The proteolysis of mitotic cyclins in mammalian cells persists from the end of mitosis until the onset of S phase.
We have studied how the cell cycle-specific oscillations of mitotic B-type cyclins are generated in mouse fibroblasts. A reporter enzyme comprising the N-terminus of a B-type cyclin fused to bacterial chloramphenicol acetyl transferase (CAT) was degraded at the end of mitosis like endogenous cyclins. Point mutations in the destruction box of this construct completely abolished its mitotic instability. When the destructible reporter was driven by the cyclin B2 promoter, CAT activity mimicked the oscillations in the level of the endogenous cyclin B2. These oscillations were largely conserved when the reporter was transcribed constitutively from the SV40 promoter. Pulse-chase experiments or addition of the proteasome inhibitors lactacystin and ALLN showed that cyclin synthesis continued after the end of mitosis. The destruction box-specific degradation of cyclins normally ceases at the onset of S phase, and is active in fibroblasts arrested in G0 and in differentiated C2 myoblasts. We were able to reproduce this proteolysis in vitro in extracts of synchronized cells. Extracts of G1 cells degraded cyclin B1 whereas p27Kip1 was stable, in contrast, cyclin B1 remained stable and p27Kip1 was degraded in extracts of S phase cells.
Brandeis
M
M
ICRF Clare Hall Laboratories, South Mimms, Herts, UK.
Hunt
T
T
D016475:3T3 Cells; D000595:Amino Acid Sequence; D000818:Animals; D018797:Cell Cycle Proteins; D002460:Cell Line; D002474:Cell-Free System; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics / Q000378:metabolism; D019926:Cyclin B; D056744:Cyclin B1; D050760:Cyclin-Dependent Kinase Inhibitor p27; D016213:Cyclins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D005786:Gene Expression Regulation; D017930:Genes, Reporter / Q000235:genetics; D007399:Interphase / Q000502:physiology*; D051379:Mice; D008869:Microtubule-Associated Proteins / Q000378:metabolism; D008938:Mitosis / Q000502:physiology*; D008969:Molecular Sequence Data; D018485:Muscle Fibers, Skeletal / Q000502:physiology; D017354:Point Mutation; D011401:Promoter Regions, Genetic / Q000235:genetics; D012333:RNA, Messenger / Q000096:biosynthesis; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D014158:Transcription, Genetic; D025521:Tumor Suppressor Proteins
C534700:Ccnb1 protein, mouse; C493961:Cdkn1b protein, mouse; D018797:Cell Cycle Proteins; D019926:Cyclin B; D056744:Cyclin B1; D016213:Cyclins; D008869:Microtubule-Associated Proteins; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D025521:Tumor Suppressor Proteins; D050760:Cyclin-Dependent Kinase Inhibitor p27; D015500:Chloramphenicol O-Acetyltransferase