Received for publication July 7, 2004.
Revised December 21, 2004.
Accepted for publication December 22, 2004.

Src Family Kinase Inhibitors Block Amphiregulin-Mediated Autocrine ErbB Signaling in Normal Human Keratinocytes

Abstract

c-Src potentiates proliferation, survival, and invasiveness in response to EGF in human mammary carcinoma cells. Tyrosine 845 of ErbB1 is phosphorylated by Src, and has been implicated in control of malignant behavior. While several lines of evidence also suggest important interactions of ErbB and Src family kinase signaling in normal epithelial cells, little is known about the mechanism of this interaction. Studying normal human keratinocytes (NHK), here we demonstrate strong expression of the Src family kinases Src, Yes, and Fyn; Src family kinase-dependent stimulation of Tyr 845 by EGF; and potent inhibition of NHK proliferation and migration by two Src family kinase inhibitors, PP1 and PD173952. EGF-stimulated ERK phosphorylation occurred at much lower concentrations of EGF than required to phosphorylate Tyr 845. Moreover, the effect of Src family kinase inhibitors on EGF-stimulated ERK phosphorylation was transient, prompting a search for other targets of Src family kinase action. By ELISA analysis, we found that three different Src family kinase inhibitors (PD173952, PP1, and SU6656) markedly inhibited elaboration of soluble amphiregulin by NHK. The ErbB inhibitor PD158780 and the MEK inhibitor U0126 also markedly inhibited NHK proliferation, migration, and amphiregulin production. Together, these observations demonstrate that one or more Src family kinases act upstream as well as downstream of ErbB1 to promote amphiregulin-dependent autocrine stimulation of NHK, and suggest that autocrine NHK proliferation is more dependent upon ERK activation than upon Tyr 845 phosphorylation.

Key words: NGF/EGF, Phosphorylation/Dephosphorylation, Src and other nonreceptor tyrosine kinases, MAP Kinase