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Prolactin decreases epidermal growth factor receptor kinase activity via a phosphorylation-dependent mechanism
1Endocrinology-Reproductive Physiology Program, University of Wisconsin, Madison, Wisconsin 53706, USA.
Abstract:
Previously, we have shown that prolactin inhibits epidermal growth factor (EGF)-induced mitogenesis in mouse mammary epithelial cells without altering the response to other growth promoting agents. This effect has been associated with reduced EGF-induced EGF receptor (EGFR) tyrosine phosphorylation, Grb-2 association, and Ras activation. Our current hypothesis is that prolactin induces an alteration in EGFR kinase activity via a phosphorylation-dependent mechanism. To test this hypothesis, we treated normal murine mammary gland cells with or without 100 ng/ml prolactin. EGFR isolated by wheat germ agglutinin affinity chromatography from nontreated cells exhibited substantial ligand-induced phosphorylation, and EGFR isolated from prolactin-treated cells displayed minimal EGF-induced EGFR phosphorylation, as well as decreased kinase activity toward exogenous substrates. The observed decrease in ligand-induced EGFR phosphorylation could not be attributed to either differential amounts of EGFR, decreased EGF binding affinity, or the presence of a phosphotyrosine phosphatase or ATPase. EGFR isolated from prolactin-treated cells exhibited increased phosphorylation on threonine. Removal of this phosphorylation with alkaline phosphatase restored EGFR kinase activity to levels observed in nontreated cells. Therefore, these results suggest that prolactin antagonizes EGF signaling by increasing EGFR threonine phosphorylation and decreasing EGF-induced EGFR tyrosine phosphorylation.
Insights
Prolactin inhibits epidermal growth factor (EGF) signaling by altering EGF receptor (EGFR) activity. It increases EGFR threonine phosphorylation, reducing EGF-induced tyrosine phosphorylation and cell growth.
Area of Science:
- Cell biology
- Molecular endocrinology
- Signal transduction
Background:
- Prolactin is known to inhibit epidermal growth factor (EGF)-induced mitogenesis in mammary epithelial cells.
- This inhibition is linked to reduced EGF receptor (EGFR) tyrosine phosphorylation, Grb-2 association, and Ras activation.
Purpose of the Study:
- To investigate the hypothesis that prolactin alters EGFR kinase activity through a phosphorylation-dependent mechanism.
- To elucidate the specific molecular events mediating prolactin's antagonistic effect on EGF signaling.
Main Methods:
- Normal murine mammary gland cells were treated with or without prolactin.
- EGFR was isolated using wheat germ agglutinin affinity chromatography.
- EGFR phosphorylation (tyrosine and threonine), EGF binding affinity, and kinase activity were assessed.
- The effect of alkaline phosphatase on EGFR activity was examined.
Main Results:
- Prolactin treatment significantly reduced EGF-induced EGFR tyrosine phosphorylation and kinase activity.
- EGFR isolated from prolactin-treated cells showed increased threonine phosphorylation.
- This decrease in activity was not due to altered EGFR levels, EGF binding, or phosphatase activity.
- Dephosphorylation of threonine residues restored EGFR kinase activity.
Conclusions:
- Prolactin antagonizes EGF signaling by increasing EGFR threonine phosphorylation.
- This increase in threonine phosphorylation leads to decreased EGF-induced EGFR tyrosine phosphorylation and subsequent inhibition of mitogenesis.