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First published online 16 January 2008
doi: 10.1242/dev.013425
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Department of Molecular Biology, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX 75390, USA.
Author for correspondence (e-mail:
Rueyling.Lin{at}UTSouthwestern.edu)
Accepted 19 November 2007
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Caenorhabditis elegans, MEX-5, Embryo, Polarity, Polo kinase
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Many of these
PAR proteins are asymmetrically localized at the cortex along the AP axis. The
asymmetric distribution of PAR proteins determines the position of the first
mitotic spindle and the asymmetric localization of key cytoplasmic
determinants (for reviews, see Cowan and
Hyman, 2004; Lyczak et al.,
2002). This results in two daughters that are different in both
size and developmental fate: the larger anterior somatic cell, AB, and the
smaller posterior germline blastomere, P1. P1 then undergoes a series of three
more asymmetric divisions, each giving rise to a germline blastomere
(sequentially P2 to P4, termed the P lineage) and a corresponding somatic cell
(Fig. 1).
PAR proteins regulate spindle position via a G-protein signaling pathway
(Colombo et al., 2003;
Hess et al., 2004), and
cytoplasmic polarity via two maternally supplied proteins, MEX-5 and MEX-6
(Schubert et al., 2000). MEX-5
and MEX-6 are closely related proteins and are both preferentially localized
toward the anterior cytoplasm of the one-cell embryo and are enriched in the
somatic daughter after the division of each germline blastomere
(Cuenca et al., 2003;
Schubert et al., 2000).
Whereas mex-5 mutants exhibit 100% embryonic lethality,
mex-6 mutant embryos are 100% viable with no observable defects
(Schubert et al., 2000).
However, many molecular defects in mex-5 mutant embryos are
dramatically enhanced when mex-6 is also mutated or depleted,
suggesting partially redundant functions for these two genes
(Schubert et al., 2000). For
simplicity, unless specifically noted, we will use MEX-5/6 to refer to MEX-5
and MEX-6.
One major function of MEX-5/6 is to restrict the localization of maternally
supplied germline proteins, such as PIE-1, POS-1 and MEX-1, to germline
blastomeres (Guedes and Priess,
1997; Mello et al.,
1996; Schubert et al.,
2000; Tabara et al.,
1999). In the one-cell embryo, as MEX-5/6 become asymmetrically
localized anteriorly, PIE-1 becomes localized posteriorly
(Cuenca et al., 2003;
Mello et al., 1996;
Schubert et al., 2000). After
cell division, PIE-1 is enriched in P1, and this pattern reiterates in each
subsequent P-lineage division. The small amount of PIE-1 segregated to the
somatic sister after each division is degraded by a ZIF-1-containing CUL-2 E3
ligase complex (DeRenzo et al.,
2003; Reese et al.,
2000). Both asymmetric distribution of PIE-1 before division, as
well as asymmetric degradation after division, require the function of MEX-5/6
(DeRenzo et al., 2003;
Schubert et al., 2000).
MEX-5/6 are themselves also substrates for this ZIF-1-containing E3 ligase
complex (DeRenzo et al.,
2003).
Before meiosis II, high levels of both PIE-1 and MEX-5/6 proteins are
detected uniformly throughout the cytoplasm of oocytes and one-cell embryos
(Fig. 1)
(Cuenca et al., 2003;
Schubert et al., 2000). This
suggests that localization of PIE-1 by MEX-5/6 is a developmentally regulated
event that initiates following meiosis II. However, it is not clear what
regulates the function of MEX-5 or MEX-6, or even what their biochemical
activity as it relates to PIE-1 localization might be. MEX-5/6 each contain
two TIS-11-like zinc fingers, a protein domain shown to function in
RNA-binding (Pagano et al.,
2007; Schubert et al.,
2000). It is not known whether RNA-binding is required for MEX-5/6
regulation of PIE-1 localization or degradation. It has been suggested that
PAR-1 protein, which regulates MEX-5/6 asymmetric localization, is also
required independently for their activity
(Cuenca et al., 2003).
Asymmetric localization of PIE-1 in the one-cell zygote, as well as
degradation in the soma, also requires the DYRK2 kinase, MBK-2
(Pellettieri et al., 2003).
MBK-2 activation occurs at meiosis II and is accompanied by changes in its
subcellular localization, and in vivo phosphorylation of two known substrates,
MEI-1 and OMA-1 (Nishi and Lin,
2005; Pellettieri et al.,
2003; Shirayama et al.,
2006; Stitzel et al.,
2006). Depletion of mbk-2 abolishes PIE-1 asymmetry and
PIE-1 degradation, but not MEX-5 asymmetry
(Pellettieri et al., 2003),
suggesting that regulation of PIE-1 by MBK-2 is either independent of MEX-5
function or downstream of MEX-5 localization.
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; Nigg,
1998). In addition, PLKs have also been shown to function in a
number of other cell biological processes
(Eckerdt et al., 2005;
Ma et al., 2003;
Seeburg et al., 2005;
Takai et al., 2005;
Wang et al., 2007). All PLKs
possess an N-terminal kinase domain and a noncatalytic, C-terminal polo box
domain (PBD), composed of two related polo boxes, each approximately 70-80
residues long (Clay et al.,
1993; Lee et al.,
1998). Both domains are essential for PLK function in vivo. The
PBD binds preferentially to phosphorylated serine or threonine at the
consensus sequence S-[pS/pT]-P/X (Elia et
al., 2003a), and is crucial for in vivo spatial and temporal
subcellular localization of polo kinases
(Lee et al., 1998;
Lee et al., 1999;
Qi et al., 2006;
Seong et al., 2002).
