Phosphorylation and transformation sensitivity of a major collagen-binding protein of fibroblasts.
Using affinity chromatography with immobilized gelatin and native type I collagen, we have identified the major collagen-binding proteins in Nonidet P-40 extracts of chick embryo fibroblasts labeled with [35S] methionine. After washing the gelatin- or collagen-Sepharose beads with high ionic strength buffer, a 47,000-dalton protein was the only major protein besides fibronectin found to bind to these affinity beads. The isoelectric point of this protein was approximately 9.0, with a closely spaced minor spot. The total amount and the synthesis of this collagen-binding protein were both decreased in Rous sarcoma virus-transformed cells. This collagen-binding protein was found to be phosphorylated by incubating intact cells with [32P]orthophosphate. Phosphoamino acid analysis revealed that serine and threonine residues were phosphorylated, but tyrosine was not. Although quantities of the 47,000-dalton protein labeled with [35S]methionine were decreased by a factor 2.5 after transformation, the incorporation of [32P]orthophosphate/unit of protein was 5-7-fold higher in transformed cells. In temperature-sensitive mutant virus-infected cells, the amount of the 47,000-dalton protein was also decreased at the temperature permissive for transformation, and the incorporation of [32P]orthophosphate/protein was also increased. These studies establish that a major membrane-associated collagen-binding protein of fibroblasts is phosphorylated and that it is altered in both total quantity and degree of phosphorylation after malignant transformation.
D000818:Animals; D001358:Avian Sarcoma Viruses; D002471:Cell Transformation, Neoplastic / Q000378:metabolism*; D002478:Cells, Cultured; D002642:Chick Embryo; D002846:Chromatography, Affinity; D005347:Fibroblasts / Q000032:analysis; D008970:Molecular Weight; D009154:Mutation; D010761:Phosphorus Radioisotopes; D010766:Phosphorylation; D038981:Receptors, Collagen; D011971:Receptors, Immunologic / Q000302:isolation & purification / Q000378:metabolism*; D013462:Sulfur Radioisotopes; D013696:Temperature
D010761:Phosphorus Radioisotopes; D038981:Receptors, Collagen; D011971:Receptors, Immunologic; D013462:Sulfur Radioisotopes