Wee1B is down-regulated and Cdc2 remains activated. Indeed, endogenous cyclin B and securin
were not degraded in Wee1B knockdown oocytes (Fig. 3, E and F). Moreover, Emi2—which
negatively regulates APC activity and is degraded
during egg activation (14)—was partially stabilized in Wee1B knockdown oocytes (Fig. 3, E
and F). Consistent with this finding, the activation of APC is impaired when constitutively active
Cdc2 is overexpressed in mitotic cells (13, 15).
Taken together, these data indicate that Wee1B-
mediated inhibition of Cdc2 is required for the
timely activation of the APC, which further promotes MPF inactivation through the degradation of cyclin B.
In the reverse experiments, Wee1B overexpres-
sion in oocytes drove pronuclear formation. How-
ever, cortical granule exocytosis did not occur,
indicating that pronuclear formation proceeds in
the absence of Ca2+ signals (fig. S6).
C WT S15A
arbitrary units (%)
100 200 300 400 500
CaMKII : − + − +
W ee1B M O
W ee1 B- W T
Oocytes with pronuclei (%)
W ee 1 B-S 15 D
(Wee1B-dead) was used (Fig. 4A). This is consistent with Wee1B having autophosphorylation
activity (8) but also indicates that CaMKII phosphorylates Wee1B. To identify the CaMKII phosphorylation site in Wee1B, we incubated several
truncated forms of Wee1B fused to maltose-binding protein (MBP) with CaMKII. Phosphorylation was detected only in the N terminus of
Wee1B (amino acids 1 to 124) (Fig. 4B). Sequence
analysis of Wee1B predicted that the S15 residue
of Wee1B is a potential CaMKII phosphorylation site (17). Indeed, disruption of this residue
(S15A) almost completely abolished Wee1B phosphorylation by CaMKII (Fig. 4C). Moreover,
the tyrosine autophosphorylation of Wee1B was
significantly increased by Ca2+ stimulation with
A23187, and this phosphorylation was blocked
by the S15A mutation (Fig. 4D). Taken together,
these results demonstrate that Wee1B is a direct
substrate of CaMKII in mouse oocytes and that
phosphorylation causes an increase in Wee1B
activity. Next, we tested whether phosphorylation of the S15 of Wee1B by CaMKII mediated
Wee1B activation during egg exit from MII.
Injection of the S15D mutant that mimics phosphorylation status induced pronuclear formation more efficiently than did wild-type Wee1B
(Fig. 4E). On the other hand, pronuclear formation was significantly decreased when the
S15A mutant was injected. Thus, we conclude
that phosphorylation of Wee1B at the S15 residue by CaMKII activates the dormant Wee1B
during egg activation.
Because Wee1B is a CaMKII substrate,
CaMKII-induced MII exit should be blocked
by Wee1B down-regulation. To test this possibility, we injected constitutively active CaMKII,
which is known to induce the completion of
meiosis, into Wee1B knockdown oocytes (18).
CaMKII-driven MII exit was significantly reduced in Wee1B knockdown oocytes but was restored in oocytes treated with roscovitine (Fig.
4F). Comparable inhibition was obtained when
nondegradable cyclin B (D90cyclin B) was overexpressed, suggesting that inhibitory phosphorylation and cyclin B degradation are equally
important. These data, along with the observations that MPF activity remains high and the
degradation of cyclin B does not properly occur when Wee1B is down-regulated, indicate
that Wee1B is activated by CaMKII. This activation is responsible for the initiation of MPF
inactivation, which is further promoted by the
degradation of cyclin B through the APC activation. Thus, we propose that M-phase exit is
tightly regulated not only by the proteolytic
degradation of cyclin B but also by the kinase-dependent inhibition of Cdc2 to ensure rapid
and irreversible exit from meiosis in mouse oocytes (Fig. 4G).
References and Notes
1. K. T. Jones, Reproduction 130, 813 (2005).
2. D. J. Lew, S. Kornbluth, Curr. Opin. Cell Biol. 8, 795