Structural studies indicate that the PBD binds intramolecularly to its own
kinase domain (Elia et al.,
2003b), inhibiting both kinase activity and the ability of PLK to
bind phosphopeptides (Elia et al.,
2003b; Jang et al.,
2002; Lee and Erikson,
1997; Mundt et al.,
1997). Upon binding of the PBD to a phosphorylated peptide
substrate, kinase activity is elevated
(Elia et al., 2003b). The PBD
alone is better than the full-length PLK in binding the phosphopeptide
substrate (Elia et al.,
2003a).
In C. elegans, there are three genes that encode polo-like
kinases, plk-1, plk-2 and plk-3, with PLK-3 protein being
significantly more diverged (Chase et al.,
2000a; Chase et al.,
2000b; Ouyang et al.,
1999). Depletion of plk-1 by RNAi results in defects in
nuclear envelope breakdown, meiotic chromosome segregation and cytokinesis,
producing embryos arrested at the one-cell stage without cleavage, consistent
with a role in cell division (Chase et al.,
2000b). The functions of plk-2 and plk-3 remain
unclear, as no abnormality was observed in embryos depleted of either gene
alone by RNAi (Rual et al.,
2004).
Here we report a novel function for PLKs in embryonic cytoplasmic polarity. We show that PLK-1 and PLK-2 interact with both MEX-5 and MEX-6 in yeast and exhibit an asymmetric cytoplasmic localization similar to MEX-5/6 in early embryos. Both interaction in yeast and asymmetric localization in embryos require the PBD of PLK-1 and PLK-2 as well as amino acid T186 of MEX-5. In addition, PLK-1 and PLK-2 are required for MEX-5/6 function in vivo, including regulating PIE-1 polarity in the one-cell embryo and degradation in somatic cells. We show that MBK-2 is required for PLK-1 cytoplasmic polarity in vivo, and that MBK-2 primes for PLK-1 at the T186 site in vitro. Our results provide a mechanism by which MEX-5/6 function is temporally regulated during the crucial oocyte-to-embryo transition.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Transgenic
strains were generated by microparticle bombardment
(Praitis et al., 2001) of the
respective plasmids: Ppie-1gfp::mex-5 T186A in
TX672(teIs35) and TX673(teIs36);
Ppie-1gfp::plk-1PBD in TX773(teIs65),
TX774(teIs66) and TX775(teIs67);
Ppie-1gfp::plk-1FL in TX787(teIs70).
For a given transgene, multiple transgenic lines were analyzed and showed
identical patterns. GZ336 (Leidel and
Gonczy, 2003), JH227 (Reese et
al., 2000) and JH1448 (axEx1125)
(Cuenca et al., 2003) contain
Ppie-1gfp::plk-1FL, Ppie-1gfp::pie-1
and Ppie-1gfp::mex-5 transgenes, respectively.
Plasmid construction
All expression clones used in this study were generated using the Gateway
technology (Invitrogen) and have been confirmed by sequencing. Germline
expression constructs were generated by introducing corresponding cDNAs into
the destination vector pID3.01B under the control of the pie-1
promoter (Reese et al., 2000).
MBK-2 and PLK-1 kinases were N-terminally tagged with maltose binding protein
(MBP) and FLAG, respectively (Nishi and
Lin, 2005). Full-length MEX-5 and MEX-6 were tagged with MBP,
whereas truncated MEX-5 and MEX-6 were tagged with 6x His, all at the
N-terminus. All site-directed mutagenesis was performed using the Quick Change
Site-directed Mutagenesis Kit (Stratagene). The kinase dead mutations used
were Y237A for MBK-2 and N166A for PLK-1. Details
regarding the cloning of these plasmids are available upon request.
Protein purification and in vitro kinase assays
MBP-tagged MBK-2, OMA-1, MEX-5 and MEX-6 were expressed in and purified
from Rosetta (DE3) pLysS (Novagen) cells as described previously
(Nishi and Lin, 2005).
His-tagged MEX-5 and MEX-6 fragments were purified using Ni2+
columns (Clontech) according to the manufacturer's instructions.
FLAG-tagged PLK-1 and MBK-2 were expressed in and purified from HEK293T
cells as described previously for FLAG-tagged GSK-3
(Nishi and Lin, 2005). MBK-2
(25 mM HEPES pH 7.6, 5 mM MgCl2, 5 mM MnCl2, 0.5 mM DTT,
10 mM β-glycerol phosphate, 30 nM ATP and 0.5 µCi
[
-32P] ATP), H.s DYRK2 (same as for MBK-2 except 50
µM ATP), H.sCdk-1 (50 mM HEPES pH 7.6, 10 mM MgCl2, 1
mM DTT, 20 mM β-glycerol phosphate, 50 µM ATP and 0.5 µCi
[-32P] ATP), and PLK-1 (same as for HsCdk1 except
10 mM β-glycerol phosphate, 1 mM NaVO4) kinase assays were
performed at 25°C for 20 minutes.
For the tandem kinase reaction, MBP::MEX-5 was preincubated with H.sDYRK2 (Upstate/Millipore) in the presence of 500 µM ATP (37°C for 30 minutes), pulled down with amylose resin (New England Biolabs), washed extensively, eluted with 50 mM maltose and added to the second kinase reaction with H.sPLK-1 (Cell Signaling Technology) (20°C for 20 minutes). Products were separated by SDS-PAGE and Coomassie stained. Incorporation of 32P was visualized by autoradiography of dried gels.
