Abstract
The mechanism(s) by which monoclonal antibodies (mAbs) against the epidermal growth factor (EGF) receptor regulate receptor function have been investigated with NIH3T3/HER14 fibroblasts expressing human EGF receptors. Bivalent 225 mAb or monovalent 225 Fab' inhibited transforming growth factor (TGF)-alpha-induced EGF receptor tyrosine phosphorylation and cell proliferation. Culture of HER14 cells with 225 mAb or 225 Fab' did not activate EGF receptor tyrosine kinase when assayed after lysis of cells in SDS sample buffer. However, when cells were cultured with bivalent 225 mAb, but not with monovalent 225 Fab', and were subsequently lysed and further incubated in Triton X-100 lysis buffer containing proteinase and phosphatase inhibitors, receptor phosphorylation was observed. Phosphorylation was confined to tyrosine residues and was inhibited by addition of genistein after lysis, indicating that it was due to the activation of protein tyrosine kinase. The activity of bivalent 225 mAb was unphysiologic, in contrast with TGF-alpha, in that receptor kinase activation occurred only after cell lysis and with delayed kinetics; serine and threonine phosphorylation did not occur; and down-regulation of EGF receptors was slower. Selective mAb-mediated phosphorylation of tyrosine residues on EGF receptors was sufficient to activate phosphorylation of a SH2 group-bearing substrate, phospholipase C-gamma, indicating that serine/threonine phosphorylation is not required for EGF receptor kinase activity. These studies provide novel insights into the capacity of bivalent mAb to modulate EGF receptor function.
Highlights
From the $Laboratoryof Receptor Biology, Memorial Sloan-Kettering Cancer Center and the$Cornell UniversityMedical College, New York, New York 10021
Selective mAb-mediated breast [12, 13], colon [14, 15], lung [16], renal [17],and phosphorylation of tyrosine residueson EGF receptors prostate cells [18]. These findings provide evidence for an was sufficient to activate phosphorylation of a SH2 group-bearing substrate, phospholipase C-7, indicating that serine/threonine phosphorylation is not required for EGF receptor kinase activityT.hese studies provide novel insights into the capacity of bivalent mAb to modulate EGF receptor function
C, fresh HER14 cells, unexposed to ligand, ation of EGF receptors on tyrosine residues is accompanied werelysed in Triton X-100 lysis buffer, and TGF-a or antibodies by increased phosphorylation of serine and threonineresidues were added afterwards at the concentrations used in A followed by incubation for 30 min at 4 "C
Summary
Physiology,The Pennsylvania State University, College ofMedicine, Hershey, PA 17033. a, transforming growth factor-a; PLC-y, phospholipase-y; mAb, monoclonal antibody; PAGE, polyacrylamide gel electrophoresis; kinase activity. A, transforming growth factor-a; PLC-y, phospholipase-y; mAb, monoclonal antibody; PAGE, polyacrylamide gel electrophoresis; kinase activity. We performed these studieswith HER14 cells, which are NIH3T3cells expressing high levelsof human EGF receptors introduced by transfection[33]. DME, Dulbecco’smodified Eagle’s medium; FBS, fetal bovine serum, have been extensively investigated and appear to be normal Sample buffer to immediately denature receptor tyrosine kinase activity or werelysedby incubation in a lysis buffer containing Triton X-100 which is commonly utilized for experiments requiring EGF receptor immunoprecipitation from cell lysates [9, 10].The results of these studies provide novel insights into the relationship between tyrosine phosphorylation and EGFreceptor function
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