RESEARCH ARTICLE
661
Development 137, 661-670 (2010) doi:10.1242/dev.042044
© 2010. Published by The Company of Biologists Ltd
Differential requirements for STRAD in LKB1-dependent
functions in C. elegans
Patrick Narbonne1, Vincent Hyenne2, Shaolin Li1, Jean-Claude Labbé2,3 and Richard Roy1,*
SUMMARY
The protein kinase LKB1 is a crucial regulator of cell growth/proliferation and cell polarity and is the causative gene in the cancerpredisposing disease Peutz-Jeghers syndrome (PJS). The activity of LKB1 is greatly enhanced following its association with the Ste20like adapter protein STRAD. Unlike LKB1 however, mutations in STRAD have not been identified in PJS patients and thus, the key
tumour suppressive role(s) of LKB1 might be STRAD independent. Here, we report that Caenorhabditis elegans strd-1/STRAD mutants
recapitulate many phenotypes typical of par-4/LKB1 loss of function, showing defects during early embryonic and dauer development.
Interestingly, although the growth/proliferation defects in severe par-4 and strd-1 mutant dauers are comparable, strd-1 mutant
embryos do not share the polarity defects of par-4 embryos. We demonstrate that most of par-4-dependent regulation of germline
stem cell (GSC) quiescence occurs through AMPK, whereby PAR-4 requires STRD-1 to phosphorylate and activate AMPK. Consistent
with this, even though AMPK plays a major role in the regulation of cell proliferation, like strd-1 it does not affect embryonic polarity.
Instead, we found that the PAR-4-mediated phosphorylation of polarity regulators such as PAR-1 and MEX-5 in the early embryo occurs
in the absence of STRD-1. Thus, PAR-4 requires STRD-1 to phosphorylate AMPK to regulate cell growth/proliferation under reduced
insulin signalling conditions, whereas PAR-4 can promote phosphorylation of key proteins, including PAR-1 and MEX-5, to specify early
embryonic polarity independently of STRD-1. Our results therefore identify a key strd-1/STRAD-independent function of par-4/LKB1 in
polarity establishment that is likely to be important for tumour suppression in humans.
INTRODUCTION
Peutz-Jeghers syndrome (PJS) is an autosomal dominant disorder
that predisposes affected individuals to tumour formation in several
organs (Giardiello et al., 1987; Jeghers et al., 1949; Peutz, 1921;
Spigelman et al., 1989). Mutations within the LKB1 locus (also
known as STK11) have been found associated with this disease
(Hemminki et al., 1998; Jenne et al., 1998), although the role of this
gene in the aetiology of PJS is still not clear. LKB1 encodes a highly
conserved serine/threonine kinase, and nearly all mutations in LKB1
that have been identified in PJS patients are predicted to disrupt its
kinase activity (Alessi et al., 2006; Launonen, 2005). Thus, LKB1
is likely to act as a tumour suppressor through phosphorylating one
or several target proteins.
LKB1 phosphorylates and activates AMPK (Hawley et al., 2003;
Woods et al., 2003), as well as 12 other kinases of the AMPK family
in vitro (Lizcano et al., 2004). Some of these LKB1 targets have
been found to regulate cell growth/proliferation and/or cell polarity
in various model systems. Indeed, AMPK was initially characterised
as a master cellular metabolic regulator (Hardie, 2007), which also
affects growth by phosphorylating TSC2 and consequently
inhibiting the TOR (target of rapamycin) pathway in response to
energetic stress (Inoki et al., 2003). Interestingly, the loss of AMPK
activity also results in polarity defects during energy-stress
conditions in Drosophila epithelia (Lee et al., 2007; Mirouse et al.,
1
Department of Biology, McGill University, Montréal, H3A 1B1 Québec, Canada.
Institut de recherche en immunologie et cancérologie (IRIC), Université de Montréal,
Montréal, H3C 3J7 Québec, Canada. 3Department of Pathology and Cell Biology,
Université de Montréal. Montréal, H3C 3J7 Québec, Canada.
2
*Author for correspondence ([email protected])
Accepted 16 December 2009
2007). Likewise, the MARK kinase PAR-1 requires LKB1dependent phosphorylation to regulate cell polarity (Alessi et al.,
2006; Martin and St Johnston, 2003; Wang et al., 2007), and PAR-1
also regulates growth/metabolism (Bessone et al., 1999; Hurov and
Piwnica-Worms, 2007). The LKB1-dependent activation of SADA/B kinases (also known as BRSK1 and BRSK2), conversely,
induces polarization and axonal outgrowth in undifferentiated
neurites (Kishi et al., 2005; Shelly et al., 2007). LKB1 also
phosphorylates the tumour suppressor dual phosphatase PTEN in
vitro (Mehenni et al., 2005), although the effect of this modification
on PTEN activity has not been well characterised and PTEN affects
cell growth/proliferation and cell polarity in many organisms (Brazil
et al., 2004; Lee et al., 2001; Martin-Belmonte et al., 2007; von Stein
et al., 2005).
The tumour suppressor function of LKB1 might thus be conferred
through its ability to negatively regulate cell growth/proliferation
and positively regulate polarity establishment/maintenance by
phosphorylating one or several of these downstream targets.
Interestingly, mutations in PTEN also result in predisposition to
cancer and an increased frequency of hamartomatous tumours:
multiple benign growths that are also common in patients with
tuberous sclerosis and PJS (Wirtzfeld et al., 2001). Thus, the
aetiology of the related cancer-predisposing syndromes resulting
from germline mutations in PTEN, TSC1/2 or LKB1 might be
related to their common functions in regulating cell
growth/proliferation and polarity in potentially overlapping
pathways.
The kinase activity and subcellular localization of LKB1 are
modified upon its association with two cofactors, a Ste20-related
adapter protein called STRAD and MO25, which together form the
highly active heterotrimeric complex (Baas et al., 2003; Boudeau et
al., 2003; Boudeau et al., 2004; Hawley et al., 2003). Surprisingly,
however, mutations in STRAD or MO25 are not associated with any
DEVELOPMENT
KEY WORDS: AMPK, LKB1, PAR-1, Peutz-Jeghers syndrome, Polarity, STRAD, C. elegans
RESEARCH ARTICLE
predisposition to cancer or PJS (Alhopuro et al., 2005; de Leng et
al., 2005). There are two isoforms of STRAD (a and b) in humans,
both of which can enhance LKB1 activity (Boudeau et al., 2003) and
thus redundancy could underlie this discrepancy. Alternatively, the
functions of LKB1 in tumour suppression could be independent of
STRAD. Consistent with this, the purified LKB1 kinase can still
phosphorylate protein targets in the absence of both STRAD and
MO25, albeit with lesser efficiency (Baas et al., 2003; Boudeau et
al., 2003).
In Caenorhabditis elegans, the LKB1 orthologue par-4 regulates
both cell growth/proliferation and cell polarity and is essential for
appropriate partitioning of developmental determinants during the
first cell division in the zygote (Kemphues et al., 1988; Watts et al.,
2000). Its precise role in establishing the anterior and posterior
domains of the first asymmetric division is still unclear, but par-4
mutants demonstrate defects in segregating P granules,
ribonucleoprotein complexes that function as germline
determinants, while also affecting division timing in the second
zygotic cell division (Morton et al., 1992). In addition to this role in
early axis specification in C. elegans, par-4/LKB1 induces
quiescence in the GSC population in response to environmental
stress, partially through activating AMPK (Narbonne and Roy,
2006). Indeed, mutations that affect either AMPK or LKB1, as well
as PTEN signalling, cause germline hyperplasia in the dauer larva,
a long-lived juvenile stage that renders animals highly resistant to
harsh environmental conditions while maintaining them in a
developmentally quiescent state (Narbonne and Roy, 2006).
To better understand how the LKB1 signalling cascade ultimately
regulates cell proliferation in C. elegans, we combined forward and
reverse genetic strategies to isolate and characterise new genes that
regulate GSC quiescence during dauer development. We describe here
the roles of strd-1, aak-1/2 and aakb-1/2, the C. elegans orthologues
of STRADa (there is no clear homologue of STRADb in C. elegans),
and of the two AMPK a-catalytic and b-regulatory subunit isoforms,
respectively, in the regulation of GSC quiescence and early embryonic
development. We show that strd-1 cooperates with par-4 to ensure
GSC quiescence during environmental stress, while also affecting
cortical integrity during early embryonic development, indicating that
PAR-4 and STRD-1 are implicated in both developmental processes.
