Protein kinase C inhibits epidermal growth factor receptor phosphorylation in enterocytes.
Epidermal growth factor (EGF) is an important proliferative signal in the gastrointestinal tract. The EGF receptor (EGFr), which transduces the mitogenic stimulus to the cell, may be regulated by a number of factors including extracellular matrix, cell-cell contact, and other peptides. As protein kinase C (PK-C) has been shown to phosphorylate and down-regulate the EGFr in certain tumor cell lines, we propose that PK-C, an important regulatory enzyme, modulates the phosphorylation of the EGFr in the IEC 6 rat enterocyte cell line. IEC 6 cells were cultured in dishes with Dulbecco's modified Eagle's medium, (DMEM)/5% fetal bovine serum (FBS), which was changed to DMEM/1% FBS 24 hr prior to all experiments. Cells (three dishes per group) were treated with the PK-C activating phorbol ester phorbol-12-myristate-13-acetate (PMA) (100 nM) or vehicle for 1 hr and challenged with EGF (50 ng/ml) or vehicle for 15 min. Cell lysates were then prepared. EGFr tyrosine phosphorylation was determined by immunoprecipitating the EGFr and immunoblotting with an antibody against phosphotyrosine. EGFr apparent molecular weight was assessed in the same lysates by Western blot with an anti-EGFr antibody. Blots were analyzed by computer densitometry. Data are expressed as mean +/- SEM; n = 3 with P value determined by t test. Exposure of cells to PMA resulted in a decrease in the EGF-stimulated EGFr phosphotyrosine content from 96 +/- 5 U in control to 66 +/- 6 U in PMA (P < 0.01). The amount of receptor did not change, 43 +/- 3 U in control vs 44 +/- 3 U in PMA (P = 0.44). Further, exposure to PMA in the absence of EGF caused a gel shift of the EGFr band consistent with a nontyrosine phosphorylation of the protein. We demonstrate that activation of PK-C results in a modification of the EGFr coincident with inhibition of EGF-stimulated receptor tyrosine kinase activity. These data support a role for PK-C in the regulation of EGFr function and hence modulation of mitogenic signals in enterocytes.
Summers
S T
ST
Department of Surgery, Veteran's Administration Medical Center, Baltimore, Maryland 21201, USA.
Bass
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BL
D000818:Animals; D002450:Cell Communication / Q000187:drug effects; D002460:Cell Line; D066246:ErbB Receptors / Q000037:antagonists & inhibitors* / Q000187:drug effects / Q000378:metabolism; D007413:Intestinal Mucosa / Q000378:metabolism*; D007422:Intestines / Q000166:cytology; D010766:Phosphorylation / Q000187:drug effects; D011493:Protein Kinase C / Q000494:pharmacology*; D051381:Rats; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D013997:Time Factors; D014443:Tyrosine / Q000378:metabolism
D014443:Tyrosine; D066246:ErbB Receptors; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate