various aspects of morphological transformation through dissociable pathways.
Highlights
In addition to its role in mitogenic signaling, c-Src is a critical regulator of both cadherin- and integrin-mediated adhesion structures [9, 10]
To clarify how different integrins regulate the various aspects of Src-mediated morphological transformation and how this relates to oncogenic transformation, we have expressed a c-Src mutant that is constitutively in an open, primed conformation (c-Src[Y530F], here referred to as SrcYF) in the context of wild type, chimeric, and mutant 1 and 3 integrin subunits in two independent 1-deficient cell lines
In summary, we show that (i) Src-mediated oncogenic and morphological transformations are distinct processes; (ii) podosome formation and cell rounding are independent aspects of Src-mediated morphological transformation; (iii) ␣v3 supports SrcYF-mediated tumor formation and protects against SrcYF-induced loss of adhesion and spreading through distinct mechanisms; and (iv) Src-induced podosome assembly in the presence of 1 requires phosphorylation of the integrin cytoplasmic domain to reduce cytoskeletal contractility (e.g. 1YYFF)
Summary
Cell Lines, Plasmids, Antibodies, and Other Materials—The 1-deficient GE11 and GD25 cells were previously described [23]. Immunofluorescence and Flow Cytometry—For immunofluorescence, cells were fixed in 4% formaldehyde, permeabilized in 0.4% Triton X-100, blocked with 2% bovine serum albumin, and incubated with anti-paxillin antibody or anti-human 3 (23C6), followed by Alexa-488-conjugated secondary antibody, rhodamine-phalloidin or TOPRO-3 staining (Molecular Probes). For flow cytometry and cell sorting, cells were trypsinized, collected in culture medium, washed with PBS, and incubated with primary antibodies in PBS containing 2% serum for 1 h at 4 °C. Rho Activity Assays—Cells were plated overnight to subconfluency before lysis in Nonidet P-40 lysis buffer (0.5% Nonidet P-40, 50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 5 mM MgCl2, 10% glycerol, supplemented with a protease inhibitor mix (SigmaAldrich)), and lysates were clarified by centrifugation at 14,000 rpm for 20 min at 4 °C. The beads were resolved in reduced sample buffer and analyzed by SDS-PAGE and Western blotting
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