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 com prising the N-terminus of a B-type cyclin fused to bacterial chloramph enicol acetyl transferase (CAT) was degraded at the end of mitosis lik e endogenous cyclins. Point mutations in the destruction box of this c onstruct completely abolished its mitotic instability. When the destru ctible reporter was driven by the cyclin B2 promoter, CAT activity mim icked 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 add ition of the proteasome inhibitors lactacystin and ALLN showed that cy clin synthesis continued after the end of mitosis, The destruction box -specific degradation of cyclins normally ceases at the onset of S pha se, and is active in fibroblasts arrested in G(0) and in differentiate d C2 myoblasts. We were able to reproduce this proteolysis in vitro in extracts of synchronized cells, Extracts of G(1) cells degraded cycli n B1 whereas p27(Kip1) was stable, in contrast, cyclin B1 remained sta ble and p27(Kip1) was degraded in extracts of S phase cells.