Cyclin B targets p34cdc2 for tyrosine phosphorylation.

Abstract

A universal intracellular factor, the 'M phase-promoting factor' (MPF), triggers the G 2 /M transition of the cell ode in all organisms. In late G 2 , it is present as an inactive complex of tyrosine-phosphorylated p34 cdc2 and unphosphorylated cyclin B cdc13 . In M phase, its activation as an active MPF displaying histone H1 kinase (H1K) originates from the concomitant tyrosine dephosphorylation of the p34 cdc2 subunit and the phosphorylation of the cylin B cdc13 subunit. We have investigated the role of cyclin in the formation of this complex and the tyrosine phosphorylation of p34 cdc2 , using highly synchronous mitotic sea urchin eggs as a model. As cells leave the S phase and enter the G 2 phase, a massive tyrosine phosphorylation of p34 cdc2 occurs. This large p34 cdc2 tyrosine phosphorylation burst does not arise from a massive increase in p34 cdc2 concentration. It even appears to affect only a fraction (non-immuno-precipitable by anti-PSTAIR antibodies) of the total p34 cdc2 present in the cell. Several observations point to an extremely close association between accumulation of unphosphorylated cyclin and p34 cdc2 tyrosine phosphorylation: (i) both events coincide perfectly during the G 2 phase; (ii) both tyrosine-phosphorylated p34 cdc2 and cyclin are not immunoprecipitated by anti-PSTAIR antibodies; (iii) accumulation of unphosphorylated cyclin by aphidicolin treatment of the cells, triggers a dramatic accumulation of tyrosine-phosphorylated p34 cdc2 ; and (iv) inhibition of cyclin synthesis by emetine inhibits p34 cdc2 tyrosine phosphorylation without affecting the p34 cdc2 concentration. These results show that, as it is synthesized, cyclin B binds and recruits p34 cdc2 for tyrosine phosphorylation; this inactive complex then requires the completion of DNA replication before it can be turned into fully active MPF. These results fully confirm recent data obtained in vitro with exogenous cyclin added to cycloheximide-treated Xenopus egg extracts.