Inhibition of the EGF-Activated MAP Kinase Signaling Pathway by Adenosine 3′,5′-Monophosphate

TLDR

Mitogen‑activated protein kinases p42/p44 are activated by EGF through a sequential cascade involving Sos, Ras, Raf‑1, MKK, and MAPK, and phosphorylation of Raf‑1 reduces its affinity for Ras, potentially contributing to cAMP‑mediated blockade. The study examined whether elevating intracellular cAMP with forskolin and 3‑isobutyl‑1‑methylxanthine inhibits the EGF‑stimulated MAPK pathway. Higher cAMP concentrations blocked Raf‑1, MKK, and MAPK activation in Rat1hER fibroblasts and caused a three‑fold increase in Raf‑1 phosphorylation at serine 43.

Abstract

Mitogen-activated protein (MAP) kinases p42 mapk and p44 mapk are activated in cells stimulated with epidermal growth factor (EGF) and other agents. A principal pathway for MAP kinase (MAPK) activation by EGF consists of sequential activations of the guanine nucleotide exchange factor Sos, the guanosine triphosphate binding protein Ras, and the protein kinases Raf-1, MAPK kinase (MKK), and MAPK. Because adenosine 3′,5′-monophosphate (cAMP) does not activate MAPK and has some opposing physiologic effects, the effect of increasing intracellular concentrations of cAMP with forskolin and 3-isobutyl-1-methylxanthine on the EGF-stimulated MAPK pathway was studied. Increased concentrations of cAMP blocked activation of Raf-1, MKK, and MAPK in Rat1hER fibroblasts, accompanied by a threefold increase in Raf-1 phosphorylation on serine 43 in the regulatory domain. Phosphorylation of Raf-1 in vitro and in vivo reduces the apparent affinity with which it binds to Ras and may contribute to the blockade by cAMP.