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Studies of the decrease of tyrosine-O-sulphated proteins in Rous sarcoma-virus-transformed rat embryo fibroblasts,
Abstract:
The sulphate activation and tyrosyl-protein sulphotransferase systems in normal 3Y1 rat embryo fibroblasts and the same cells transformed by Schmidt Ruppin subgroup-A-Rous sarcoma virus (SRA-3Y1) were examined. Employing metabolic [35S]sulphate-labelling followed by PEI (polyethyleneimine)-cellulose thin-layer chromatography of the labelled cell lysates, it was found that the steady-state level of 'active' sulphate, adenosine 3'-phosphate 5'-phosphosulphate, was drastically lower in SRA-3Y1 cells compared with their normal counterparts. When the sulphate activating enzymes were tested, it appeared that the activities in 3Y1 homogenates were 2-2.5 times greater than those in SRA-3Y1 homogenates. An endogenous sulphation assay for tyrosyl-protein sulphotransferase revealed that activities in 3Y1 and SRA-3Y1 homogenates were comparable. Nearly identical patterns were observed with both sets of cells when [35S]sulphated proteins generated in the endogenous assay were separated by two-dimensional gel electrophoresis. It therefore seems that the tyrosyl-protein sulphotransferase(s) are unimpaired in SRA-3Y1 cells. While the lower (approx. 8 times) sulphate uptake remains the major cause for the decrease of tyrosine-O-sulphated proteins in SRA-3Y1 cells [Liu & Lipmann, (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 3695-3698], the 2-2.5-fold lower sulphate activating enzyme activities also contribute to some extent to the difference between the SRA-3Y1 and 3Y1 cells.
Insights
Schmidt Ruppin subgroup-A-Rous sarcoma virus (SRA-3Y1) transformation reduces active sulphate levels in rat fibroblasts. Lower sulphate uptake and decreased sulphate-activating enzyme activity contribute to reduced tyrosine-O-sulphated proteins in SRA-3Y1 cells.
Area of Science:
- Cell Biology
- Biochemistry
- Virology
Background:
- Tyrosine-O-sulfation is a post-translational modification crucial for protein function.
- Rous sarcoma virus transformation alters cellular metabolism and protein modification pathways.
- Understanding these alterations is key to comprehending viral oncogenesis.
Purpose of the Study:
- To investigate the impact of Rous sarcoma virus transformation on sulphate activation and tyrosyl-protein sulphotransferase activity in rat fibroblasts.
- To determine the contribution of altered sulphate metabolism to the decrease in tyrosine-O-sulphated proteins in transformed cells.
Main Methods:
- Metabolic [35S]sulphate labelling of normal (3Y1) and transformed (SRA-3Y1) rat fibroblasts.
- Polyethyleneimine (PEI)-cellulose thin-layer chromatography to quantify adenosine 3'-phosphate 5'-phosphosulphate (active sulphate).
- Enzyme assays for sulphate-activating enzymes and tyrosyl-protein sulphotransferase.
- Two-dimensional gel electrophoresis to analyze [35S]sulphated proteins.
Main Results:
- SRA-3Y1 cells exhibited significantly lower steady-state levels of active sulphate compared to 3Y1 cells.
- Sulphate-activating enzyme activities were 2-2.5 times lower in SRA-3Y1 homogenates than in 3Y1 homogenates.
- Tyrosyl-protein sulphotransferase activity and protein sulphation patterns were comparable between normal and transformed cells.
- Lower sulphate uptake (approx. 8-fold) is the primary cause for reduced tyrosine-O-sulphated proteins, with reduced sulphate-activating enzyme activity also contributing.
Conclusions:
- Rous sarcoma virus transformation impairs sulphate activation pathways in rat fibroblasts.
- While tyrosyl-protein sulphotransferase activity remains unaffected, reduced sulphate uptake and lower sulphate-activating enzyme activity contribute to decreased tyrosine-O-sulphated proteins in SRA-3Y1 cells.
- These metabolic alterations likely play a role in the cellular changes induced by viral transformation.
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