Synergistic activation of signalling to extracellular signal-regulated kinases 1 and 2 by epidermal growth factor and 4 beta-phorbol 12-myristate 13-acetate.

Signal transduction pathways are often embedded in complex networks, which result from interactions between pathways and feedback circuitry. In order to understand such networks, qualitative information on which interactions take place and quantitative data on their strength become essential. Here, we have investigated how the multiple interactions between the mitogen-activated protein kinase cascade and protein kinase C (PKC) affect the time profile of extracellular signal-regulated kinase (ERK) phosphorylation upon epidermal growth factor (EGF) stimulation in normal rat kidney fibroblasts. This profile is a major determinant for the cellular response that is evoked. We found that EGF stimulation leads to a biphasic ERK-PP pattern, consisting of an initial peak and a relaxation to a low quasi-steady state-phase. Costimulation with the EGF and PKC activator, 4 beta-phorbol 12-myristate 13-acetate (PMA) resulted in a similar pattern, but the ERK-PP concentration in the quasi-steady state-phase was synergistically higher than after stimulation with either EGF or PMA only. This resulted in prolonged signalling to ERK. PMA increased the EGF concentration sufficient to obtain half-maximum ERK phosphorylation. These data suggest that PKC amplifies EGF-induced signalling to ERK, without increasing its sensitivity to low EGF concentrations. Furthermore, PKC inhibition did not affect the ERK-PP time profile upon EGF stimulation and a cellular phospholipase A2 (cPLA(2)) inhibitor did not decrease the synergistic effect of EGF and PMA. This indicates that the positive feedback loop from ERK to Raf via cPLA(2) and PKC does not contribute significantly to signalling from EGF to ERK in normal rat kidney cells. Taken together, we provide a quantitative description of which reported interactions in this network affect the time profile of ERK phosphorylation.