Developmental Biology 288 (2005) 139 – 149
www.elsevier.com/locate/ydbio
DYRK2 and GSK-3 phosphorylate and promote the timely degradation of
OMA-1, a key regulator of the oocyte-to-embryo transition in C. elegans
Yuichi Nishi, Rueyling Lin *
Department of Molecular Biology, University of Texas Southwestern Medical Center, 6000 Harry Hines Blvd., Dallas, TX 75390-9148, USA
Received for publication 31 August 2005, revised 27 September 2005, accepted 27 September 2005
Available online 11 November 2005
Abstract
Oocyte maturation and fertilization initiates a dynamic and tightly regulated process in which a non-dividing oocyte is transformed into a
rapidly dividing embryo. We have shown previously that two C. elegans CCCH zinc finger proteins, OMA-1 and OMA-2, have an essential and
redundant function in oocyte maturation. Both OMA-1 and OMA-2 are expressed only in oocytes and 1-cell embryos, and need to be degraded
rapidly after the first mitotic division for embryogenesis to proceed normally. We report here a distinct redundant function for OMA-1 and OMA-2
in the 1-cell embryo. Depletion of both oma-1 and oma-2 in embryos leads to embryonic lethality. We also show that OMA-1 protein is directly
phosphorylated at T239 by the DYRK kinase MBK-2, and that phosphorylation at T239 is required both for OMA-1 function in the 1-cell embryo
and its degradation after the first mitosis. OMA-1 phosphorylated at T239 is only detected within a short developmental window of 1-cell embryos,
beginning soon after the proposed activation of MBK-2. Phosphorylation at T239 facilitates subsequent phosphorylation of OMA-1 by another
kinase, GSK-3, at T339 in vitro. Phosphorylation at both T239 and T339 are essential for correctly-timed OMA-1 degradation in vivo. We propose
that a series of precisely-timed phosphorylation events regulates both the activity and the timing of degradation for OMA proteins, thereby
allowing restricted and distinct functions of OMA-1 and OMA-2 in the maturing oocyte and 1-cell embryo, ensuring a normal oocyte-to-embryo
transition in C. elegans.
D 2005 Elsevier Inc. All rights reserved.
Keywords: OMA-1; C. elegans; DYRK; GSK-3; Protein degradation; Embryos; Phosphorylation
Introduction
In vertebrates, upon stimulation oocytes undergo maturation
and are then arrested in meiotic metaphase II (Masui and
Markert, 1971). Fertilization activates meiosis II-arrested eggs
to reenter the cell cycle and initiate embryonic development
(review by Runft et al., 2002). Egg activation is triggered by a
rapid rise in cytosolic Ca++ concentration, which has been
shown in many systems to be both necessary and sufficient for
the fertilized egg to reenter the cell cycle (reviews by BenYosef and Shalgi, 1998; Carroll, 2001; Runft et al., 2002). The
ability to release Ca++ in response to fertilization changes
during oogenesis, and reaches a maximum late during oocyte
maturation (review by Carroll et al., 1996). These observations
suggest that successful activation at fertilization relies on
changes occurring during oocyte maturation and that a
* Corresponding author.
E-mail address: [email protected] (R. Lin).
0012-1606/$ - see front matter D 2005 Elsevier Inc. All rights reserved.
doi:10.1016/j.ydbio.2005.09.053
precisely regulated transition from oocyte to embryo helps to
ensure proper embryonic development. In addition to a rise in
Ca++ level, an equally important event during this transition is
the turnover or inactivation of oocyte- and egg-specific
proteins (DeRenzo and Seydoux, 2004). However, little is
known about inactivation of oocyte or egg specific proteins or
the molecular mechanisms coordinating these events with the
initiation of embryonic development.
C. elegans reproduces primarily as a self-fertilizing hermaphrodite. In a young adult, oocytes mature every 23 min and
are normally immediately fertilized by sperm stores (see Fig. 1)
(Detwiler et al., 2001; McCarter et al., 1999; Miller et al.,
2001). Fertilization triggers the completion of the two rounds
of meiosis generating the oocyte pronucleus and extrusion of
the two polar bodies (McCarter et al., 1999; McNally and
McNally, 2005; Ward and Carrel, 1979). In C. elegans, there is
no obvious arrest between either oocyte maturation and
fertilization or meiosis I and meiosis II (McCarter et al.,
1999), and therefore a stage equivalent to the egg of other
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Y. Nishi, R. Lin / Developmental Biology 288 (2005) 139 – 149
Fig. 1. Schematic diagram of a wildtype C. elegans hermaphrodite gonad,
oocytes, and the first mitotic cycle. Germ nuclei (white circles) are in a syncitial
cytoplasm of the gonad arm before being enclosed by membranes to form
developing oocytes. The oocyte adjacent to the spermatheca (maturing oocyte)
undergoes maturation, and is then immediately ovulated and fertilized. Small
black ovals: polar bodies (anterior end of embryo).
systems does not exist. However, changes characteristic of egg
activation, including an increase in cytosolic Ca++ levels and
completion of meioses, do occur upon fertilization (McCarter
et al., 1999; McNally and McNally, 2005; Samuel et al., 2001).
Upon completion of the meiotic divisions, the oocyte
pronucleus decondenses, migrates toward the now decondensed sperm pronucleus, and fuses with it. After the two
pronuclei fuse, the zygote then enters metaphase of the first
mitotic division without a preceding interphase (Fig. 1).
Centrosomes contributed by the sperm form the microtubuleorganizing center that cues the embryonic anterior –posterior
polarity (Cowan and Hyman, 2004; Wallenfang and Seydoux,
2000). In this article, the term oocyte-to-embryo transition will
refer to the progression from initiation of oocyte maturation,
through fertilization and activation, to telophase of the first
mitotic division. We will use the term ‘‘1-cell embryo’’ to refer
to embryos from immediately after fertilization to the
completion of the first mitotic division.
Complex regulation of the oocyte-to-embryo transition is
suggested by the dependence of one event upon successful
completion of a previous event. For example, completion of
oocyte maturation is required for fertilization to occur (Detwiler
et al., 2001), which in turn is required for the completion of
meiosis by the oocyte nucleus (McNally and McNally, 2005;
Ward and Carrel, 1979). Likewise, formation of sperm asters, the
cue for embryonic polarity, depends upon successful completion
of oocyte meiosis (Wallenfang and Seydoux, 2000). The fact that
C. elegans fertilization and embryonic development occur
immediately following oocyte maturation also suggests that
molecular events regulating the oocyte-to-embryo transition
must be very precisely timed. While it remains unclear exactly
how these events are coordinated, regulated protein degradation
appears to play a key role (DeRenzo and Seydoux, 2004;
Pellettieri et al., 2003). First, formation of the mitotic spindle is
dependent on the degradation of two meiotic microtubulesevering proteins, MEI-1 and MEI-2, whose functions are
required for the meiotic spindle (Mains et al., 1990; Srayko et al.,
2000). Second, normal embryonic development requires the
rapid degradation of OMA-1 and OMA-2 after the first mitotic
division (Lin, 2003). OMA-1 and OMA-2, two closely related
CCCH zinc finger proteins that function redundantly in oocyte
maturation, are expressed only in oocytes and the 1-cell embryo
and are rapidly degraded soon after the first mitotic division
(Detwiler et al., 2001; Lin, 2003). Their correctly timed
degradation is essential for proper embryonic development, as
an oma-1 gain-of-function mutation (zu405) that leads to OMA1 persistence results in embryonic lethality (Lin, 2003). Third,
the asymmetric segregation of PIE-1, MEX-1 and POS-1, three
other CCCH zinc finger proteins specific to the germ cell
precursors (Guedes and Priess, 1997; Mello et al., 1996; Reese et
al., 2000; Tabara et al., 1999), is, in part, regulated by the
degradation of these proteins in non-germ cell precursors (Reese
et al., 2000).
MEI-1/2, OMA-1/2, PIE-1, and POS-1 have very different
spatiotemporal expression and degradation patterns, and yet
all require the DYRK kinase family member MBK-2 for their
regulated degradation (Pang et al., 2004; Pellettieri et al.,
2003; Quintin et al., 2003). Depletion of mbk-2 either
genetically or by RNAi results in pleiotropic defects in the
early embryo, including defects in the degradation of these
proteins (Pang et al., 2004; Pellettieri et al., 2003; Quintin et
al., 2003). DYRK kinases have been implicated in a number
of important biological processes, including neuronal function
(Fotaki et al., 2002; Raich et al., 2003; Tejedor et al., 1995),
Down syndrome (Altafaj et al., 2001; Smith and Rubin, 1997;
Smith et al., 1997) and cancer (Lee et al., 2000; Miller et al.,
2003). However, our understanding of cellular functions of
DYRK kinases remains limited due to the lack of identified in
vivo substrates.
Since a number of processes in the oocyte-to-embryo
transition depend on mbk-2, it has been suggested as a key
regulator of the transition (Pellettieri et al., 2003). However, how
MBK-2 regulates this transition at a molecular level is unclear
because no in vivo substrate has yet been identified. MBK-2
might regulate a number of diverse cellular processes directly via
phosphorylation of distinct substrates. Alternatively, MBK-2
might phosphorylate a single substrate that then regulates a
number of different downstream processes. The subcellular
localization of GFP::MBK-2 undergoes a dramatic rearrangement in embryos prior to meiosis II, changing from a uniform to
Y. Nishi, R. Lin / Developmental Biology 288 (2005) 139 – 149
a punctate pattern at the cortex (Pellettieri et al., 2003). It has
been proposed that this change in subcellular localization
coincides with MBK-2 activation (Pellettieri et al., 2003).
However, while the degradation of MEI-1 occurs soon after
the proposed activation of MBK-2 (Clark-Maguire and Mains,
1994; Pintard et al., 2003), the degradation of OMA-1 and PIE-1
does not occur until first mitotic anaphase and the 2-cell stage,
respectively (Lin, 2003; Reese et al., 2000). How this temporal
difference in degradation kinetics is achieved is also unclear.
Here, we identify OMA-1 as an in vivo substrate for MBK-2.
We show that OMA-1 degradation requires direct phosphorylation by MBK-2 at T239, and that this phosphorylation is
diminished in the oma-1(zu405) gain-of-function mutant embryo, resulting in OMA-1 persistence. OMA-1 is phosphorylated at T239 only within the short developmental window
following completion of meiosis II until OMA-1 is normally
degraded. We also show that OMA-1 is phosphorylated by
another kinase, GSK-3, at T339 in vitro and that this phosphorylation is also crucial for OMA-1 degradation in vivo. Sequential
phosphorylation is suggested by the observation that phosphorylation of T339 by GSK-3 in vitro is facilitated by amino acid
substitutions that mimic phosphorylated T239. Finally, we show
that OMA-1 and OMA-2 have a redundant function in 1-cell
embryos that is required for embryonic development and that
this function is distinct from their role in oocyte maturation. We
propose a model whereby sequential phosphorylation by MBK2 and GSK-3 converts OMA-1 and OMA-2 from being key
regulators of oocyte maturation to key regulators of 1-cell
embryo development, as well as ensuring their timely degradation after the first mitotic cycle.
141
Cells were sonicated in (20 mM Tris pH 7.5, 200 mM NaCl, 1 mM EDTA, 1
mM DTT, Complete protease inhibitor [Roche]), treated with 0.1 U/Al DNase
and 0.01 Ag/Al RNaseA, and fusion protein purified by amylose resin (NEB).
All substrates were eluted with 10-mM maltose, whereas resin-bound MBK-2
kinases were used directly for kinase assay. 1% TritonX-100 was added to
solubilize MBK-2 during purification.
Sixty hours after transfection, HEK293T cells were lysed (10 mM NaPO4
pH 7.2, 150 mM NaCl, 1% NP40, 1 mM EDTA, Complete protease inhibitor
[Roche], phosphatase inhibitor cocktail I [Calbiochem], 1 mM NaVO4), and
FLAG::GSK-3 was immunoprecipitated using protein G beads (Pierce) and
anti-FLAG M2 antibody, and eluted with 1 mM FLAG peptide (Sigma).
Recombinant Rabbit GSK-3h was purchased from New England Biolabs.
MBK-2 (25 mM HEPES pH 7.6, 5 mM MgCl2, 5 mM MnCl2, 0.5 mM DTT,
30 nM cold ATP and 0.5 ACi [g-32P] ATP) and GSK-3 (20 mM Tris – Cl pH 7.2,
10 mM MgCl2, 5 mM DTT, 30 nM cold ATP and 0.5 ACi [g-32P] ATP) kinase
assays were performed at 25-C for 15 and 10 min, respectively. Products were
separated by SDS-PAGE and incorporation of 32P was visualized by
autoradiography.
Antibody production and immunofluorescence
Anti-T239-P antibody was generated by immunizing rabbits with the peptide
RLPEP4 HPLEMFARPST(PO4)PDEPAAK in which threonine 239 is phosphorylated. The antibodies were precleared with the non-phosphorylated peptide
counterpart before being affinity-purified using the phosphorylated peptide
(Bethyl Laboratory, Inc). In ELISA assays, the affinity-purified anti-T239-P
antibody reacts with phosphorylated RLPEP4 with at least 100-fold higher
affinity compared to the unphosphorylated RLPEP4 or unrelated peptides. This
affinity-purified antibody fails to detect any protein in worm extracts or
recombinant OMA-1 phosphorylated by MBK-2 as assayed by western blots.
Immunofluorescence of embryos using anti-T239-P was performed as
described (Lin et al., 1998) except with mild squashing for 5 min in 4%
paraformaldehyde. Antibody dilutions: anti-T239-P, 1/300, anti-PIE-1, 1/10, antiMEX-5 (Schubert et al., 2000), 1/2, Alexa488 goat-anti-rabbit IgG, 1/250,
Alexa568 goat-anti-mouse IgG, 1/250.
Analysis of embryos and imaging
Materials and methods
Strains
N2 was used as the wildtype strain. Genetic markers are: LGII, rrf3(pk1426); LGIII, unc-119(ed3), ruIs32[unc-119(+) P pie-1 ::GFP::H2B]; LGIV,
oma-1(te21), oma-1(te33), oma-1(zu405), mbk-2(pk1427); LGV, oma-2(te50),
oma-2(te51), nT1(IV;V). Transgenic strains were generated by microparticle
bombardment (Praitis et al., 2001). teIs20 (P oma-1 DNoma-1::gfp); teIs21
(P oma-1 DNoma-1 T239A ::gfp); teIs23 (P oma-1 DNoma-1 T239A S302A ::gfp); teIs22
(P oma-1 DNoma-1 S302A ::gfp); teIs61 (P oma-1 DNoma-1 T339A ::gfp). The expression patterns of each transgene were verified in at least three independent lines.
Plasmid construction
All expression clones were generated using the Gateway technology
(Invitrogen). The P oma-1 gfp destination vector pRL781 contains 2.1 kb
upstream, and 1 kb downstream of OMA-1 coding sequence. Desired oma-1
cDNA fragments were recombined into pRL781, generating OMA-1 protein
with GFP at the C-terminus. pMALp2X- and pcDNA3.1-derived vectors were
used for N-terminally tagged MBP and FLAG, respectively, fusion proteins. The
gsk-3 RNAi feeding vector was derived from pDONRdT7 (Reddien et al., 2005).
All site-directed mutagenesis was performed using the Quick Change kit
(Stratagene). The kinase dead mutations used were Y237A for MBK-2 and Y196F
for GSK-3.
In vitro kinase assays
All substrates and kinases except GSK-3 used in this study were MBPtagged. MBP fusions were produced in Rosetta (DE3) pLysS (Novagen) cells.
Imaging of immunofluorescence and live embryos was performed as
described previously (Rogers et al., 2002). The filter wheels (Ludl Electronic
Product) and shutter controller are driven by a custom software package (os4d
1.0, freely available upon request to [email protected]).
RNAi screen for genes required for OMA-1 degradation
Feeding RNAi for oma-2 and gsk-3 were performed at 25-C by placing L2
worms onto RNAi bacteria and scoring 24 – 30 h later (Detwiler et al., 2001;
Timmons and Fire, 1998).
RNAi clones from the Ahringer RNAi feeding library that gave rise to
either sterile or embryonic lethal progeny (Fraser et al., 2000; Kamath et al.,
2003; Simmer et al., 2003), or that were enriched in the germline (Reinke et al.,
2000) were selected to conduct the screen using TX492 [teIs1 ( P oma-1 oma1::gfp); rrf-3(pk1426)] (Simmer et al., 2002; Lin, 2003). TX492 L1 were
placed on RNAi plates for 3 days at 20-C before scoring. Any clone resulted in
an OMA-1::GFP pattern deviated from the wild-type pattern was retested.
Results
MBK-2 phosphorylates OMA-1 in vitro, primarily at T239
To investigate whether MBK-2 has a direct role in OMA-1
degradation, we began by determining whether MBK-2 can
phosphorylate OMA-1 in vitro. It has been shown, in vitro, that
mammalian DYRK1 and DYRK2 prefer to phosphorylate a
serine or threonine with the following consensus sequence:
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Y. Nishi, R. Lin / Developmental Biology 288 (2005) 139 – 149
RXXS/TP (Campbell and Proud, 2002; Himpel et al., 2000).
The arginine at the 3 position is essential, whereas the proline
at the +1 position is highly favored (Campbell and Proud,
2002; Himpel et al., 2000). Replacing the P at the +1 position
with the hydrophobic amino acid valine still permits phosphorylation by DYRK2 in vitro, although the K m is increased
more than 10-fold (Campbell and Proud, 2002). Examination
of the OMA-1 and OMA-2 sequences revealed two such
DYRK consensus sites in each protein (T239 and S302 for
OMA-1; T227 and S291 for OMA-2, Fig. 2A).
We tested whether MBK-2 can directly phosphorylate
OMA-1 or OMA-2 in vitro and, if so, whether it does so via
these putative substrate sites. Using bacterially expressed
kinase and substrates, we found that wild-type, but not
kinase-dead, MBK-2 phosphorylates both OMA-1 and OMA-
2 in vitro (Fig. 2B). In a parallel reaction, MBK-2 did not
phosphorylate C. elegans cyclin B (Fig. 2B), a protein that, like
OMA-1, is rapidly degraded in the 1-cell embryo but whose
degradation is known to be independent of MBK-2 (Pellettieri
et al., 2003).
To investigate whether MBK-2 phosphorylates OMA-1 at
specific sites, we performed kinase assays using OMA-1
proteins in which T239, S302, or both, had been mutated to
alanine (Fig. 2B). While changing S302 to alanine resulted in
only a minor decrease in the overall phosphorylation of
OMA-1, changing T239 to alanine abolished most of the
phosphorylation signal. OMA-1 with both T239A and S302A
substitutions had a level of phosphorylation very close to
background. This result suggests that, in vitro, MBK-2
phosphorylates OMA-1 primarily at T239 but can also
phosphorylate S302. In addition, it appears that these two
sites account for the vast bulk of OMA-1 phosphorylation by
MBK-2.
The oma-1 gain-of-function mutation, zu405, which results
in prolonged persistence of OMA-1 protein past the 1-cell
stage, changes the proline at the +1 position of the T239
consensus site to a leucine (Lin, 2003). As the change of
proline at the +1 position to valine was shown to increase the
K m dramatically for the DYRK2 kinase (Campbell and Proud,
2002), the zu405 mutation would be predicted to also affect
phosphorylation of OMA-1 by the MBK-2 kinase. We directly
tested this prediction using the in vitro kinase assay (Fig. 2C).
OMA-1 with P240L and P240L S302A changes resulted in
reductions in OMA-1 phosphorylation similar to those observed with T239A and T239A S302A changes, respectively. This
result suggests that P240 is important for phosphorylation of
OMA-1 at T239 by MBK-2 in vitro and that MBK-2 might
therefore have a direct role in vivo in regulating OMA-1
degradation.
Phosphorylation of OMA-1 at T239 is detected in embryos after
MBK-2 activation and before OMA-1 degradation
Fig. 2. MBK-2 phosphorylates OMA-1 and OMA-2 in vitro. (A) Sequence
alignment of OMA-1, OMA-2 and a known DYRK2 substrate, human eIF2B.
Critical residues for DYRK2 phosphorylation are shown in blue and red. *: the
residue phosphorylated by DYRK2 in eIF2B and corresponding residues in
OMA-1 and OMA-2. (B and C) Kinase assays conducted using indicated
substrates and MBK-2 WT (wildtype) or KD (kinase dead mutant). Upper
panels: 32P incorporation visualized by autoradiography. Lower panel:
Coomassie stained gel. The positions of MBK-2, OMA-1, OMA-2, and
CYB-1 (C. elegans cyclin B) are indicated by >, *, @, and #, respectively.
To determine whether OMA-1 phosphorylation at T239
occurs in vivo, we raised antibodies that specifically recognize
this epitope (anti-T239-P). We performed immunofluorescence
with the anti-T239-P antibody to investigate the in vivo
spatiotemporal distribution of T239-phosphorylated OMA-1.
While OMA-1 (and OMA-2) is expressed at a high level in
oocytes, no signal was detected in oocytes or any part of the
gonad with the anti-T239-P antibody (data not shown). We did,
however, detect a high cytoplasmic signal in a specific subset
of 1-cell embryos (Fig. 3A), with no signal detected in embryos
past the 1-cell stage. Precise staging of the embryos showed
that the staining was detected only after meiosis II and before
anaphase of the first mitotic division (0/9 at meiosis I, 0/4 at
meiosis II, 25/25 after meiosis II to first mitotic metaphase, 0/2
at first mitotic anaphase, 0/11 at 2-cell stage). This time
window corresponds to the period soon after the proposed
activation of MBK-2 (Pellettieri et al., 2003) until endogenous
OMA-1 degradation (Lin, 2003). This observation is the first
molecular evidence correlating MBK-2 redistribution with its
Y. Nishi, R. Lin / Developmental Biology 288 (2005) 139 – 149
143
all strongly suggest that MBK-2 phosphorylates OMA-1 at
T239 in vivo, thereby promoting OMA-1 degradation.
MBK-2-dependent phosphorylation of OMA-1 is not detected
in oma-1(zu405) embryos
Fig. 3. OMA-1 is phosphorylated at T239 in vivo. Embryos co-stained with antiT239-P (left column), anti-PIE-1 (middle column) and DAPI (right column). (A)
Wildtype embryos at various developmental stages from meiosis I to the first
mitotic anaphase. (B) mbk-2 (pk1427) and oma-1(zu405); oma-2 (RNAi)
mutant embryos at the pronuclear migration stage. Scale bar: 10 Am.
activation. These data also support the proposal that phosphorylation of OMA-1 at T239 is carried out by MBK-2 in vivo.
Phosphorylation of OMA-1 at T239 depends on MBK-2
The onset of T239 phosphorylation in 1-cell embryos follows
the proposed activation of MBK-2 (Pellettieri et al., 2003). We
then asked whether the phosphorylation of OMA-1 at T239 in
vivo is dependent on the MBK-2 kinase by staining embryos
derived from homozygous mbk-2(pk1427) worms (Raich et al.,
2003). One hundred percent of these embryos have early
cleavage defects, and appear as 1-cell or multicellular embryos
due to a defect in cytokinesis (Pellettieri et al., 2003). These
embryos exhibit high OMA-1 protein levels, as shown
previously (Pellettieri et al., 2003) and confirmed here (data
not shown). Strikingly, none of the mbk-2(pk1427) embryos
examined (n > 500) exhibited detectable anti-T239-P staining
(Fig. 3B). Meanwhile, every one of these mbk-2(pk1427)
embryos was correctly co-stained with the control PIE-1
antibody (Fig. 3B) (Pellettieri et al., 2003). Taken together,
the in vitro phosphorylation of OMA-1 by MBK-2, the close
timing of proposed MBK-2 activation and T239 phosphorylation, and the dependence of phosphorylation at T239 on MBK-2
The oma-1(zu405) P240L mutation diminished the in vitro
phosphorylation at T239 by MBK-2 (Fig. 2C). To further
investigate a direct role in vivo for MBK-2 in phosphorylation
of OMA-1 at T239, we examined T239 phosphorylation in oma1(zu405) embryos by immunofluorescence. Interpretation of
antibody-staining results is complicated by the fact that
sequences surrounding T227 in OMA-2 and T239 in OMA-1
are nearly identical (Fig. 2A), making it likely that anti-T239-P
recognizes phosphorylated epitopes in both OMA-1 and OMA2. Consistent with anti-T239-P antibody recognizing both
OMA-1 and OMA-2, the 1-cell staining was still detected in
either oma-1(te33) or oma-2(te51) null mutant embryos (data
not shown). In an attempt to eliminate antibody staining
deriving from OMA-2, we performed antibody staining in
oma-1(zu405) embryos depleted of oma-2 by RNAi. We have
shown previously that oma-2(RNAi) depletes OMA-2 protein
while leaving OMA-1 levels unaffected (Detwiler et al., 2001).
