Biochemical characterization of human carcinoma surface antigen associated with protein kinase activity.
MAb 50H.19 immunoprecipitates two proteins from lysates of human carcinoma cell lines, and embryonic fibroblasts intrinsically labelled with 3H-leucine, 35S-methionine, or a 3H-amino acid mixture; a major component of Mr = 22,000 (22 kd component) and a minor component of Mr = 24,000 (24 kd component). Oligomeric forms of the proteins are not observed under reducing or non-reducing conditions. Both proteins are expressed on the plasma membrane, and are glycoproteins. We investigated the relationship between the proteins in terms of their glycosylation and derivation from precursors. The 22 kd component is O-glycosylated as demonstrated by 3H-galactose incorporation, insensitivity to tunicamycin (TM), and its stepwise generation from a 20.5 kd precursor. The 24 kd protein is N-glycosylated, as shown by 3H-mannose incorporation, and by the total inhibition of its synthesis in the presence of TM. Further evidence for its N-glycosylation is provided by the appearance of a 23 kd precursor in lysates from the osteogenic sarcoma cell line SKOSC pulse-labelled for 5 min, a time preceding O-glycosylation of the 20.5 kd protein. Furthermore, mild alkali treatment of the immune complex leads to a loss of approximately 1,000 daltons in each glycoprotein confirming the O-glycosylated nature of the 22 kd component, and suggesting that the 24 kd component is additionally O-glycosylated. Both glycoproteins undergo an apparent increase of molecular weight of about 500 daltons when run in the non-reduced form on SDS polyacrylamide gels under standard electrophoretic conditions, suggesting they contain a similar degree of intra-chain disulphide bonding. Confirmatory evidence that the two components share a common polypeptide backbone is provided by the appearance of only the 20.5 kd component in lysates from SKOSC cells pulse-labelled for 5 min in the presence of TM.
D000911:Antibodies, Monoclonal / Q000276:immunology; D000951:Antigens, Neoplasm / Q000032:analysis*; D000954:Antigens, Surface / Q000032:analysis*; D004591:Electrophoresis, Polyacrylamide Gel; D006023:Glycoproteins / Q000032:analysis; D006801:Humans; D008970:Molecular Weight; D010766:Phosphorylation; D011494:Protein Kinases / Q000032:analysis*
D000911:Antibodies, Monoclonal; D000951:Antigens, Neoplasm; D000954:Antigens, Surface; D006023:Glycoproteins; D011494:Protein Kinases