Transient epidermal growth factor (EGF)-dependent suppression of EGF receptor autophosphorylation during internalization.
To study the activity of the epidermal growth factor (EGF) receptor during EGF-directed internalization, liver epithelial cells were exposed to EGF at 37 degrees C for various periods of time, washed, and homogenized at 0 degrees C. EGF receptor autophosphorylation was assessed in homogenates using [gamma-32P]ATP. Autophosphorylation was stimulated 3- to 6-fold in homogenates of cells incubated with EGF (100 ng/ml) for 15 min but was at or below basal levels in homogenates of cells treated with EGF for 2.5-5 min. This was surprising because immunoblotting revealed that EGF receptor phosphotyrosine (P-Tyr) content in intact cells was near maximal from 30 s to 5 min after EGF treatment. Excess EGF (1 microgram/ml), added after homogenization but prior to the assay, increased autophosphorylation in homogenates of cells that had not been treated with EGF, but failed to increase activity in homogenates of cells treated with EGF in culture for 2.5-5 min. Suppression of tyrosine phosphorylation of an exogenous kinase substrate was also observed at times paralleling the suppression of EGF receptor autophosphorylation. The transient suppression of receptor autophosphorylation in the cell-free assay was not explained by persistent occupation of autophosphorylation sites by phosphate added in the intact cells. The sites were greater than 80% dephosphorylated during the homogenization. Additionally phosphatase inhibition that prevented the normal loss of EGF receptor P-Tyr in intact cells at 15 min did not affect the pattern of early (2.5-5 min) suppression and later (15 min) stimulation of autophosphorylation measured in the cell-free assay. The suppression was not explained by activation of protein kinase C in that depletion of greater than 95% of cellular protein kinase C activity by an 18-h incubation of cells with 10 microM 12-O-tetradecanoylphorbol 13-acetate (TPA) did not affect the early suppression of autophosphorylation in EGF-treated cells. Moreover, under the conditions tested, activation of protein kinase C by short-term treatment (0.5-10 min) with TPA or angiotensin II did not appreciably alter subsequent autophosphorylation in the cell-free assay. In contrast, a 30 degrees C preincubation of homogenates from cells with suppressed EGF receptor autophosphorylation led to the recovery of the ability of EGF to stimulate EGF receptor autophosphorylation. These results suggest that a rapid reversible protein kinase C-independent process prevents detection of EGF receptor kinase activity during an early phase of EGF-dependent receptor internalization.
Lineberger Cancer Research Center, University of North Carolina, Chapel Hill 27599.
B K
BK
McCune
C A
CA
Prokop
H S
HS
Earp
D000804:Angiotensin II / Q000494:pharmacology; D000818:Animals; D015153:Blotting, Western; D002460:Cell Line; D004815:Epidermal Growth Factor / Q000494:pharmacology*; D004848:Epithelium / Q000378:metabolism; D066246:ErbB Receptors / Q000187:drug effects / Q000378:metabolism*; D007700:Kinetics; D008099:Liver / Q000378:metabolism; D010766:Phosphorylation; D019000:Phosphotyrosine; D011493:Protein Kinase C / Q000378:metabolism; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014443:Tyrosine / Q000031:analogs & derivatives / Q000032:analysis
D000804:Angiotensin II; D019000:Phosphotyrosine; D014443:Tyrosine; D004815:Epidermal Growth Factor; D066246:ErbB Receptors; D011505:Protein-Tyrosine Kinases; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate