Stress-activated protein kinases are negatively regulated by cell density

Stimulation by UV irradiation, TNF alpha, as well as PDGF or EGF activates the JNK/SAPK signalling pathway in mouse fibroblasts, This results in the phosphorylation of the N-terminal domain of c-Jun, increasing its transactivation potency. Using an antibody that specifically recognizes c-Jun phosphorylated at Ser63, we show that culture conflueucy drastically inhibited c-Jun N-terminal phosphorylation due to the inhibition of the JNK/SAPK pathway. Transfection experiments demonstrate that the inhibition occurs at the same level as, or upstream of, the small G-proteins cdc42 and Rad. In contrast, the classical MAPK pathway was insensitive to conflueucy, The inhibition of JNK/SAPK activation depended on the integrity of the actin microfilament network. These results were confirmed and extended in monolayer wounding experiments. After PDGF, EGF or UV stimulation, c-Jun was predominantly phosphorylated in cells bordering the wound, which are the cells that move to occupy the wounded area. Thus, modulation of the stress-dependent signal cascade by conflueucy will restrict c-dun N-terminal phosphorylation in response to mitogenic or chemotactic agents to cells that border a wounded area.