In 1-cell oma-1(zu405);oma-2(RNAi) embryos (n > 70), no
anti-T239-P staining was detected (Fig. 3B). This result
demonstrates the specificity of this anti-T239-P antibody for
OMA-1 and OMA-2 proteins in the 1-cell embryo. Although
we are unable to completely exclude the possibility that the Pto-L change interferes with the epitope recognition of T239-P by
the anti-T239-P antibody, the absence of detectable antibody
staining, along with the in vitro phosphorylation result,
strongly suggests that the zu405 mutation diminishes phosphorylation at T239.
GSK-3 kinase is also required for OMA-1 degradation in vivo
In the course of an RNAi screen using an OMA-1::GFP
strain as a reporter aimed at identifying other genes regulating
OMA-1 degradation, we discovered that the C. elegans gsk-3
homolog is required for OMA-1 degradation. Depletion of gsk3 by RNAi results in a severe delay in the degradation of
OMA-1 protein, similar to that observed in mbk-2(RNAi)
embryos (Fig. 4A). However, unlike mbk-2(RNAi) or mbk2(pk1427) embryos, gsk-3(RNAi) embryos do not have gross
defects in early embryonic divisions (Schlesinger et al., 1999).
GSK-3 phosphorylation of OMA-1 in vitro is affected by the
phosphorylation state at T239
To investigate whether GSK-3, like MBK-2, plays a direct
role in OMA-1/OMA-2 degradation, we first asked whether
GSK-3 can phosphorylate OMA-1 or OMA-2 in vitro.
Phosphorylation of many proteins by GSK-3 first requires that
a serine or threonine four residues C-terminal be phosphorylated by a priming kinase (Biondi and Nebreda, 2003; Fiol et
al., 1987). GSK-3 dependent phosphorylation of an initial S/T
target can then prime additional GSK-3 dependent phosphor-
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Y. Nishi, R. Lin / Developmental Biology 288 (2005) 139 – 149
Fig. 4. GSK-3 phosphorylates OMA-1 primarily at T339 and is required for
OMA-1 degradation. (A) gsk-3 depletion results in stabilization of OMA1::GFP. Embryos in the uterus arrange in a developmental progression from left
to right. OMA-1 is mostly degraded by the 2-cell stage (arrows) in non-RNAi
embryos but is still detected in gsk-3(RNAi) embryos up to the several hundredcell stage. (B) Sequence alignment of OMA-1 and OMA-2 surrounding T339 in
OMA-1 and T327 in OMA-2 (asterisk). All putative GSK-3 substrate sites are in
red and the aspartate at +4 position is blue. (C) Kinase assays using the
indicated substrates and GSK-3 WT (wildtype) or KD (kinase dead mutant).
Reactions were separated and 32P incorporation was visualized as described in
Fig. 2. (D) 1-cell gsk-3(RNAi) embryo co-stained with anti-T239-P (left
column), anti-MEX-5 (middle column), and DAPI (right column). Scale bar:
20 Am.
ylation of S/T located 4 residues N-terminal. Although
preferred substrate sequences for C. elegans GSK-3 have not
been characterized, they are likely to be similar to those found
for vertebrate GSK-3 kinases, given the high sequence
similarity between these proteins (Schlesinger et al., 1999).
Examination of OMA-1 and OMA-2 sequences identifies
several potential GSK-3 phosphorylation sites (Fig. 4B):
S331, S335, and T339 for OMA-1, and S319, S323, and T327 for
OMA-2. Sequences surrounding these sites are nearly identical
between OMA-1 and OMA-2, and the residues at the +4
position relative to T339 in OMA-1 and T327 in OMA-2 are
aspartate (D), an amino acid known to be able to mimic
phosphorylated serine or threonine residues. D343 and D331
could therefore prime GSK-3 phosphorylation of T339 in
OMA-1 and T327 in OMA-2, respectively.
Using recombinant GSK-3 kinase, both OMA-1 and OMA2 can be phosphorylated in vitro (Fig. 4C and data not shown).
OMA-1 can be phosphorylated with similar specificity with
either bacterially-expressed rabbit GSK-3 or worm GSK-3
isolated from transfected HEK293T cells (data not shown).
Mutating the most C-terminal candidate GSK-3 phosphorylation sites (T339 of OMA-1 and T327 of OMA-2) to alanines
reduced but did not abolish phosphorylation by GSK-3 in vitro
(Fig. 4C). This was not due to phosphorylation of nearby sites
because OMA-1 with seven additional mutations that changed
all threonine and serine residues surrounding T339 (from S331 to
S347) to alanines was still phosphorylated to a similar extent as
OMA-1 T339A (data not shown). However, mutating T239, in
addition to T339, to alanine abolished most, if not all,
phosphorylation by GSK-3 in our assay (Fig. 4C). In fact,
mutation of T 239 to alanine alone diminished OMA-1
phosphorylation by GSK-3 to a near background level.
Conversely, when T239 was mutated to aspartate or glutamate,
phosphorylation of OMA-1 by GSK-3 was reduced only
slightly (Fig. 4C). We conclude from the kinase assay that
phosphorylation at T239 appears to facilitate in vitro phosphorylation at T339 by GSK-3. This is contrary to the in vitro
phosphorylation of T239 by MBK-2, which is unaffected by
whether position 339 is a threonine, alanine, or aspartate (data
not shown). It is interesting to note that, although only T339,
and not T239, is a predicted GSK-3 substrate site, it would
appear that both sites can be phosphorylated by GSK-3 in vitro.
We did not, however, detect a significant difference in antiT239-P signal between gsk-3(RNAi) and non-RNAi embryos
(Fig. 4D), suggesting that GSK-3 does not phosphorylate T239
in vivo in a significant way. This result, plus the observation
that anti-T239-P staining is abolished in mbk-2 embryos,
demonstrates that T239 is phosphorylated in vivo primarily, if
not exclusively, by MBK-2. Taken together, our results suggest
that, in vivo, phosphorylation at T239 by MBK-2 likely
facilitates phosphorylation at T339 by GSK-3.
Phosphorylation of OMA-1 at both T239 and T339 is essential
for OMA-1 degradation in vivo
To evaluate functional importance of T239, S302 and T339
phosphorylation in OMA-1 degradation, we examined the
stability of OMA-1 protein in which phosphorylation at one or
more of these three sites is prevented. Failure to degrade OMA1 protein (as in oma-1(zu405) embryos) results in embryonic
lethality (Lin, 2003), precluding the use of a functional protein
for such analysis. Thus, all analyses were performed using a
truncated OMA-1 protein in which the sequence N-terminal to
the first zinc finger is deleted (OMA-1DN). Expression of
OMA-1DN::GFP in wildtype worms did not cause any lethality
or abnormal phenotype (data not shown). In addition, OMA1DN::GFP was degraded following the first mitotic division,
albeit at a slightly slower rate compared to full-length OMA-1
(Fig. 5). Importantly, OMA-1DN::GFP proteins with either
T239 or T339 changed to alanine demonstrated a very severe
defect in OMA-1 degradation (Fig. 5), with OMA-1DN::GFP
persisting in embryos as late as the 100-cell stage (data not
shown). GFP was detected throughout the entire embryo with a
higher abundance in germline precursors (Fig. 5). Consistent
with S302 being a secondary site for MBK-2 in vitro, the S302A
change resulted in only a minor defect in OMA-1 degradation
Y. Nishi, R. Lin / Developmental Biology 288 (2005) 139 – 149
Fig. 5. Phosphorylation at T239 and T339 is required for OMA-1 degradation
in vivo. All fusion proteins have the first 110 amino acids of OMA-1 deleted.
OMA-1 S302A, OMA-1 T239A, OMA-1 T239A S302A, and OMA-1 T339A
express OMA-1::GFP with the indicated amino acid changes. The arrangement of embryos and arrows are as described in Fig. 4A. Arrowheads point to
12 – 16-cell embryos. Scale bar: 30 Am.
145
derived from the anterior versus posterior portions of the
embryo. Cells in most of these mutant embryos are found in
two distinct clumps: an anterior clump which appears to be
derived from the anterior cell, AB, of the two-cell stage
embryo, and a posterior clump derived from the two-cell stage
posterior cell, P1 (Fig. 6). In most cases, the anterior clump
consists of several hundred cells, whereas the posterior clump
remains as a large single cell with multiple disorganized nuclei.
Second, depletion of oma-2 by RNAi in the oma-1(zu405)
background at a permissive temperature results in 100% dead
embryos (n > 500). This is in contrast to the 50% lethality at
the same temperature for embryos produced by oma-1(zu405)
animals (n > 500). The oma-1(zu405);oma-2(RNAi) animals do
not appear to have any defect in oocyte maturation (data not
shown). The phenotype of arrested embryos resembles but is
not identical to that of zu405 embryos (Lin, 2003). While
excess pharynx is evident in almost all oma-1(zu405);oma2(RNAi) embryos, no excess intestinal cells were observed
(data not shown). Further characterizations of this phenotype
will be reported elsewhere. Because OMA-2 is only expressed
in oocytes and the 1-cell embryo, the enhanced lethality of
oma-1(zu405) embryos at a permissive temperature would
appear to be a result of OMA-2 depletion in the 1-cell embryos.
However, the requirement for OMA-2 in the 1-cell stage is only
and did not exacerbate the OMA-1T239A defect in vivo (Fig.
5). These results are consistent with phosphorylation of both
T239, by MBK-2, and T339, by GSK-3, being essential for
correct timing of OMA-1 degradation in vivo.
OMA-1 and OMA-2 have a redundant role in embryogenesis
Cyclin B and MEI-1/2 are degraded soon after their
functions in oocyte maturation and meiotic division, respectively (Clark-Maguire and Mains, 1994; Liu et al., 2004;
Sonneville and Gonczy, 2004) In contrast, OMA-1 and OMA2 persist until anaphase of the first mitotic division,
approximately 1 h after completion of oocyte maturation
(Lin, 2003). This marked delay led us to investigate whether
OMA-1 and OMA-2 have any function in one-cell embryos.
Animals homozygous for loss-of-function mutations in both
oma-1 and oma-2 genes are sterile (Detwiler et al., 2001), and
therefore we could not directly address the potential function of
OMA-1 or OMA-2 in the 1-cell embryo. However, two
observations offer strong support for the notion that OMA-1
and OMA-2 have functions in the 1-cell embryo required for
proper embryogenesis and that, as in oocyte maturation, these
functions are redundant. First, worms homozygous for reduction-of-function oma-1(te21) and oma-2(te50) alleles (Detwiler
et al., 2001) are not fully penetrant for the oocyte maturation
defect and can produce embryos. However, they produce only
dead embryos that have complex phenotypes, including fragile
eggshells and severe cleavage defects (100%, n > 500). Using
an H2B::GFP reporter, we observed polar bodies in most of
these embryos, indicating that these embryos have been
fertilized and have completed meiosis (Fig. 6). Although the
phenotypes of these dead embryos vary, the majority presented
a dramatic difference in cell division defects between cells
Fig. 6. oma-1(te21);oma-2(te50) embryos have severe defects in early
cleavage. DIC (left column) or GFP::H2B fluorescence (right column)
micrographs of embryos derived from wildtype or oma-1(te21);oma-2(te50).
Genotype for each embryo is shown to the left. AB and P1 blastomeres in the 2cell stage wildtype embryo are indicated. Arrows point to polar bodies that
mark the anterior end of the C. elegans embryo. Scale bar: 30 Am.
146
Y. Nishi, R. Lin / Developmental Biology 288 (2005) 139 – 149
revealed in the oma-1(zu405) genetic background, as no
abnormal phenotype was observed in oma-1(+) embryos
depleted of oma-2 either genetically or by RNAi (Detwiler et
al., 2001). Our interpretation from the above two observations
is: (1) OMA-1 and OMA-2 have a redundant function in the 1cell embryo distinct from their redundant function in oocyte
maturation; and (2) while the mutant OMA-1 P240L protein can
substitute for the wild-type OMA-1 protein in maturing
oocytes, it cannot substitute for the wildtype OMA-1 function
in 1-cell embryos.
Discussion
We report here that the transition from oocyte to embryo in
C. elegans requires the functions of OMA-1 and OMA-2 in
both the maturing oocyte and the 1-cell embryo. For embryonic
development to proceed normally, the OMA-1 protein must be
rapidly degraded immediately after the first mitotic metaphase
(Lin, 2003). We show that this precisely-timed degradation
requires combined, and likely sequential, phosphorylation by
MBK-2 and GSK-3 at two distinct sites on the OMA-1 protein,
with GSK-3 phosphorylation facilitated by prior MBK-2
phosphorylation. Although no gain-of-function oma-2 mutation is available, we believe that OMA-2 degradation is subject
to a similar regulation and is essential for embryogenesis based
on the in vivo expression pattern of OMA-2, in vitro and in
vivo phosphorylation by MBK-2, and the high degree of
sequence similarity between OMA-1 and OMA-2.
maturation to a regulator for events in the 1-cell embryo, in
addition to facilitating phosphorylation by GSK-3. The
phenotype of oma-1(zu405);oma-2(RNAi) embryos argues that
the zu405 mutation does not simply result in an excess of wildtype OMA-1 protein (Lin, 2003). Rather, it is consistent with
zu405 being a neomorphic allele that has a modified function
different from the function of the wild-type allele.
Possible function for OMA proteins in embryos
The phenotype of oma-1(te21);oma-2(te50) embryos suggests that OMA protein function is required primarily for the
proper development of the P1 cell. Many key regulators
important for proper embryonic development in C. elegans are
differentially localized between AB and P1 at the first
embryonic division (Kemphues and Strome, 1997; Schnabel
and Priess, 1997). It is possible that the function of OMA-1 and
OMA-2 in the 1-cell embryo leads to the restriction of a key
developmental regulator to either P1 or AB. Depletion of OMA
proteins could result in the absence of a P1 factor or the ectopic
activity of an AB factor, respectively, in P1, and therefore, the
P1 lineage defects. Although many P1- or AB-restricted factors
have been characterized to date, depletion of any individually,
or many in combination, did not result in a phenotype
resembling that shown here for oma-1(te21);oma-2(te50),
(Bowerman et al., 1992; Guedes and Priess, 1997; Kawasaki
et al., 1998; Kuznicki et al., 2000; Mello et al., 1992; Tabara et
al., 1999). The molecular target(s) of OMA-1 and OMA-2 in
the 1-cell embryo is currently being investigated.
Redundant function in the 1-cell embryo?
Dual regulation of OMA-1 by MBK-2 and GSK-3
Our data strongly suggest that OMA-1 and OMA-2 have a
redundant function in 1-cell embryos, and that their function in 1cell embryos is distinct from their redundant function in oocyte
maturation. Our result that oma-1(zu405);oma-2(RNAi) embryos
produce 100% dead embryos with no apparent defects in oocyte
maturation further suggests that OMA-1 P240L can substitute for
wild-type OMA-1, but only in oocytes and not in embryos. We
could not rule out the possibility that the embryonic defects
observed in oma-1(te21);oma-2(te50) and oma-1(zu405);oma2(RNAi) embryos were due to the reduced function of OMA-1
and OMA-2 in the oocyte, leading to imperfect maturation.
However, we find it difficult to reconcile imperfect oocyte
maturation with such different phenotypes in the two genetic
backgrounds, as well as such different defects in the AB- versus
P1-derived lineages in oma-1(te21);oma-2(te50) embryos.
The differential ability of OMA-1P240L to substitute for
wild-type OMA-1 protein in oocyte maturation versus 1-cell
embryos is very interesting. Because the P240L mutation
prevents phosphorylation at T239 by MBK-2 in vitro, and
likely in vivo, this suggests that phosphorylation at T239 is
important for OMA-1 function in the 1-cell stage embryo.
Consistent with our result that T239 is not phosphorylated in
oocytes, the P240L mutation does not inhibit OMA-1 function
in oocytes. An intriguing, albeit highly speculative, possibility
is that phosphorylation of OMA-1 at T239 by MBK-2 is
involved in switching OMA-1 from a regulator for oocyte
Although phosphorylation of OMA-1 at both T239 and T339
is essential for its correct temporal degradation in vivo,
phosphorylation at each site might serve different purposes.
We propose that a series of co-dependent events, including
phosphorylation of OMA-1 and OMA-2, regulates their
differential activities in both oocytes and 1-cell embryos, as
well as the timing of their degradation (Fig. 7). We propose that
MBK-2 phosphorylation at T239 serves three purposes: (1) it
limits degradation of OMA proteins to only after completion of
oocyte maturation. MBK-2 does not appear to be activated
until just before meiosis II, which is supported by our results
here. Therefore, OMA-1 phosphorylation at T239 does not
occur until after the OMA proteins have completed their
functions in oocyte maturation. (2) As mentioned above, we
propose that phosphorylation at T239 switches OMA-1 from a
regulator of oocyte maturation to a regulator of events in the 1cell embryo. (3) It permits the subsequent phosphorylation of
T339 by GSK-3 and eventual OMA degradation. While
phosphorylation of OMA-1 at T239 occurs soon after meiosis
II, OMA-1 is not degraded until approximately 40 min later, in
contrast to the rapid degradation of another candidate substrate
for MBK-2, MEI-1 (Clark-Maguire and Mains, 1994; Pintard
et al., 2003). We propose that additional levels of regulation,
including phosphorylation by GSK-3, delay the degradation of
OMA proteins such that they can complete their functions in
Y. Nishi, R. Lin / Developmental Biology 288 (2005) 139 – 149
147
Similarly, the mechanism by which phosphorylation at T239
and T339 promotes OMA-1 degradation is not known.
Degradation of OMA-1 is proteasome dependent (Y.N. and
R. L., unpublished results). It is possible that phosphorylation
at T339 (and/or T239) creates a recognition motif for an E3
ubiquitin ligase to ubiquitinate OMA-1. Alternatively, phosphorylation at T339 might facilitate additional phosphorylation
events that affect OMA-1 degradation.
It is interesting to note that the phenotypes of embryos
carrying the oma-1(zu405) mutation, in which OMA-1 degradation is drastically delayed, are very similar to that of embryos
depleted of gsk-3 by RNAi (Lin, 2003; Maduro et al., 2001;
Schlesinger et al., 1999). Both strains have a non-penetrant
defect in the Wnt signaling pathway that specifies the endoderm
precursor, a fate transformation in the C blastomere allowing it to
produce endoderm, and defects in the degradation of other
maternal factors (Lin, 2003; Maduro et al., 2001; Schlesinger et
al., 1999; Y.N. and R. L., unpublished results). One intriguing
possibility is that the primary role of GSK-3 in early C. elegans
development is to degrade OMA-1 (and likely OMA-2) protein.
This possibility is currently under investigation.
DYRK kinase and protein degradation
Fig. 7. Model for regulation of OMA-1 degradation by MBK-2 in early
embryos. A schematic diagram as shown in Fig. 1. OMA-1 expression (green)
is highest from oocyte maturation to the first mitotic division, following
which it is rapidly degraded. Yellow hatched lines denote T239-phosphorylated OMA-1. The big red arrow indicates the proposed time for MBK-2
activation.
the 1-cell embryo. In addition, it is possible that GSK-3
phosphorylation is also required for the OMA proteins to carry
out their 1-cell embryo specific function.
In vertebrates, GSK-3 is inactivated by an inhibitory
phosphorylation at its N-terminus (Ser21 in GSK3a and Ser9
in GSK3h) that binds to and inhibits the kinase domain by
serving as a pseudosubstrate (Cross et al., 1995; Frame et al.,
2001; Sutherland et al., 1993). The sequence surrounding these
inhibitory phosphorylation sites is not conserved in C. elegans
GSK-3, and therefore it is not clear whether its activity is
subjected to a similar regulation in vivo. It is possible that in C.
elegans, GSK-3 is inactive in the 1-cell embryo until immediately before OMA-1 degradation. Alternatively, GSK-3 might
be constitutively active in the C. elegans embryo, but the
sequential phosphorylation by MBK-2 and GSK-3 is enough to
delay the degradation of OMA-1 until the end of the first mitosis.
GSK-3 and protein degradation
Although we did not demonstrate that GSK-3 phosphorylates OMA-1 in vivo, our results strongly suggest that GSK-3
phosphorylates and promotes OMA-1 degradation in vivo.
Phosphorylation by GSK-3 has been linked to the degradation
of key regulators in both the Wnt and hedgehog signaling
pathways, h-catenin (Aberle et al., 1997) and Ci (Jia et al.,
2002; Price and Kalderon, 2002), respectively. However, the
precise mechanism by which phosphorylation of h-catenin and
Ci triggers their degradation remains to be elucidated.
Depletion of mbk-2 affects the degradation of many maternal
proteins (Pang et al., 2004; Pellettieri et al., 2003; Quintin et al.,
2003). Within this group, degradation of PIE-1 and POS-1 is also
defective in the oma-1(zu405) mutant (Lin, 2003). Because there
are no obvious potential substrate sites for DYRK2 kinase
(Campbell and Proud, 2002) in PIE-1 or POS-1, the observed
degradation defect for these two proteins could be an indirect
effect of OMA-1 persistence. However, the degradation of MEI1 occurs prior to the degradation of OMA-1 in wildtype embryos
(Clark-Maguire and Mains, 1994; Lin, 2003) and oma-1(zu405)
embryos do not resemble the mei-1 gain-of-function phenotype.
It is therefore unlikely that MBK-2 regulates MEI-1 degradation
indirectly through OMA-1. The pleiotropic defects associated
with the mbk-2 mutant and the complex subcellular localization
of MBK-2 protein suggest that MBK-2 might directly regulate
multiple proteins. It remains unclear why MBK-2 undergoes a
dramatic change in subcellular location at the time of activation
and how the cortically localized MBK-2 then phosphorylates
substrates at diverse subcellular locations.
Human DYRK1B has been shown to phosphorylate and
destabilize cyclin D1 in vivo (Zou et al., 2004). As a nondegradable form of cyclin D1 is capable of transforming murine
fibroblasts and promoting tumor growth in immune compromised mice, regulated degradation of cyclinD1 by DYRK1B
phosphorylation is important for proper control of cell divisions
(Alt et al., 2000). We extend this observation by demonstrating
that MBK-2 phosphorylation regulates the stability, and likely
activity, of key players in the oocyte-to-embryo transition.
Furthermore, we show that the degradation of OMA proteins
requires a combined regulation by MBK-2 and GSK-3.
Phosphorylation at T239 might induce a conformational change
in OMA-1 that makes T339 accessible for GSK-3. In vitro,
DYRK kinases have been shown to phosphorylate specific
148
Y. Nishi, R. Lin / Developmental Biology 288 (2005) 139 – 149
residues of several cytoplasmic proteins, priming for GSK-3
kinase-dependent phosphorylation (Chen-Hwang et al., 2002;
Woods et al., 2001). It is an intriguing possibility that MBK-2
regulates the activity and/or degradation of multiple proteins by
facilitating phosphorylation by GSK-3 or additional kinases.
Acknowledgments
The authors would like to thank Scott Robertson for his
comments on the manuscript, Scott Cameron and Leon Avery
for useful discussions, Geraldine Seydoux for the mbk-2 cDNA
and strain, Jim Priess for antibodies, Scott Cameron for a
vector, Edward Kipreos for cyb-1 cDNA, Peter Reddien for
pDONRdT7, Jim Waddle for his custom software, os4d, and
Manisha Patel for technical support. This work is supported by
an NIH grant (HD37933) to R. L.
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