Transformation of chicken embryo fibroblasts by v-src uncouples beta1 integrin-mediated outside-in but not inside-out signaling.
Adhesion of cells to extracellular matrix is mediated by integrin family receptors. The process of receptor-ligand binding is dependent on metabolic energy and is regulated by intracellular signals, termed inside-out signals. The strength of the initial alpha5beta1-mediated adhesion of v-src-transformed chicken embryo fibroblasts (v-srcCEF) was similar to that of normal CEF. A chemically cross-linked fibronectin substrate was able to restore cell spreading and the ability of v-srcCEF to assemble a fibronectin matrix. Over time, v-srcCEF showed decreased adhesion due to the reduction of alpha5beta1-fibronectin bonds consequent on the reduction of substrate-bound fibronectin due to the secretion of proteases by v-srcCEF. Excess synthesis of hyaluronic acid by v-srcCEF also reduced the alpha5beta1-fibronectin bonds and contributed to cell detachment at later times in culture. Thus, the adhesion defects were not due to a failure of alpha5beta1 function and adhesion of the v-srcCEF was alpha5beta1 dependent. Integrin-mediated adhesion also produces signals that affect cell proliferation and cell differentiation. An early consequence of these "outside-in" signals was the phosphorylation of FAK Y397 in direct proportion to the number of alpha5beta1-fibronectin bonds formed. In contrast, v-srcCEF had an increased level of phosphorylation on five different tyrosines in FAK, and none of these phosphorylation levels were sensitive to the number of alpha5beta1-fibronectin bonds. In the absence of serum, CEF proliferation was sensitive to changes in alpha5beta1-mediated adhesion levels. Transformation by v-src increased the serum-free proliferation rate and made it insensitive to alpha5beta1-mediated adhesion. Thus, the v-srcCEF were insensitive to the normal outside-in signals from alpha5beta1 integrin.
Datta
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Department of Microbiology, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA.
Shi
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Boettiger
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D000818:Animals; D002448:Cell Adhesion; D002454:Cell Differentiation; D002455:Cell Division; D002465:Cell Movement; D002471:Cell Transformation, Neoplastic; D002478:Cells, Cultured; D002642:Chick Embryo; D003432:Cross-Linking Reagents / Q000494:pharmacology; D016895:Culture Media, Serum-Free / Q000494:pharmacology; D004797:Enzyme-Linked Immunosorbent Assay; D005347:Fibroblasts / Q000166:cytology* / Q000378:metabolism*; D005353:Fibronectins / Q000378:metabolism; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; D006801:Humans; D006820:Hyaluronic Acid / Q000494:pharmacology; D019012:Integrin beta1 / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D015688:Oncogene Protein pp60(v-src) / Q000378:metabolism*; D010766:Phosphorylation; D017434:Protein Structure, Tertiary; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D017469:Receptors, Fibronectin / Q000378:metabolism; D015398:Signal Transduction; D013997:Time Factors
D003432:Cross-Linking Reagents; D016895:Culture Media, Serum-Free; D005353:Fibronectins; D019012:Integrin beta1; D017469:Receptors, Fibronectin; D006820:Hyaluronic Acid; D011505:Protein-Tyrosine Kinases; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; D015688:Oncogene Protein pp60(v-src); C496050:PTK2 protein, human