EFFECTS OF EPIDERMAL GROWTH-FACTOR ON THE TYROSINE PHOSPHORYLATION OFMITOGEN-ACTIVATED PROTEIN-KINASES IN MONOLAYER-CULTURES OF PORCINE GRANULOSA-CELLS

We have examined porcine granulosa cells (pGCs) for the presence of im munodetectable mitogen-activated protein (MAP) kinases (extracellular signal-regulated kinases, ERK) and have further studied the effects of epidermal growth factor (EGF) an the activation of these kinases. Cel l lysates prepared from untreated monolayer cultures of pGCs were subj ected to Western immunoblotting analysis using monoclonal antibodies t o ERK1, ERK2 and pan-specific ERK. MAP kinases were detected having mo l wts of 87K (ERK87), 54K (ERK54), 44K (ERK1), and 42K (ERK2). Treatme nt of pGCs with increasing concentrations (1-10 ng/ml) of EGF for 10 m in resulted in electrophoretic mobility shifts of ERK1 and ERK2 sugges ting hyperphosphorylation. Immunoprecipitation with an antiphosphotyro sine antibody (PY20), followed by Western analysis using pan-ERK, reve aled a marked concentration-dependent increase in tyrosine phosphoryla tion of ERK2 in response to EGF treatment. The mobility shift and tyro sine phosphorylation of ERK2 was observed as early as 1 min after trea tment with 10 ng/ml EGF. In-gel myelin basic protein (MBP) kinase assa ys revealed significant MBP kinase activity associated with ERK1 and E RK2 in total cell lysates and ERK2 in PY20 immunoprecipitates. Althoug h ERK1 displayed a moderate mobility shift in response to EGF, tyrosin e phosphorylation of this MAP kinase was not appreciably increased by EGF. Furthermore, PY20 immunoprecipitates demonstrated minimal MBP kin ase associated with ERK1 in response to EGF treatment. Electrophoretic migration, tyrosine phosphorylation, and MBP kinase activity of the E RK54 and ERK87 was not effected regardless of EGF concentration or dur ation of treatment. These data demonstrate for the first time that pGC s contain immunodetectable MAP kinases. EGF, in a concentration- and t ime-dependent manner, increases tyrosine phosphorylation and MBP kinas e activity (i.e. activation) of ERK2, and to a lesser degree ERK1, sug gesting that the activation of MAP kinase may mediate the mitogenic ac tion of EGF in pGCs.