Generation of PLK-2 antibody
Rabbit Anti-PLK-2 antibody (Bethyl Laboratory) was generated against a
peptide corresponding to the first 16 amino acids of PLK-2, a region
significantly diverged from PLK-1.
RNA interference
RNAi for pie-1 was performed by injection. For plk-1 and
plk-2, similar results were obtained with feeding and injection RNAi.
Feeding RNAi was performed at 25°C by placing L2 worms onto RNAi bacteria
and scoring 24-30 hours later (Timmons and
Fire, 1998). Because plk-2(RNAi) alone has no
phenotype, a mild plk-1(RNAi);plk-2(RNAi)
effect was obtained by diluting plk-1(RNAi) bacteria with
plk-2(RNAi) bacteria to various ratios. The
mex-6-specific RNAi clone contains sequence corresponding to amino
acids 1-75 of the MEX-6 protein, chosen for sequence divergence relative to
MEX-5. The plk-1 3'UTR feeding RNAi clone contains a 350 bp
sequence beginning 5 bp downstream of the stop codon.
Rescue assay
Transgenic strains carrying
axEx1125(Ppie-1gfp::mex-5) or
teIs36(Ppie-1gfp::mex-5 T186A) were
crossed to JJ1238 [unc-30(e191)
mex-5(zu199) IV/nT1 (IV;V)] and F2
unc-30(e191) mex-5(zu199) progeny that expressed GFP were
singled out to 25°C plates and assayed for the number of viable progeny
produced. Each unc-30(e191) mex-5(zu199) animal produced
approximately 150 embryos in our assay. teIs35 is an integrated
transgene and animals carrying one or two copies exhibit different expression
levels. Only animals carrying two copies of teIs35 were scored for
rescue. Rescued lines were placed on mex-6-specific RNAi plates and
resulting embryos analyzed. RNAi with the mex-6-specific RNAi clone
results in no abnormal or dead embryos from wild-type N2 animals, but 100%
dead embryos that resemble
mex-5(zu199);mex-6(ah6) mutant embryos
from mex-5(zu199) animals.
The plk-1 3'UTR RNAi clone does not interfere with the expression of GFP::PLK-1 or GFP::PLK-1PBD and results in near 100% dead embryos in wild-type N2 that exhibit a phenotype resembling a weak phenotype obtained by RNAi with plk-1 coding sequence (double nucleated blastomeres). No significant rescue was observed with either GFP::PLK-1FL-, or GFP::PLK-1PBD-expressing worms.
Immunofluorescence
Immunofluorescence protocols for antibodies specific for PLK-1
(Lin et al., 1998), PLK-2
(Lin et al., 1998), PIE-1
(Mello et al., 1996), MEX-5
(Schubert et al., 2000) and
GFP (Molecular Probes, rabbit A-11122)
(Lin et al., 1998) are as
described. Dilutions used: PLK-1, 1/2000; PLK-2, 1/500; PIE-1, 1/10; MEX-5, no
dilution; GFP, 1/200. We could not reproducibly detect nuclear PIE-1 signal in
one- and two-cell wild-type embryos by anti-PIE-1 antibody. All experiments
showing nuclear PIE-1 in one-cell embryos utilized a GFP::PIE-1-expressing
strain.
Analysis of embryos and imaging
Imaging of immunofluorescence and live embryos was performed using an
Axioplan microscope equipped with epifluorescence, differential interference
contrast (DIC) optics, and a MicroMax-512EBFT CCD camera as described
previously (Rogers et al.,
2002).
Yeast two-hybrid assay
Yeast two-hybrid assays were performed using the GAL4-based transcription
system in AH109 (Clontech), with PLK-1 and PLK-2 expressed as baits and MEX-5
and MEX-6 expressed as preys. Transformants were spotted onto SD-Leu-Trp-His
plates containing various concentrations of 3-amino triazole (3AT, 0-50 mM),
and their growth at 30°C 3 days after spotting was scored and imaged. A
higher 3AT concentration represents a more stringent assay and normally
detects a stronger interaction.
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). With this antibody, we observed, in addition to the
reported localization patterns, strong cytoplasmic staining in newly
fertilized embryos following the completion of meiosis
(Fig. 2A-C). This cytoplasmic
staining was asymmetric along the AP axis, with the highest levels at the
anteriormost portion of the embryo (Fig.
2B,C). After the first mitotic division, anti PLK-1 cytoplasmic
staining was higher in the somatic cell AB than in the germline blastomere P1
(Fig. 2D). This asymmetric
pattern, that is, asymmetric cytoplasmic localization before division and
enrichment in the somatic cell after division, reiterated at the three
subsequent germline blastomere divisions
(Fig. 2E,F, and data not
shown). The level of cytoplasmic staining was dynamic throughout the cell
cycle, decreasing during mitosis when a high level of staining was observed on
centrosomes and chromosomes. This made it difficult to compare the level of
cytoplasmic staining in later cells that have very different cell division
rates. Therefore, the results reported here focus on embryos up to the
four-cell stage.
