Structural and functional features of a cell surface phosphoglycoprotein associated with tumorigenic phenotype in human fibroblast x HeLa cell hybrids.
Monoclonal antibodies have been raised against a dimeric cell surface antigen (p75/150) which is specifically associated with the tumorigenic phenotype in human fibroblast X HeLa hybrids. During biosynthesis, a precursor molecule (p70/140), was associated with microsomal membranes in vivo but possessed no detectable cytoplasmic domains. At this stage, each p70 monomer contained 3 "high-mannose" type N-linked glycans which were subsequently processed into endoglycosidase H-insensitive complex oligosaccharides on the mature cell surface forms. Cleavage of this cell surface form with endoglycosidase F yielded non-N-glycosylated polypeptides of Mr = 60,000/120,000. All the monoclonal antibodies identified similar non-N-glycosylated polypeptides in cells grown in the presence of tunicamycin. p75/150 could be weakly labeled with [3H]palmitic or myristic acid. In vivo, p75/150 was found to be phosphorylated on serine residues. Immunoprecipitates of p75/150 from HeLa or tumorigenic hybrid cell lysates exhibited protein kinase activity in vitro, which phosphorylated p75/150 itself, also on serine residues. We were unable to detect this kinase activity in normal fibroblasts and in the nontumorigenic hybrid cells. Furthermore, we were unable to detect p75/150 or its precursors by either cell surface labeling, metabolic labeling, or Western blotting in nontumorigenic cell hybrids; p75/150 thus represents a tumor-specific marker in this system. Tryptic peptides of highly purified p75/150 have been generated, but their amino acid sequences did not reveal any significant homology with previously described proteins.
Sutherland
D R
DR
Bicknell
D C
DC
Downward
J
J
Parker
P
P
Waterfield
M D
MD
Baker
M A
MA
Greaves
M F
MF
Stanbridge
E J
EJ
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000276:immunology; D000912:Antibodies, Neoplasm / Q000276:immunology; D000936:Antigen-Antibody Complex / Q000032:analysis; D000954:Antigens, Surface / Q000032:analysis* / Q000096:biosynthesis / Q000276:immunology; D002471:Cell Transformation, Neoplastic / Q000032:analysis; D005347:Fibroblasts / Q000032:analysis; D006023:Glycoproteins / Q000032:analysis / Q000096:biosynthesis / Q000276:immunology; D006026:Glycoside Hydrolases; D006367:HeLa Cells / Q000032:analysis; D006801:Humans; D006822:Hybrid Cells / Q000032:analysis* / Q000187:drug effects; D007122:Immunoelectrophoresis; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase; D010455:Peptides / Q000032:analysis; D010750:Phosphoproteins / Q000032:analysis* / Q000096:biosynthesis / Q000276:immunology; D010766:Phosphorylation; D011498:Protein Precursors / Q000032:analysis; D011499:Protein Processing, Post-Translational; D014415:Tunicamycin / Q000494:pharmacology
D000911:Antibodies, Monoclonal; D000912:Antibodies, Neoplasm; D000936:Antigen-Antibody Complex; D000954:Antigens, Surface; D006023:Glycoproteins; D010455:Peptides; D010750:Phosphoproteins; D011498:Protein Precursors; D014415:Tunicamycin; D006026:Glycoside Hydrolases; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase