MPF activation

Studies in several laboratories showed that dephosphorylation of tyrosine 15/ threonine 14 in cyclin B/Cdc2 was the final step in activation of MPF, just prior to progesterone-stimulated germinal vesicle (nuclear envelope) breakdown (GVBD) in MI. Changes in the phosphorylation state of a protein reflect changes in the relative balance between the protein kinase(s) and protein phosphatase(s) acting on the protein. In the case of tyrosine-phosphorylated cyclin B/Cdc2, the phosphatase is Cdc25C and the kinase is Myt1. In this laboratory a major effort has been devoted over the last 10 years to understanding how Cdc25C is activated during maturation. The general approach has been to work backwards up the pathway to elucidate how Cdc25C is activated. The mechanism has importance not only for maturation but also for the DNA replication checkpoint in the somatic cell cycle. Izumi et al (1992) and Kumagai & Dunphy (1992) demonstrated that Cdc25C is activated by phosphorylation on Ser/Thr residues. Although cyclin B/Cdc2 can perform this reaction in vitro, forming a positive feedback loop, Cdc25C activation occurs earlier than cyclin B/Cdc2 activation and can be obtained in microcystin-treated extracts in the absence of Cdc2 (Izumi & Maller 1995). The upstream activating kinase was found to be the Xenopus homologue of the polo-like kinase Plx1 (Kumagai & Dunphy 1996), and the kinetics of Plx1 activation fit with a role as an upstream activator of Cdc25C (Qian et al 1998a). Two other experiments strongly support Plx1 as an upstream

Progesterone Secretion by Follicle Cells

Fertilization Releases Metaphase Arrest

M BT

Prophase Extract

Immature Oocyte G2

CSF-arrested Extract

Metaphase Arrest Mediated by CSF

Cycling Extract

Cycling Extract

CSF-arrested Extract

MPF Activity

Meiosis I Meiosis II M M

Fertilization Mitosis I Mitosis I S M S M

FIG. 1. Schematic diagram of early Xenopus development. The figure shows relative levels of MPF (cyclin B/Cdc2) activity from the beginning of oocyte maturation until after fertilization.

activator of MPF. First, constitutively active Plxl is able to induce oocyte maturation and Cdc2 activation in the absence of progesterone (Qian et al 1998b). Second, extracts from resting oocytes that activate Cdc25C upon addition of the heat-stable inhibitor of PKA (PKI, see below) are unable to do so if Plxl has been immunodepleted. Plxl is thus both necessary and sufficient for Cdc25C activation.

Qian et al (1998b) demonstrated that injection of active Cdc25C into resting oocytes leads to Plxl activation, implying the existence of a positive feedback loop between a component downstream of Cdc25C and Plxl itself or a component of the upstream pathway regulating Plxl. Inasmuch as inhibition of Cdc2 by p2lCipl blocks Plxl activation (O. Haccard, personal communication), it has been suggested that Plxl activation is normally downstream of Cdc2 and not upstream. This possibility appears to be excluded by the kinetics of Plxl activation during oocyte maturation and the immunodepletion experiments described above. In addition, very early events in maturation, such as c-Mos synthesis, are also blocked by Cipl injection (Frank-Vaillant et al l999), making it impossible to evaluate the order of maturation events in Cipl-injected oocytes. These considerations, along with evidence that Plxl itself is not directly phosphorylated or activated by Cdc2, have focused attention on upstream components regulating Plxl.

Qian et al (l998a) found that Plxl, like its mammalian counterpart, is activated by phosphorylation. A large-scale purification effort for an activating kinase led to purification of a novel protein kinase termed xPlkkl (Xenopus polo-like kinase kinase) (Qian et al l998a). These results defined the Plxl activation pathway as a polo-like kinase cascade. Dephosphorylation experiments demonstrated that xPlkkl itself is also activated by phosphorylation, suggesting that yet another kinase lies upstream of xPlkkl. More recent data show that xPlkkl can be phosphorylated and activated by itself as well as by Plxl, forming a positive feedback loop between Plxl and xPlkkl (E. Erikson & Y.-W. Qian, unpublished results).

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