A GFP reporter containing the PLK-1 polo box domain recapitulates PLK-1 localization by immunofluorescence
We believe that the observed asymmetric cytoplasmic staining represented
PLK-1 localization in vivo for the following three reasons. First, in
plk-1(RNAi) embryos, the cytoplasmic staining, as well as
centrosomal and chromosomal stainings, were not detected (0%, n=30;
not shown), indicating that all observed stainings were
plk-1-dependent. Second, we generated an antibody against PLK-2 and
detected a similar, albeit much fainter, asymmetric staining pattern
(Fig. 2M,N). PLK-1 and PLK-2
have a partially redundant function (see below), which can be attributed to
this cytoplasmic asymmetry. Third, fusion proteins in which GFP was fused to
the PLK-1 PBD (GFP::PLK-1PBD, amino acids 340-648) recapitulated
the anti-PLK-1 staining pattern, including centrosomal, chromosomal and
asymmetric cytoplasmic stainings (Fig.
2G-L). GFP fused to full-length PLK-1 (GFP::PLK-1FL),
however, did not recapitulate the anti-PLK-1 staining pattern. As previously
reported, a GFP::PLK-1FL reporter exhibited only weak centrosomal
and chromosomal signals (Leidel and
Gonczy, 2003). We found that while we could obtain transgenic
lines expressing higher levels of GFP::PLK-1FL, still only a small
proportion of GFP fluorescence was associated with centrosomes and
chromosomes. In only a small percentage of embryos (
5%,
n>200), we could detect that the cytoplasmic GFP is slightly
higher in the somatic cell compared with the germline blastomere. Our results
suggest that the subcellular localization of PLK-1 in early C.
elegans embryos is mediated through the PBD domain. We will refer to this
asymmetric cytoplasmic staining as PLK-1 cytoplasmic asymmetry or simply PLK-1
asymmetry.
|
),
suggesting a possible functional relationship. We found that MEX-5/6 are
redundantly required for cytoplasmic PLK-1 asymmetry. Simultaneous depletion
of both mex-5 and mex-6 resulted in a fully penetrant defect
in the observed PLK-1 asymmetry (n
300,
Fig. 3C,M,Q,R). Depletion of
mex-6 alone by either genetic mutation or RNAi did not result in any
observable defect in PLK-1 polarity (n>200,
Fig. 3B,L). Inactivation of
mex-5 alone by either genetic mutation or RNAi resulted in a weaker,
but still scorable, PLK-1 asymmetry (n>200,
Fig. 3A,K,R). However, MEX-5
localization remained asymmetric in plk-1(RNAi) embryos (see
more below and Fig. 7),
suggesting that MEX-5/6 regulates the localization of PLK-1 and not vice
versa.
Localization of MEX-5/6 to the anterior cytoplasm depends on PAR proteins
(Cuenca et al., 2003;
Schubert et al., 2000). We
observed a loss of PLK-1 cytoplasmic asymmetry in all par mutants
tested: par-1 (n=38), par-2 (n=25),
par-3 (n=41), par-4 (n=15), par-5
(n=22) and par-6 (n=7)
(Fig. 3D-I,R). In embryos
depleted of pie-1, a gene downstream of MEX-5/6, PLK-1 cytoplasmic
asymmetry was unaffected (n>100,
Fig. 3J). Importantly, the
centrosomal and chromosomal localization of PLK-1 during mitosis was not
disrupted in mutants with abolished PLK-1 cytoplasmic asymmetry
(n>100, Fig. 3N-R
and data not shown).
Heterotrimeric G proteins also regulate polarity in early C.
elegans embryos (Colombo et al.,
2003; Hess et al.,
2004). They are asymmetrically activated at the cortex and are
required for the asymmetric positioning of the spindle. The asymmetric
activation of G proteins also depends on PAR proteins, but is independent of
MEX-5/6. We found no evidence for an involvement of genes in the G-protein
signaling pathway in PLK-1 cytoplasmic asymmetry. We observed wild-type PLK-1
asymmetry in goa-1(RNAi); gpa-16(RNAi)
(Colombo et al., 2003),
lin-5(ev571tsw) (Lorson
et al., 2000) or let-99(or204ts)
(Tsou et al., 2002) embryos
(n>30 for each strain; data not shown).
Taken together, these results demonstrate that regulation of PLK-1 asymmetry lies downstream of MEX-5/6 but upstream of PIE-1. In addition, PLK-1 asymmetry, like MEX-5 asymmetry, is established independently of G-protein signaling.
PLK-1 and PLK-2 physically interact with MEX-5, and this interaction depends on T186 of MEX-5 and the polo box domain of PLK
The similar cytoplasmic asymmetry exhibited by PLK-1 and MEX-5/6 in early
embryos, as well as the dependency of PLK-1 asymmetry on MEX-5/6, suggests a
physical interaction that polarizes cytoplasmic PLK-1 in vivo. Indeed, we
detected interaction between MEX-5 or MEX-6, and PLK-1 or PLK-2, using yeast
two-hybrid assays (Fig. 4A).
The polo box domains of PLK-1 and PLK-2 are both necessary and sufficient for
this interaction. No interaction was observed using the N-terminal, kinase
domain and only weak interaction was detected with full-length PLK-1 or PLK-2.
For simplicity, unless otherwise noted, we will refer to PLK-1 and PLK-2 as
PLK-1/2.
