Requirement for receptor-intrinsic tyrosine kinase activities during ligand-induced membrane ruffling of KB cells. Essential sites of src-related growth factor receptor kinases.
We have prepared site-specific antibodies toward human insulin, insulin-like growth factor-I, and epidermal growth factor receptors with chemically synthesized peptides derived from the cDNA-predicted amino acid sequences of these receptors. Two classes of antibodies were produced toward each receptor: one toward the carboxyl termini and the other against the kinase domains containing sequences homologous to the tyrosyl phosphorylation site of the product of src gene (pp60v-src). Both classes of antibodies specifically immunoprecipitated the appropriate 125I-ligand-receptor complexes and [35S]methionine-labeled receptors with almost equal potencies. Antibodies toward the kinase domains inhibited both autophosphorylation and tyrosine kinase activity of the corresponding receptors in a cell-free system, whereas antibodies toward the carboxyl termini did not. Microinjection of the kinase-inhibitory antibodies into the cytoplasm of human epidermoid carcinoma KB cells blocked the ability of the corresponding ligand to induce membrane ruffling. In contrast, these inhibitory antibodies did not block the ability of noncorresponding ligands to induce the same response. Furthermore, control immunoglobulin and antibodies toward the carboxyl termini did not block this biological response. These results support a role for the tyrosine-specific protein kinase activities of these growth factor receptors in mediating their biological effects and suggest that the regions homologous to the tyrosyl phosphorylation site of pp60v-src are important for these kinase activities both in cell-free and intact cell systems.
Izumi
T
T
Third Department of Internal Medicine, Faculty of Medicine, University of Tokyo, Japan.
Saeki
Y
Y
Akanuma
Y
Y
Takaku
F
F
Kasuga
M
M
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal; D002462:Cell Membrane / Q000201:enzymology / Q000378:metabolism / Q000648:ultrastructure*; D006801:Humans; D007624:KB Cells; D007700:Kinetics; D008024:Ligands; D008970:Molecular Weight; D015688:Oncogene Protein pp60(v-src); D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D011972:Receptor, Insulin; D011956:Receptors, Cell Surface / Q000276:immunology / Q000378:metabolism; D012191:Retroviridae Proteins / Q000378:metabolism
D000911:Antibodies, Monoclonal; D008024:Ligands; D011956:Receptors, Cell Surface; D012191:Retroviridae Proteins; D011505:Protein-Tyrosine Kinases; D011972:Receptor, Insulin; D015688:Oncogene Protein pp60(v-src)