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Sci Rep
2020 Jun 02;101:8924. doi: 10.1038/s41598-020-65009-w.
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Two-Step Mechanism of Cyclin B Degradation Initiated by Proteolytic Cleavage with the 26 S Proteasome in Fish.
Tokumoto T
,
Hossain MF
,
Jyoti MMS
,
Ali MH
,
Hossain MB
,
Acharjee M
,
Rezanujjaman M
,
Tokumoto M
.
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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.
Figure 1. Proteolytic cleavage of E. coli-produced goldfish cyclin Bs by the 26 S proteasome purified from immature goldfish oocytes. (A) Amino acid sequence of the N-terminal region of goldfish cyclin B. The site cleaved by the 26 S proteasome (C-terminus of K57) and sites mutated from lysine to arginine residues are indicated by dots. The destruction box and lysine-rich stretch are also indicated. (B) Proteolytic cleavage of cyclin Bs by purified 26 S proteasome. Cyclins (5 μg/ml) were incubated for 60 min at room temperature with (+) or without (−) purified 26 S proteasome (60 μg/ml) in reaction buffer (100 mM Tris-HCl, 5 mM MgCl2, 0.04 mM ATP, pH 7.6). The samples were combined with Laemmli’s SDS sample buffer and separated by SDS-PAGE. Cyclin B was detected by immunoblotting against an anti-goldfish cyclin B (B63) monoclonal antibody. The position to which the cleaved cyclin B migrated is indicated by an asterisk.
Figure 2. Degradation of goldfish cyclin Bs in Xenopus egg extracts. (A) E. coli-produced goldfish cyclins were added to a Xenopus egg extract at a final concentration of 5 μg/ml. The incubations proceeded in the absence (−Ca2 + ) or presence (+Ca2 + ) of 0.4 mM CaCl2 for the indicated times. Cyclin degradation was terminated by transferring a portion of the reaction mixture into SDS sample buffer at the indicated times. Cyclin B was detected with the B63 antibody. The position of the cleaved cyclin B is indicated by an asterisk. (B) Effect of protease and proteasome inhibitors on cyclin B degradation in Xenopus egg extracts. E. coli- produced goldfish cyclin Δ0 or Δ57 was added to Xenopus egg extract at a final concentration of 5 μg/ml. Incubations proceeded after addition of (+Ca2 + ) of 0.4 mM CaCl2 in the presence of various inhibitors at 50 μM for 60 min except absence of (−Ca2 + ). The sample without incubation was included as Control. ATP- indicates the addition of ATP-depletion system (1 μg/ml hexokinase and 10 mM glucose). Cyclin B was detected by the B63 antibody.
Figure 3. Ubiquitination of cyclin B after proteolytic cleavage by the 26 S proteasome. (A) Proteolytic cleavage and ubiquitination of native cyclin B. The MPF complex in mature goldfish oocytes was prepared using suc1-beads35. The beads were washed with buffer (50 mM Tris-HCl, 20% glycerol, 10 mM 2-mercaptoethanol, 0.1 mM ATP, pH 7.5) and shaken in the absence (−) or presence (+) of 60 μg/ml of the 26 S proteasome at room temperature with agitation. Samples were treated with SDS sample buffer (2 lanes at left side), or 26 S proteasomes were washed out with buffer (2 lanes at right side). Then the beads were shaken in the ubiquitination system (goldfish recombinant E1, E2-C, APC11 and ubiquitin) at room temperature with agitation (Ubiquitination system + ). Samples were treated with SDS sample buffer. All samples were immunoblotted against anti-goldfish cyclin B polyclonal antibodies42. The truncated cyclin B produced by the 26 S proteasome proteolytic cleavage is indicated by an asterisk. Protein bands of ubiquitinated cyclin B are indicated by a square bracket (Ub-cycB). (B) A model of the two-step degradation of cyclin B upon fish fertilization. Lysine residues that are target sites for proteolytic cleavage by the 26 S proteasome (K57) and ubiquitination (K77) are indicated. Ubiquitin is indicated as Ub.
Figure 4. Cell division arrest assay using 2-cell-stage Xenopus embryos. Purified recombinant cyclin Bs at a final concentration of 5 μg/ml were microinjected into one side of 2-cell-stage Xenopus embryos. The embryos were incubated until stages 7–8 and photographed. The formation of a blastomere stopped the cleavage, is indicated by the arrowhead
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