The crystal structure of human PLK1 shows that W414 and
V415 of the first polo box, and H538 and K540
of the second polo box, are crucial for phosphopeptide binding. Amino acid
changes W414A/V415A or H538A/K540M
in human PLK1 abolished phosphopeptide binding without altering the structure
of the polo domain (Elia et al.,
2003b; Lee et al.,
1999; Seong et al.,
2002). The corresponding changes to C. elegans
PLK-1PBD (W417A/V418A and
H542A/K544M) also abolished binding with MEX-5/6 in
yeast (Fig. 4A), suggesting
that PLK-1 binds to MEX-5/6 in yeast via phosphorylated epitopes. Sequence
analysis of MEX-5 identified a putative polo docking site (TSSTPL)
surrounding threonine 186 (threonine 190 of MEX-6). Changing MEX-5
T186 or MEX-6 T190 to alanine abolished or dramatically
reduced the interaction between MEX-5/6 and the two PBDs in the yeast
two-hybrid assay (Fig. 4A). We
detected no interaction between MEX-5 T186A with either
PLK-1PBD or PLK-2PBD. A weak interaction, detected only
on 20 mM but not 50 mM 3AT plates, was observed between MEX-6 T190A
and PLK-1PBD or PLK-2PBD
(Fig. 4A and data not shown).
These results support a model whereby PLK-1/2 bind to MEX-5/6 through their
polo box domains, resulting in PLK-1/2 asymmetry in the cytoplasm of early
C. elegans embryos.
|
). Worms
homozygous for zu199 can grow to adulthood but produce dead embryos.
We assayed the ability of MEX-5 T186A to rescue the embryonic
lethality of embryos produced by mex-5(zu199) mothers. We
scored the number of viable larvae produced by mex-5(zu199)
mothers that express no transgenic protein, GFP::MEX-5 or GFP::MEX-5
T186A in embryos. We observed that 0% (n=62), 38%
(n=94) and 4% (n=48) of mex-5(zu199) worms
expressing no transgenic protein, GFP::MEX-5 and GFP::MEX-5 T186A,
respectively, produced viable larvae (Fig.
4C). The percentages of viable larvae per mother were low, with no
more than 22% for GFP::MEX-5-expressing, and lower than 5% for GFP::MEX-5
T186A-expressing mex-5(zu199) worms. Despite weak
rescue, however, embryos expressing either transgenic protein could grow up to
adulthood and rescue lines could be maintained. This result suggests that
T186 is important, but not essential, for the function of MEX-5 in
vivo.
We also assayed the function of GFP::MEX-5 T186A by examining
its ability to regulate asymmetric PIE-1 distribution before, and degradation
after, cell division. Both wild-type MEX-5 and MEX-6 can polarize PIE-1 before
division. Pre-division PIE-1 polarity is abolished only when both
mex-5 and mex-6 are mutated or depleted
(Schubert et al., 2000). By
contrast, MEX-5 has a predominant function versus MEX-6 in regulating PIE-1
degradation in somatic cells. PIE-1 degradation is defective in at least some
somatic cells in 58% of mex-5(zu199) and 0% of
mex-6(ah6) single mutant embryos, but 100% of
mex-5(zu199);mex-6(ah6) double mutant
embryos (DeRenzo et al., 2003;
Schubert et al., 2000). We
show that expressing wild-type GFP::MEX-5 in mex-5(zu199)
embryos reduced the degradation defect to 15% (n=150). By contrast,
approximately 50% (n=150) mex-5(zu199) embryos
expressing GFP::MEX-5 T186A still exhibited a PIE-1 degradation
defect (Fig. 4B). The weak
rescue by GFP::MEX-5 T186A of the PIE-1 degradation defect is
consistent with its weak rescue of embryonic viability. When mex-6
was depleted by RNAi in mex-5(zu199) embryos expressing
GFP::MEX-5 T186A, defects in both pre-division PIE-1 polarity
(n>200) and PIE-1 degradation in somatic cells (n>300)
were enhanced to 100%. It has been shown that MEX-5/6 function was required
for their own degradation in somatic cells
(DeRenzo et al., 2003). We
observed that while GFP::MEX-5 T186A was polarized properly, its
degradation was completely abolished in
mex-5(zu199);mex-6(RNAi) embryos
(Fig. 4B). The preceding rescue
result and these molecular analyses demonstrate that GFP::MEX-5
T186A has a dramatically reduced MEX-5 function.
|
). In C. elegans, there are over 30 kinases in this
superfamily, and therefore we focused on the CMGC kinases known to be required
for early C. elegans embryogenesis
(Boxem et al., 1999;
Kamath et al., 2003;
Pang et al., 2004;
Pellettieri et al., 2003;
Quintin et al., 2003;
Schlesinger et al., 1999;
Simmer et al., 2003). We
examined the localization of GFP::PLK-1PBD in
cdk-1(RNAi), mpk-1(ga111ts),
mpk-1(RNAi), gsk-3(RNAi) and
mbk-2(RNAi) embryos. Weak RNAi (see Materials and methods)
was performed for cdk-1 and mbk-2 in order to allow
embryonic divisions, as strong depletion of either gene results in embryos
arresting at the one-cell stage (Boxem et
al., 1999; Pellettieri et al.,
2003).
We detected no defect in GFP::PLK-1PBD cytoplasmic polarity when
either gsk-3 or mpk-1 was inactivated (n>300;
Fig. 5). However, in
cdk-1(RNAi) and mbk-2(RNAi) embryos, we
observed a loss of GFP::PLK-1PBD cytoplasmic asymmetry without
observable defect in the asymmetric localization of MEX-5 (n>150;
Fig. 5).
mbk-2(RNAi) embryos were affected most severely, exhibiting
a nearly fully penetrant polarity defect. This result suggests that MBK-2 and
CDK-1 function in PLK-1 polarity downstream of MEX-5 localization, a property
consistent with a priming kinase. We believe that the
cdk-1(RNAi) phenotype is indirect for the following two
reasons. First, it has been shown that CDK-1 is required for MBK-2 activation
(Stitzel et al., 2006).
