Abstract
The product of the c-mos proto-oncogene functions not only as an initiator of oocyte maturation but also as a component of cytostatic factor that causes the natural arrest of the unfertilized egg at the second meiotic metaphase. It has been shown that Mos can phosphorylate and activate mitogen-activated protein (MAP) kinase kinase (MAPKK) in vitro, leading to activation of MAP kinase. In this study, by using an anti-MAPKK antibody that can specifically inhibit Xenopus MAPKK activity, we have shown that MAPKK mediates the cytostatic factor activity of Mos. Coinjection of this anti-MAPKK antibody with the bacterially expressed Mos protein into a two-cell embryo prevented the Mos-induced cleavage arrest as well as the Mos-induced MAP kinase activation. The analysis of individual embryos indicated that the degree of the cleavage arrest was correlated with the extent of the MAP kinase activation in the Mos- and the Mos/antibody-injected embryos. These observations suggest the involvement of a signal transmission pathway consisting of Mos, MAPKK, and MAP kinase in the metaphase arrest.
Highlights
Theproductof the c-mos proto-oncogene functions The product of the c-mos proto-oncogene, a 39-kDa germ-cellnot onlyas an initiator of oocyte maturation buatlso as specific serinelthreonine protein kinase, aisn active component a componentof cytostaticfactor thatcauses the natural of cytostatic factor (CSF) [7]
During this meiotic matu- kinase activating factor can undergo ration process, a key event is the activation of maturation- autophosphorylation on serine, threonine, and tyrosine resipromoting factor (MPF)’ that causes germinal vesicle break- dues (23,271 and phosphorylate thkeinase-deficient mutant of down and chromosome condensation [1].MPF, a complex of the MAP kinase on the regulatory tyrosine and threonine residues (MAP=, serinelthreonine protein kinase~ 3 4 “a~nd‘ c~yclin B, is ubiqui- [22,24,28,29,30]
Immunoblot analysis revealed that this antiMAPKK antibody reacted with the 45-kDa MAPKK band in total extracts of embryos at the blastula stage(Fig. 1,lanes 2 and 5).This antibody reacted strongly with bacterially expressed recombinant MAPKK (Fig. 1,lanes 3 and 6)but not at all with bacterially expressed Mos
Summary
Reagents-The neutralizing antibody against Xenopus MAPKK was prepared by injecting mice with bacterially expressed glutathione Stransferase-MAPKK as described previously [49]. Stage VI oocytes were sorted by hand, washed twice in EB (20 mM HEPES pH7.2,0.25 M sucrose, 0.1 M NaCI, 2.5 mM MgCI,) and transferred to a 1.5-ml tube containing EB with aprotinin, pepstatin, chymostatin, and leupeptin (each 10 pg/ml) and with cytochalasin B Excess EB was removed, and oocytes were crushed by centrifugation a t 15,000 x g for 15 min at 2 "C. For assays of MAP kinase, groups of 10embryos or individual embryos were homogenized in 200 or 50 pl of XB (20 mM Tris-CI (pH 7.5), 60 m P-glycerophosphate, 10 mM MgCI,, 10 mM EGTA, 2 m dithiothreitol, 1 mM Na,VO,, 1mM phenylmethylsulfonyl fluoride, 20 pg/ml aprotinin), respectively. Homogenates were clarified by centrifugation at 15,000 x g for 15 min a t 2 "C
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