REGULATION OF P42 MITOGEN-ACTIVATED-PROTEIN KINASE-ACTIVITY BY PROTEIN PHOSPHATASE 2A UNDER CONDITIONS OF GROWTH-INHIBITION BY EPIDERMAL GROWTH-FACTOR IN A431 CELLS

Epidermal growth factor (EGF), which plays an important role in the gr owth regulation of a large variety of normal and tumor cells, has been shown to display an ambivalent dose-dependent effect on the prolifera tion of epithelial cells overexpressing EGF receptor. In a previous st udy aimed at dissecting the biochemical events leading to this dual ac tion in A431 cells which over express EGF receptor, we have reported a relationship between the dual stimulator/inhibitor effect of EGF and the activity of the serine/threonine p42 mitogen-activated protein (MA P) kinase. Indeed, a growth stimulatory concentration of EGF is shown to lead to a moderate but persistent activation of p42 MAP kinase. Con versely, an early peak of MAP kinase activation, that rapidly falls be low the basal level, is observed in the presence of a growth-inhibitor y concentration of EGF To assess the mechanism of the p42 MAP kinase i nactivation under circumstances of negative growth regulation by EGF, we have investigated the role of the serine/threonine phosphatase 2A i n this process. A constitutive phosphatase 2A activity was observed in untreated cells, that decreases rapidly in response to both high and low EGF concentrations. However, after this early inactivation, the ph osphatase 2A activity was completely reversed concurrently with MAP ki nase inactivation, after 40 min of treatment with 10 nM EGF. Conversel y, in cells treated with 1 pM EGF, phosphatase 2A activity remained be low the control level during all the time of the treatment, in associa tion with a sustained MAP kinase activation. These results suggest tha t MAP kinase inactivation is closely related to phosphatase 2A activat ion. We then investigated the effect of the serine/threonine phosphata se inhibitor okadaic acid on the MAP kinase inactivation and observed that okadaic acid, at a concentration reported to specifically inhibit phosphatase 2A activity, totally reverses the MAP kinase inactivation induced by long-term treatment with 10 nM EGF. Additionally, we have shown that the protein synthesis inhibitor cycloheximide fails to affe ct the EGF-induced MAP kinase regulation, indicating that mitogen-indu ced protein phosphatases are not, or are only slightly, required in th is regulation. In conclusion, our data demonstrate that the ambivalent action of EGF on the proliferation of A431 cells is associated with d ifferential mechanisms of p42 MAP kinase regulation catalysed by the s erine/threonine phosphatase 2A.