Therefore, cdk-1(RNAi) would also affect processes requiring
MBK-2 activity. We observed a similar abolishment of PLK-1 polarity in embryos
depleted by RNAi of cul-2, another gene required for MBK-2 activity
(Stitzel et al., 2006) (data
not shown). Second, we detected no phosphorylation of MEX-5 in our assay using
human CDK1/cyclin (Qi et al.,
2006) (data not shown), whereas MBK-2 can phosphorylate MEX-5 at
T186 in vitro (Fig.
6A). T186 is not the only MEX-5 residue phosphorylated
by MBK-2 in our assay, although it is the only residue within a fragment of
MEX-5 encompassing amino acids 117-270. When T186 was mutated to
either aspartate or alanine within MEX-5117-270, phosphorylation by
MBK-2 was abolished. Taken together, these results support MBK-2 being the
priming kinase that phosphorylates MEX-5 at T186.
|
|
). Therefore, we asked whether PLK-1 phosphorylates MEX-5, and
if so, whether PLK-1-dependent phosphorylation is affected by the
phosphorylation state at T186 of MEX-5. We assayed phosphorylation
of MEX-5 by PLK-1 either with or without prephosphorylation at
T186. We found that human DYRK2 (HsDYRK2) phosphorylated
T186 in MEX-5 117-270 with the same specificity as
C. elegans MBK-2 purified from mammalian tissue culture cells, but
did so much more efficiently (Fig.
6; not shown). Under optimal conditions, nearly 100% of
MEX-5117-270 was shifted to a slower migrating band in a
polyacylamide gel (Fig. 6A; not
shown). We performed a tandem kinase assay, first with HsDYRK2 and excess cold ATP, followed by PLK-1 and radioactive ATP, to ask whether prior phosphorylation of MEX-5 at T186 enhanced subsequent phosphorylation by PLK-1. We used human PLK1 (HsPLK1) here because of its superior activity over C. elegans PLK-1 isolated from mammalian tissue culture cells. We detected a robust and specific phosphorylation of MEX-5 by HsPLK1 (as assayed by the incorporation of radioactive ATP) that was dependent on both T186 and HsDYRK2 (Fig. 6B). When MEX-5 T186A or no HsDYRK2 was used, only very low levels of radioactive MEX-5 were detected (Fig. 6B). These results suggested that: (1) prephosphorylation of MEX-5 by HsDYRK2 dramatically enhances phosphorylation of MEX5 by HsPLK-1; and (2) although HsDYRK2 phosphorylated multiple sites on MEX-5, only phosphorylation at T186 is important for HsPLK1 docking and phosphorylation of MEX-5. This result strongly supports the conclusion that MBK-2 phosphorylates MEX-5 at T186, priming for PLK-1 interaction and phosphorylation in vivo.
PLK-1 and PLK-2 are required for MEX-5 and MEX-6 function in vivo
We also characterized localization of MEX-5 and PIE-1 in embryos depleted
of plk-1, plk-2 or both. We observed three phenotypes associated with
plk-1(RNAi), depending on the strength of the RNAi effect,
which we classified as either strong, medium or mild. Strong RNAi resulted in
sterile adults. Medium RNAi resulted in embryos arrested at the one-cell stage
without cleavage, as previously reported
(Chase et al., 2000b). These
embryos failed to complete meiosis and did not have polar bodies. Mild RNAi
resulted in a very penetrant phenotype (80% of embryos examined,
n>100) of double-nucleated blastomeres, probably the result of
failure in oocyte pronucleus nuclear envelope breakdown
(Chase et al., 2000b). These
defects were not enhanced when plk-2 was also depleted
(Chase et al., 2000b). We
observed that the one-cell arrested embryos that resulted from medium strength
plk-1/2 RNAi still exhibited asymmetrically localized GFP::MEX-5
(Fig. 7A,B,E,F,I,J,Q). This
result is consistent with our above observation that MEX-5 T186
(and presumably PLK-1 binding) was not required for MEX-5 localization, but
regulates MEX-5 function in vivo. However, we could not rule out that a low
level of PLK-1/2 remains in our medium-strength RNAi embryos that is
sufficient to polarize GFP::MEX-5.
|
). We
asked therefore whether PIE-1 is polarized properly along the AP axis when
plk-1, plk-2 or both are depleted in embryos by performing RNAi in
either wild type or a GFP::PIE-1-expressing strain. We observed that both
endogenous PIE-1 and GFP::PIE-1 were not polarized in embryos derived from
plk-1(RNAi);plk-2(RNAi) animals, but both
appeared normally polarized in embryos depleted of plk-1 or
plk-2 alone (Fig.
7C,D,G,H,K,L,Q; data not shown).
Second, MEX-5/6 activity has also been correlated with the extent of PIE-1
accumulation in pronuclei (Cuenca et al.,
2003). In the wild-type one-cell embryo, PIE-1 is detected in the
sperm-derived pronucleus, but is not detected, or is detected at a very low
level, in the oocyte-derived pronucleus
(Fig. 7C,D). Although the
significance of this low PIE-1 protein in the oocyte-pronucleus is not known,
it has been shown that it requires MEX-5/6 function. In genetic backgrounds in
which MEX-5/6 activity is low, PIE-1 was detected in both pronuclei
(Cuenca et al., 2003). We
observed that embryos derived from
plk-1(RNAi);plk-2(RNAi), but not
plk-1(RNAi) or plk-2(RNAi) animals, often
exhibited a high and equal level of nuclear GFP::PIE-1 in both pronuclei
(Fig. 7G,H,K,L; data not
shown), consistent with
plk-1(RNAi);plk-2(RNAi) embryos having
reduced MEX-5/6 activity.
