Effects of epidermal growth factor on the tyrosine phosphorylation of mitogen-activated protein kinases in monolayer cultures of porcine granulosa cells

We have examined porcine granulosa cells (pGCs) for the presence of immunodetectable mitogen-activated protein (MAP) kinases (extracellular signal-regulated kinases, ERK) and have further studied the effects of epidermal growth factor (EGF) on the activation of these kinases. Cell lysates prepared from untreated monolayer cultures of pGCs were subjected to Western immunoblotting analysis using monoclonal antibodies to ERK1, ERK2 and pan-specific ERK. MAP kinases were detected having mol wts of 87K (ERK87), 54K (ERK54), 44K (ERK1), and 42K (ERK2). Treatment of pGCs with increasing concentrations (1-10 ng/ml) of EGF for 10 min resulted in electrophoretic mobility shifts of ERK1 and ERK2 suggesting hyperphosphorylation. Immunoprecipitation with an antiphosphotyrosine antibody (PY20), followed by Western analysis using pan-ERK, revealed a marked concentration-dependent increase in tyrosine phosphorylation of ERK2 in response to EGF treatment. The mobility shift and tyrosine phosphorylation of ERK2 was observed as early as 1 min after treatment with 10 ng/ml EGF. In-gel myelin basic protein (MBP) kinase assays revealed significant MBP kinase activity associated with ERK1 and ERK2 in total cell lysates and ERK2 in PY20 immunoprecipitates. Although ERK1 displayed a moderate mobility shift in response to EGF, tyrosine phosphorylation of this MAP kinase was not appreciably increased by EGF. Furthermore, PY20 immunoprecipitates demonstrated minimal MBP kinase associated with ERK1 in response to EGF treatment. Electrophoretic migration, tyrosine phosphorylation, and MBP kinase activity of the ERK54 and ERK87 was not effected regardless of EGF concentration or duration of treatment. These data demonstrate for the first time that pGCs contain immunodetectable MAP kinases. EGF, in a concentration- and time-dependent manner, increases tyrosine phosphorylation and MBP kinase activity (i.e. activation) of ERK2, and to a lesser degree ERK1, suggesting that the activation of MAP kinase may mediate the mitogenic action of EGF in pGCs.