The tyrosine kinase activity of the C-erbB-2 gene product (p185) is required for growth inhibition by anti-p185 antibodies but not for the cytotoxicity of an anti-p185-ricin-A chain immunotoxin.
Our previous studies have demonstrated that 7 of 10 IgG antibodies against distinct epitopes on the extracellular domain of the c-erbB-2 gene product (p185) inhibit the anchorage-independent growth of SKBr3 human breast-cancer cells that overexpress this transmembrane tyrosine kinase growth-factor receptor. Two of 7 growth-inhibitory antibodies also block the binding and function of the gp30 and p75 c-erbB-2 ligands. In this report we have studied phosphorylation of p185 and different intracellular substrates after binding of antibodies that do or do not inhibit tumor-cell growth. A correlation has been found between antibodies that inhibit growth and the intensity of tyrosine phosphorylation of p185. At late intervals, serine phosphorylation of at least 3 intracellular substrates is increased preferentially by growth-inhibitory antibodies. To test the importance of p185 kinase activity more critically, NIH3T3 cells were transfected with an expression vector containing the full-length human c-erbB-2 gene (cell line 17313), c-erbB-2 with deletion of the kinase region from codons 751-979 (cell line 9309) or c-erbB-2 with deletion of most of the intracellular domain from codons 684-1255 (cell line 9310). Unconjugated antibodies inhibited anchorage-independent growth of 17313 cells as well as SKBr3 cells, but did not inhibit growth of either 9309 or 9310 cells. In contrast, the cytotoxic effect of anti-p185-ricin A chain (RTA) conjugates was comparable for 17313, 9309 and 9310. The tyrosine-kinase activity of p185 is required for growth inhibition mediated by unconjugated anti-p185 antibodies, but not for the cytotoxic activity of anti-p185-RTA immunotoxins.
Xu
F J
FJ
Department of Medicine, Duke University Medical Center, Durham, NC 27710.
Boyer
C M
CM
Bae
D S
DS
Wu
S
S
Greenwald
M
M
O'Briant
K
K
Yu
Y H
YH
Mills
G B
GB
Bast
R C
RC
D016475:3T3 Cells / Q000201:enzymology / Q000502:physiology; D000818:Animals; D000906:Antibodies / Q000494:pharmacology*; D001943:Breast Neoplasms / Q000188:drug therapy / Q000473:pathology; D002448:Cell Adhesion; D002455:Cell Division / Q000187:drug effects; D006801:Humans; D000922:Immunotoxins / Q000633:toxicity*; D007424:Intracellular Fluid / Q000378:metabolism; D051379:Mice; D009154:Mutation; D010766:Phosphorylation / Q000187:drug effects; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000276:immunology* / Q000378:metabolism*; D012276:Ricin / Q000633:toxicity*; D014162:Transfection; D014407:Tumor Cells, Cultured / Q000187:drug effects
D000906:Antibodies; D000922:Immunotoxins; D011505:Protein-Tyrosine Kinases; D018719:Erb-b2 Receptor Tyrosine Kinases; D012276:Ricin