The proteolysis of mitotic cyclins in mammalian cells persists from the end of mitosis until the onset of S phase

Michael Brandeis, Tim Hunt*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

250 Scopus citations

Abstract

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 Go 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 p27(Kip1) was stable, in contrast, cyclin B1 remained stable and p27(Kip1) was degraded in extracts of S phase cells.

Original languageAmerican English
Pages (from-to)5280-5289
Number of pages10
JournalEMBO Journal
Volume15
Issue number19
DOIs
StatePublished - 1 Oct 1996
Externally publishedYes

Keywords

  • Cell cycle control
  • Lactacystin
  • P27(Kipl)
  • Proteasome
  • Ubiquitin

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