tyrosine-o-sulfate and Cell-Transformation--Viral

In a recent paper, we reported the loss of large amounts of protein-bound tyrosine sulfate after infection of rat fibroblasts by avian sarcoma viruses. The analogy to the reported loss of surface fibronectin on malignant transformation, which contained sulfate of unknown location, called our attention to this compound. In a previous paper, we briefly reported on isolation from the supernatant fraction of rat fibroblasts infected by Fujinami sarcoma virus fibronectin that yielded tyrosine-O-sulfate on Pronase hydrolysis. In this paper, we confirm and enlarge on this observation. Highly purified fibronectin was obtained from the supernatant fraction secreted by Fujinami sarcoma virus infected rat fibroblasts that contained 1.52 residues of sulfated tyrosine per protein molecule after exhaustive Pronase hydrolysis. Assuming some loss during work up, this probably indicates 2 residues of the tyrosine sulfated per fibronectin molecule.

Topics: Animals; Cell Line; Cell Transformation, Viral; Extracellular Matrix; Fibronectins; Pronase; Protein Processing, Post-Translational; Rats; Retroviridae; Sulfates; Tyrosine

Protein sulfation in baby hamster kidney cells (BHK) and their polyoma virus transformants (PY-BHK) was studied comparatively. On in vivo labeling, [35S]-sulfate was incorporated into the 50K protein and proteins in the 100-180K range, represented by the 155K protein. The incorporation into both the 50K and 155K protein was elevated 2-3 fold in PY-BHK cells compared to in BHK cells. Tyrosine-O-sulfate was the only identifiable sulfated amino acid in both proteins. On in vitro labeling with [35S]3'-phosphoadenosine 5'-phosphosulfate (PAPS), at least 6 radioactive protein bands were discernible on gel electrophoresis. Of these, sulfation of the 57K and 60K proteins was elevated in PY-BHK cells compared to in BHK cells, whereas sulfation of the 39K protein was depressed in PY-BHK cells. Tyrosine-O-sulfate was the only identifiable sulfated amino acid in these proteins.

Topics: Animals; Cell Line; Cell Transformation, Viral; Cricetinae; Electrophoresis, Polyacrylamide Gel; Genes, Viral; Kidney; Phosphoadenosine Phosphosulfate; Polyomavirus; Proteins; Sulfuric Acid Esters; Sulfuric Acids; Tyrosine

Our interest was aroused by the recent report by Huttner [ Huttner , W. B. (1982) Nature (London) 299, 273-276] on general sulfation of tyrosine residues of proteins in normal and malignantly transformed tissues. Here we report on the reduction of sulfation in embryonic rat fibroblasts, line 3Y1, infected with Rous sarcoma virus or Fujinami sarcoma virus. In view of the instability of tyrosine O-sulfate in strong acid, the protein sulfation was tested for after incubation with [35S]sulfate and exhaustive Pronase hydrolysis. We found in general a reduction of sulfation in transformed tissue. It was greatest in the fibroblasts permanently transformed with Rous sarcoma virus. When fibroblasts transformed by the temperature-sensitive Fujinami sarcoma virus, line ts225 -3Y1, were used for comparison of sulfation at nonpermissive and permissive temperatures, the latter showed a strong reduction. Furthermore, we tested these cells for the uptake of inorganic [35S]sulfate. Uptake appeared highly reduced in the permanently infected fibroblasts, but ts225 -3Y1 grown at permissive and nonpermissive temperatures exhibited no difference. Uptake at both temperatures was comparable to uptake by normal 3Y1 cells. A recently much investigated cell surface protein, fibronectin, was reported to be lost on malignant transformation and to contain sulfate in an undetermined location. We found that ts225 -3Y1 cells grown at permissive temperature released fibronectin that contained tyrosine O-sulfate.

Topics: Animals; Avian Sarcoma Viruses; Biological Transport; Cell Transformation, Viral; Cells, Cultured; Mutation; Neoplasm Proteins; Rats; Sulfates; Tyrosine