Third, MEX-5/6 function is required for the degradation of PIE-1 in somatic
cells (DeRenzo et al., 2003;
Schubert et al., 2000). To
assay for the activity of MEX-5 in the degradation of PIE-1 in somatic cells
requires that embryos undergo divisions beyond the four-cell stage. With mild
plk-1/2(RNAi), we obtained embryos that developed further
than the one-cell stage, permitting the assay of PIE-1 levels in somatic
cells. We observed a slightly higher level of PIE-1 in somatic cells in many
plk-1(RNAi);plk-2(RNAi) embryos
(Fig. 7M,N,O,P). The defect is
not as strong as in
mex-5(RNAi);mex-6(RNAi) embryos, but is
consistent with a decrease in MEX-5/6 activity.
All together, our analyses are consistent with a reduced MEX-5/6 activity in plk-1(RNAi);plk-2(RNAi) embryos.
|
DISCUSSION |
|---|
|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Partial redundancy between PLK-1 and PLK-2
While polo kinases are key regulators in cell divisions, they also have
been shown to regulate processes unrelated to cell division. Polo kinases
regulate synapse remodeling in the mammalian nervous system by promoting the
degradation of spine-associated Rap guanosine triphosphatase-activating
protein (SPAR) (Kauselmann et al.,
1999; Pak and Sheng,
2003; Seeburg et al.,
2005). In addition, a recent paper showed that polo phosphorylates
Pon, which is important for asymmetric localization of Numb and inhibition of
progenitor self-renewal in Drosophila neuroblasts
(Wang et al., 2007). Our
results here demonstrate another novel, non-cell division role for polo
kinases - regulation of cytoplasmic polarity in early C. elegans
embryos. We show here that, in addition to cytokinesis and chromosome
segregation, PLK-1 has a redundant role with PLK-2 in regulating MEX-5/6
function. The subcellular localization of PLK-2, although detected only weakly
with our PLK-2 antibody, resembles that of PLK-1. Therefore the partial
redundancy of PLK-1 and PLK-2 cannot be explained by their subcellular
localization patterns. We propose that PLK-1 and PLK-2 function together in
vivo, with PLK-1 contributing more than PLK-2 to net PLK activity. In this
scenario, depletion of plk-1 alone by RNAi would reveal defects only
in those processes requiring highest PLK activity levels (such as cytokinesis
and chromosome segregation). Processes requiring a low PLK activity (such as
MEX-5/6 activation) would not be affected unless both plk-2 and
plk-1 are depleted.
The partial redundancy between PLK-1 and PLK-2 is reminiscent of that
between MEX-5 and MEX-6 (Schubert et al.,
2000). This similarity suggests the possibility that PLK-1
regulates MEX-5, whereas PLK-2 regulates MEX-6 activity. However, our data
argue against this possibility. First, both PLK-1 and PLK-2 bind to both MEX-5
and MEX-6 in yeast. Second, the asymmetric localization pattern of PLK-1 and
GFP::PLK-1PBD was disrupted only when both mex-5 and
mex-6 were depleted or mutated. Therefore, we propose that PLK-1 and
PLK-2 regulate both MEX-5 and MEX-6 in vivo and the difference in
plk-1(RNAi) and plk-2(RNAi) phenotypes is
probably due to their different expression levels in vivo.
How might PLK-1 and PLK-2 regulate MEX-5 and MEX-6 function?
Currently there are two models, not mutually exclusive, for how the PBD
regulates in vivo substrate phosphorylation by polo kinase (reviewed by
Lowery et al., 2005). In the
`processive phosphorylation' model, PBD binding brings the kinase domain to
the bound protein, which polo phosphorylates at another site. In the
`distributive phosphorylation'model, PDB binding brings the kinase domain to
the vicinity of its substrate(s), which is not the same protein to which the
PBD is bound. How might PLK-1/2 enhance MEX-5/6 function? It is possible that
PLK-1/2 enhances MEX-5/6 function by directly phosphorylating them
(progressive phosphorylation model), thereby changing the binding affinity
and/or specificity of MEX-5/6 toward RNA targets or interacting proteins.
Alternatively, it is possible that MEX-5/6 serve simply as a support for
PLK-1/2, which provides MEX-5/6 with an acquired enzymatic activity
(distributive phosphorylation model). Phosphorylation has been shown to
facilitate substrate recognition for many proteasome-mediated protein
degradations (for a review, see Harper,
2002). PLK-1/2 might phosphorylate substrates, ZIF-1 or another
component in the E3 ligase complex (DeRenzo
et al., 2003) that degrades PIE-1, POS-1 and MEX-1, allowing their
spatially and temporally regulated degradation. Experiments are underway to
distinguish between these two models.
PLK-1/2 and MEX-5/6 interaction
Our results demonstrate that MBK-2 phosphorylates T186 of MEX-5
(and T190 of MEX-6), providing a docking site for PLK-1/2, and that
PLK-1/2 binding enhances MEX-5/6 function. We believe that phosphorylation at
T186 promotes, but is not absolutely required for, PLK-1 binding.
