Calcium-mediated inactivation of the MAP kinase pathway in sea urchin eggsat fertilization

We have evaluated the regulation of a 43-kDa MAP kinase in sea urchin eggs. Both MAP kinase and MEK (MAP kinase kinase) are phosphorylated and active in unfertilized eggs while both are dephosphorylated and inactivated after fertilization, although with distinct kinetics. Reactivation of MEK or the 43-kDa MAP kinase prior to or during the first cell division was not detect ed. Confocal immunolocalization microscopy revealed that phosphorylated (ac tive) MAP kinase is present primarily in the nucleus of the unfertilized eg g, with some of the phosphorylated form in the cytoplasm as well. Incubatio n of unfertilized eggs in the MEK inhibitor U0126 (0.5 muM) resulted in the inactivation of MEK and MAP kinase within 30 min. Incubation in low concen trations of U0126 (sufficient to inactivate MEK and MAP kinase) after ferti lization had no effect on progression through the embryonic cell cycle. Mic roinjection of active mammalian MAP kinase phosphatase (MKP-3) resulted in inactivation of MAP kinase in unfertilized eggs, as did addition of MKP-3 t o lysates of unfertilized eggs. Incubation of unfertilized eggs in the Ca2 ionophore A23187 led to inactivation of MEK and MAP kinase with the same k inetics as observed with sperm-induced egg activation. This suggests that c alcium may be deactivating MEK and/or activating a MAP kinase-directed phos phatase. A cell-free system was used to evaluate the activation of phosphat ase separately from MEK inactivation. Unfertilized egg lysates were treated with U0126 to inactivate MEK and then Ca2+ was added. This resulted in inc reased MAP kinase phosphatase activity. Therefore, MAP kinase inactivation at fertilization in sea urchin eggs likely is the result of a combination o f MEK inactivation and phosphatase activation that are directly or indirect ly responsive to Ca2+. (C) 2001 Academic Press.