Intra-M Phase-promoting Factor Phosphorylation of Cyclin B at the Prophase/Metaphase Transition

Abstract

in vivo phosphorylation as shown by phosphopeptide maps of in vivo and in vitro phosphorylated cyclin B. We demonstrate that Cdc2 itself is the cyclin B kinase; cyclin B phosphorylation requires Cdc2 activity both in vivo (sensitivity to vitamin K 3 , a Cdc25 inhibitor) and in vitro (copurification with Cdc2-cyclin B, requirement of Cdc2 dephosphorylation, and sensitivity to chemical inhibitors of cyclin-dependent kinases). Furthermore, cyclin B phosphorylation occurs as an intra-M phase-promoting factor reaction as shown by the following: 1) active Cdc2 is unable to phosphorylate cyclin B associated to phosphorylated Cdc2, and 2) cyclin B phosphorylation is insensitive to enzyme/substrate dilution. We conclude that, at the prophase/metaphase transition, cyclin B is mostly phosphorylated by its own associated Cdc2 subunit.Cell cycle events are regulated by the cyclin-dependent kinases (CDKs) 1 (for reviews, see Refs. 1-5). Cyclins are responsible for kinase activation, substrate specificity, and intracellular localization (6). The key regulator of the G 2 /M transition is the M phase-promoting factor (MPF), a complex constituted of the catalytic subunit p34 cdc2 (7-9) and the regulatory subunit cyclin B cdc13 (6, 10 -11). Activation of MPF at the onset of mitosis is associated with modifications of phosphorylation of its two subunits (11-13). In late prophase, Cdc2 is phosphorylated on three residues: Thr-14, Tyr-15, and Thr-161. Thr-161 phosphorylation is catalyzed by the Cdc2-activating kinase identified as a complex between Cdk7 (MO15), cyclin H, and MAT1 (14). This phosphorylation is necessary for Cdc2 activity (15, 16). The Thr-14 and Tyr-15 residues of Cdc2 are located in the ATP-binding pocket of the kinase (17). Following cyclin B binding to Cdc2 (6,12,18), Cdc2 becomes phosphorylated on Thr-14 by the Myt1 kinase (19,20) and on Tyr-15 by the Wee1/Mik1 or Myt1 kinases (20 -22). In yeast, only Tyr-15 is phosphorylated in G 2 (23). Cdc2 activation at prophase/metaphase transition requires dephosphorylation of both Thr-14 and Tyr-15. These dephosphorylations occur in two successive steps: first Thr-14 and then . The Cdc25 dual-specificity phosphatase dephosphorylates both residues (reviewed in Ref. 25). The Pyp3 tyrosine phosphatase acts on Tyr-15 in fission yeast (26). A positive feedback loop originating from Cdc2 leads to the autocatalytic amplification of the complex (27,28). This is possibly partially generated by the singly phosphorylated form of Cdc2, dephosphorylated on Thr-14/ phosphorylated on Tyr-15 (24).Simultaneously with Cdc2 dephosphorylation, cyclin B becomes phosphorylated as described in a variety of models such as yeast (6), starfish oocytes (13), sea urchin eggs (11), goldfish oocytes (29), Xenopus oocytes (30), and human cells (31). The residues phosphorylated in cyclin B 1 have been identified in Xenopus: Ser-2, Ser-94, 33). Mutational studies (29,32,33) have suggested that cyclin B phosphorylation is required neither for activity of the Cdc2 kinase, for Cdc2 binding, nor for cyclin B d...

Localization and Dynamics of Cdc2-Cyclin B during Meiotic Reinitiation in Starfish Oocytes

“…The location of the initial entry suggests that the first molecules to be phosphorylated are near the centrosomes. In starfish, cyclin B may be phosphorylated by an intermolecular action of Cdc2-cyclin B (Borgne et al, 1999). Our observation of entry of cyclin B-GFP suggests that activation of Cdc2-cyclin B first occurs in the region of the centrosomes.…”

Pre-M Phase-promoting Factor Associates with Annulate Lamellae inXenopusOocytes and Egg Extracts

“…Because our extracts were made at a time corresponding to the beginning of mitosis, most CyclinB is nonphosphorylated and present in the inactive pre-MPF complex, containing inactive phosphorylated Cdc2. CyclinB is intraphosphorylated by activated Cdc2/CyclinB (Borgne et al, 1999) and the appearance of the upper form of CyclinB2 has been shown to strictly correlate with GVBD/metaphase in maturing Xenopus oocytes (Kobayashi et al, 1991). The appearance of the phosphorylated (upper) form can be considered to be a marker of MPF activation.…”

Constitutive activation of the shh–ptc1 pathway by a patched1 mutation identified in BCC

“…In addition, cyclin B1 is regulated by phosphorylation during G2 phase. Specifically, the cytoplasmic retention signal (CRS) domain of cyclin B1 is phosphorylated on four Ser residues, resulting in nuclear localization of MPF and access to critical nuclear substrates for G2-M progression (Li et al, 1995(Li et al, , 1997Borgne et al, 1999;Hagting et al, 1999).…”