PP2A and MKP–1 Mediate Crosstalk Between EGF–Linked MAPK Pathways

Abstract

Abstract: : Purpose: Epidermal growth factor (EGF) in corneal epithelial cells elicits transient increases in p38 and Erk1/2 activity. However, it is unclear whether this type of response is due to crosstalk between these pathways through the protein phosphatases, MKP–1 and PP2A. Methods: Serum starved rabbit corneal epithelial cells (RCEC) were exposed to 10 ng/ml EGF. Immunoblots were performed to characterize time dependent changes in p38 and Erk1/2 activities in the presence and absence of either 100 µM sodium orthovanadate, 10 nM okadaic acid, 20 µM SB203580 or 20 µM U0126. Coimmunoprecipitation between PP2A and Erk1/2 was performed to evaluate their interaction. MKP–1 siRNA knockdown was performed to evaluate the role of this phosphatase in mediating crosstalk. Results:EGF (10 ng/ml) induced transient increases in Erk1/2 and p38 activities that reached maximal values after 15 and 30 min, respectively. These increases were in each case enhanced after 1 h approximately 2–fold in cells pre–incubate with either U0126 or SB203580. Similarly, with stably transfected d/n Erk1 and d/n p38 mutant cell lines EGF–induced similar increases in p38 and Erk1/2 activities, respectively. With okadaic acid or sodium orthovanadate, 15 min incubation with EGF increased Erk1/2 and p38 phosphorylation levels about 4– and 5–fold, respectively. Two procedures documented that PP2A interacts with Erk1/2: immunoprecipitates of total Erk were enriched with PP2A; Conversely, PP2A immunoprecipitates were enriched with Erk1/2. siRNA knockdown of MKP–1 protein expression partially inhibited crosstalk between the p38 and ERK limbs.Conclusions: EGF–induced increases in PP2A and MKP–1 activity and expression account for why increases in p38 and Erk1/2 activities are transient in RCEC. Furthermore, these increases provide insight into how crosstalk modulates EGF–induced stimulation of the p38 and ERK limbs.

Keywords: cell-cell communication • cornea: epithelium • signal transduction