First, weaker binding was still detected between
PLK-1PBD/PLK-2PBD and MEX-6 T190A in yeast.
We assume that the observed strong binding with wild-type MEX-5/6 was
facilitated by a yeast kinase phosphorylating T186 and
T190 in vivo. The candidate yeast kinases include CDC28, the CDK1
homolog, and YAK1, which shares weak similarity to MBK-2. Second, while
prephosphorylation at T186 by MBK-2 dramatically enhances MEX-5
phosphorylation by HsPLK1, we still detected a low level of
radioactive MEX-5 with MEX-5 T186A or in the absence of
HsDYRK2. Third, T186 is not essential for all MEX-5
function, as some, albeit few, embryos expressing only GFP::MEX-5
T186A survive, whereas embryos completely devoid of MEX-5 do not.
This is consistent with other studies showing that priming phosphorylations
enhance binding and phosphorylation of polo substrates by polo kinases, but
are not absolutely required (Jackman et
al., 2003; Kumagai and Dunphy,
1996; Nakajima et al.,
2003; Yarm, 2002;
Zhou et al., 2003).
The observation that full-length PLK-1/2 did not recapitulate the
subcellular localization pattern observed with antibody staining is
intriguing. One possible explanation is that overexpressed
GFP::PLK-1FL exceeds the available phosphorylated docking sites. We
do not believe this to be the case, because the level of GFP was observed to
be equal, if not higher, in GFP::PLK-1PBD-expressing lines.
Alternatively, in vivo localization of PLK1/2 might be subjected to another
level of regulation, in addition to the availability of phosphorylated docking
sites. It has been shown that the mutual inhibitory interaction between the
PBD and the kinase domain of PLK1 results in an approximately ten-fold
reduction in phosphopeptide binding (Elia
et al., 2003b). It is possible that a regulation that weakens this
mutual inhibitory interaction is required in vivo and that
GFP::PLK-1FL and GFP::PLK-2FL were not regulated
properly. Consistent with this possibility, we showed that
GFP::PLK-1FL did not rescue embryos depleted of endogenous PLK-1
(Materials and methods).
Priming by MBK-2
For most proteins that provide a phosphorylated polo docking site,
cyclin-dependent kinases (CDK) have been shown to be the priming kinase
(van Vugt and Medema, 2005).
Here we show that MBK-2 is the priming kinase for PLK-1 at MEX-5
T186 both in vitro and in vivo and that CDK-1 regulates MEX-5/6 and
PLK-1/2 interaction probably indirectly through regulating MBK-2. The finding
that MBK-2 is the priming kinase for PLK-1 at T186 is somewhat
surprising. First, DYRK kinases have not been previously shown to prime for
polo kinases. Second, the amino acid sequence surrounding MEX-5
T186 (LTSSTP) does not constitute a typical MBK-2
consensus site, which is R (X)1-3S/TP
(Campbell and Proud, 2002;
Himpel et al., 2000). Our in
vitro data suggest that MBK-2 phosphorylates at least one more site on MEX-5,
in addition to T186. This additional site is likely to be
T390, because its surrounding sequence (RATTP) resembles
the MBK-2 consensus phosphorylation site. However, our data shown in
Fig. 6 demonstrate clearly that
only phosphorylation at T186 by HsDYRK2 primes for
HsPLK1 in vitro.
Although at first glance MBK-2 seems an unlikely kinase to prime for PLK-1 at MEX-5 T186, it makes excellent sense with respect to early C. elegans embryogenesis. PLK-1 is detected in oocytes and is active during meiotic divisions, regulating chromosome segregation and nuclear envelope breakdown. However, MEX-5-dependent processes are first observed only following completion of both meiotic divisions. As MBK-2 activation does not occur until meiosis II, MBK-2 phosphorylation-dependent PLK-1 binding would provide a precise developmental regulation of MEX-5 function, ensuring that MEX-5 does not become prematurely activated.
Upon activation at meiosis II, MBK-2 has been shown to phosphorylate three
other proteins, MEI-1, OMA-1 and OMA-2, promoting their developmentally
regulated degradation (Nishi and Lin,
2005; Shirayama et al.,
2006; Stitzel et al.,
2006). OMA-1 and OMA-2 are closely related Tis11
zinc-finger-containing proteins that were initially identified as being
redundantly required for oocyte maturation
(Detwiler et al., 2001).
Precisely timed degradation of OMA-1 (and probably OMA-2) is crucial for
embryonic development (Detwiler et al.,
2001; Lin, 2003;
Nishi and Lin, 2005;
Shirayama et al., 2006). We,
and others, have shown that phosphorylation of OMA-1 by MBK-2 occurs primarily
at T239 (Nishi and Lin,
2005; Shirayama et al.,
2006; Stitzel et al.,
2006). Interestingly, the OMA-1 sequence surrounding
T239 also resembles the consensus polo domain interaction sequence
S-[pS/pT]-P. Consistent with a model that PLK-1 regulates OMA-1 via an MBK-2
primed phosphorylation site, our previous studies have suggested that MBK-2
phosphorylation not only regulates OMA-1 degradation but might also regulate
its function (Nishi and Lin,
2005). As DYRK2 kinases have been implicated in many biological
processes, our findings suggest the possibility that additional non-mitotic
functions for polo kinases will be identified in worms and other developmental
systems.
|
ACKNOWLEDGMENTS |
|---|
|
Footnotes |
|---|
Present address: Harvard University, 16 Divinity Avenue BL1048, Cambridge,
MA 02138, USA
|
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