Related Experiment Videos
Increased concentration of an apparently identical cellular protein in cells transformed by either Abelson murine
Abstract:
Abelson murine leukemia virus (A-MuLV)-transformed cells, simian virus 40 (SV40)-transformed cells, and chemically transformed cells all have increased levels of a 50,000-molecular-weight host cell protein. The protein was detected with sera raised to the A-MuLV-transformed and chemically transformed cells and was tightly bound to T-antigen in extracts of SV40-transformed cells. Partial protease digests showed that the proteins from all three sources were indistinguishable. The three proteins were phosphorylated in cells, and the linkage of phosphate to the A-MuLV-associated P50 was to a serine residue. By immunofluorescence methods, P50-related protein was found on the surface of both normal lymphoid cells and A-MuLV-transformed lymphoid cells, but cell fractionation showed that the majority of P50 was free in the cytoplasm of the transformed cells. Immunofluorescence also showed that P50 was found in granules in the cytoplasm of both untransformed and SV40-transformed fibroblasts. Other cells gave indistinct patterns. Cocapping experiments showed that the A-MuLV-specified P120 protein is weakly associated with the surface P50-related protein of lymphoid cells, but no association of P120 and P50 could be demonstrated by immunoprecipitation methods. Although a monoclonal antiserum to P50 was used in many of these studies, the identity of the bulk P50 protein with the molecules that are reactive at the cell surface requires further study.
Insights
A 50,000-molecular-weight host cell protein (P50) is elevated in Abelson murine leukemia virus (A-MuLV)-transformed, simian virus 40 (SV40)-transformed, and chemically transformed cells. This phosphorylated protein shows distinct cellular localization patterns, with surface expression on lymphoid cells and cytoplasmic granules in fibroblasts.
Area of Science:
- Molecular biology
- Virology
- Cell biology
Background:
- Transformed cells, including those induced by Abelson murine leukemia virus (A-MuLV), simian virus 40 (SV40), and chemical agents, exhibit common alterations in host cell protein expression.
- A specific 50,000-molecular-weight (P50) host cell protein is consistently found at increased levels across these diverse transformation models.
Purpose of the Study:
- To characterize the 50,000-molecular-weight host cell protein (P50) found in various transformed cell lines.
- To investigate the biochemical properties, cellular localization, and potential associations of P50 with viral antigens and other cellular proteins.
Main Methods:
- Detection of P50 using antisera raised against transformed cell extracts.
- Partial protease digestion to compare P50 from different cell sources.
- Phosphorylation analysis of P50.
- Immunofluorescence microscopy to determine cellular localization.
- Cell fractionation to isolate cytoplasmic and surface proteins.
- Cocapping and immunoprecipitation experiments to assess protein associations.
Main Results:
- Proteins from A-MuLV-, SV40-, and chemically transformed cells were indistinguishable by protease digestion, indicating they are the same protein.
- P50 is phosphorylated in cells, with phosphate linked to serine in the A-MuLV-associated form.
- Immunofluorescence revealed P50 on the surface of normal and A-MuLV-transformed lymphoid cells, but primarily in the cytoplasm of transformed cells.
- P50 was observed in cytoplasmic granules in both untransformed and SV40-transformed fibroblasts.
- Cocapping suggested a weak association between A-MuLV-specified P120 and surface P50 on lymphoid cells, but immunoprecipitation failed to confirm this association.
Conclusions:
- The 50,000-molecular-weight host cell protein (P50) is a common marker associated with cellular transformation by various agents.
- P50 exhibits differential cellular localization depending on cell type and transformation status.
- While a weak association with viral proteins like P120 is suggested, the precise role and identity of surface-reactive P50 require further investigation.