To complete meiosis II, cyclin B is degraded in a short period by the inactivation of M-phase promoting factor (MPF). Previously, we showed that the destruction of cyclin B was initiated by the ubiquitin-independent proteolytic activity of the 26 S proteasome through an initial cut in the N-terminus of cyclin (at K57 in the case of goldfish cyclin B). We hypothesized that this cut allows cyclin to be ubiquitinated for further destruction by the ubiquitin-dependent proteolytic pathway, which leads to MPF inactivation. In this study, we aimed to identify the ubiquitination site for further degradation. The destruction of cyclin B point mutants in which lysine residues in a lysine-rich stretch following the cut site of cyclin B had been mutated was analyzed. All the lysine point mutants except K57R (a point mutant in which K57 was substituted with arginine) were susceptible to proteolytic cleavage by the 26 S proteasome. However, the degradation of the K77R and K7677R mutants in Xenopus egg extracts was significantly slower than the degradation of other mutants, and a 42 kDa truncated form of cyclin B was detected during the onset of the degradation of these mutants. The truncated form of recombinant cyclin B, an N-terminal truncated cyclin BΔ57 produced as cut by the 26 S proteasome, was not further cleaved by the 26 S proteasome but rather degraded in Xenopus egg extracts. The injection of the K57R, K77R and K7677R cyclin B proteins stopped cleavage in Xenopus embryos. From the results of a series of experiments, we concluded that cyclin B degradation involves a two-step mechanism initiated by initial ubiquitin-independent cleavage by the 26 S proteasome at lysine 57 followed by its ubiquitin-dependent destruction by the 26 S proteasome following ubiquitination at lysine 77.
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