Abstract
The epidermal growth factor receptor (EGFR) is regulated by at least two mechanisms involving protein kinase C (PKC), inhibition of EGF binding and inhibition of EGF-stimulated tyrosine kinase activity. In this study we investigated whether mitogen-activated protein kinase (MAPK) mediates the inhibitory effects of PKC on EGFR binding or kinase activity by pretreating NIH3T3 and Chinese hamster ovary cells expressing the EGFR with PD98059, an inhibitor of MAPK/extracellular signal-regulated kinase kinase (MEK). We also determined whether substitution of cysteine for threonine at residue 669, the site of MAPK phosphorylation of the EGFR, alters the inhibition of kinase activity by PKC. The results indicate that 1) PKC down-regulates EGFR tyrosine kinase activity by an MEK-dependent mechanism presumably involving MAPK; 2) the inhibition by PKC is not a direct result of phosphorylation of the EGFR by PKC or MAPK; 3) activation of MAPK is not sufficient to regulate EGFR kinase activity; and 4) PKC-mediated down-regulation of EGF binding and EGFR kinase activity occur by different mechanisms. These data are consistent with a model for regulation of the EGFR by other receptors whereby their activation of PKC, in conjunction with MAPK, results in the phosphorylation of a protein(s) that modulates EGFR kinase activity.
Highlights
Kinase C (PKC), inhibition of EGF binding and inhibi- inhibition of EGF binding and inhibition of EGF-stimulated tion of EGF-stimulated tyrosine kinase activity
The results indicate that 1) PKC down-regulates epidermal growth factor receptor (EGFR) tyrosine kinase activity by an MEK-dependent mechanism presumably involving MAPK; 2) the inhibition by PKC is not a direct result of phosphorylation of the EGFR by PKC or MAPK; 3) activation of MAPK is not sufficient to regulate EGFR kinase activity; and 4) PKCmediated down-regulation of EGF binding and EGFR kinase activity occur by different mechanisms
1 The abbreviations used are: EGFR, epidermal growth factor receptor; EGF, EGF receptor; CHO, Chinese hamster ovary cells; PKC, protein kinase C; MAP, mitogen-activated protein; MAPK, MAP kinase; of the EGF receptor, we focused on other kinases that are activated by PKC
Summary
Ʈ To whom correspondence should be addressed: Ben May Inst., MC 6027, University of Chicago, 5841 S. ERK, extracellular signal-regulated kinase; MEK, MAP kinase/ERK kinase; DMEM, Dulbecco’s modified Eagle’s medium; PAGE, polyacrylamide gel electrophoresis; PDBu, phorbol 12,13-dibutyrate; TBST, Trisbuffered saline and 0.2% Tween 20; MBP, myelin basic protein. The results indicate that the MAPK signaling pathway is a key step in EGFR regulation, but the effect does not appear to be mediated through direct phosphorylation of the receptor.
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