However, we show that the requirement for strd-1 differs in each
situation. Indeed, although the severity of the GSC proliferation
defects of par-4 and strd-1 mutants are similar, the establishment of
the polarity axis in the zygote and viability of the embryos are
essentially not compromised in strd-1 mutants. Thus, our results
together suggest that PAR-4 requires STRD-1 to activate AMPK and
induce GSC quiescence under nutrient stress, whereas the essential
function of PAR-4 that consists in activating PAR-1 and other targets
to regulate embryonic polarity, is largely independent of STRD-1. The
latter strd-1/STRAD-independent function of par-4/LKB1 is likely to
be crucial for tumour suppression in humans.
MATERIALS AND METHODS
C. elegans genetics
Nematodes were grown using standard procedures on NGM (Brenner, 1974)
or MYOB plates (Church et al., 1995), fed E. coli of the strains OP50 or
HB101 and maintained at 15°C unless otherwise mentioned. The Bristol N2
strain was used as wild type.
The following alleles, transgenes and rearrangements were used: LG III:
daf-2(e1370), daf-7(e1372), aakb-2(rr88), aak-1(tm1944), strd-1(rr91, rr92,
rr111, ok2275, ok2283), dpy-18(e364), unc-69(e587), unc-119(ed3); LG IV:
mex-5(rr38); LG V: par-1(b274, zu310), dpy-21(e428), par-4(it33, it47,
it57), ozDf2, sqt-3(sc8), nT1, qIs56[lag-2p::GFP; unc-119(+)]; LG X:
aakb-1(tm2658), aak-2(ok524), rrEx211[HS::STRD-1; unc-119(+)].
Development 137 (4)
The strd-1(ok2275) deletion is predicted to completely remove the Cterminus of the protein, from within intron 3 (see Fig. S1 in the
supplementary material), truncating the protein similar to strd-1(ok2283)
(Narbonne and Roy, 2009).
Mapping and cloning of rr88, rr91, rr92 and rr111
daf-2(e1370); qIs56[lag-2p::GFP; unc-119(+)] animals were mutagenized
with EMS and screened for mutants with dauer germline hyperplasia
(Narbonne and Roy, 2006). Three alleles were isolated (rr88, rr91, rr92) and
out-crossed four times prior to subsequent analysis. rr88, rr91 and rr92 all
showed moderate linkage to daf-2 (–9.1 M.U.) on LGIII. Complementation
analysis indicated that rr91 and rr92 were allelic. Linkage analysis was used
to map rr88 and rr91 between unc-69 (+2.3 M.U.) and dpy-18 (+8.7 M.U.)
on LGIII with rr91 to the left of rr88.
Within this interval, we identified two strong candidates. Among these,
aakb-2 (Y47D3A.15) at +7.6 M.U. on LGIII, which is a C. elegans AMPactivated kinase beta regulatory subunit homologue. We identified a single
base pair substitution (GC to AT) at position 1 of the predicted codon 88 in
rr88, thereby introducing a premature stop codon (Narbonne and Roy,
2009). We cloned a segment of genomic DNA from wild-type animals
containing the aakb-2 gene flanked by ~850 bp upstream and 2 kb
downstream sequence, and micro-injected it (20 ng/ml) together with
pRF4[rol-6(su1006)] (Mello and Fire, 1995) into daf-2 rr88 mutants. Three
independent lines were obtained, in each of which the dauer germline
hyperplasia of rr88 mutant dauer animals was partially rescued (data not
shown). As repetitive transgenes are generally silenced in the C. elegans
germ line (Kelly et al., 1997), complete transgenic rescue was not expected.
Between unc-69 and dpy-18 and to the left of aakb-2, located at +5.75
M.U., resides strd-1, the closest C. elegans homologue of the Ste20-related
adapter protein (STRAD) a, a cofactor for optimal LKB1 activity in
mammalian cells (Baas et al., 2003; Boudeau et al., 2003; Hawley et al.,
2003). As par-4/LKB1 is involved in the regulation of dauer-dependent GSC
quiescence (Narbonne and Roy, 2006), we performed RT-PCR of strd-1 for
both alleles, which were found to carry typical EMS-induced GC to AT
substitutions (see Fig. S1 in the supplementary material) (Narbonne and
Roy, 2009). To test whether the impairment of strd-1 causes dauer germline
hyperplasia, we performed strd-1(RNAi) on daf-2 mutants. This indeed
consistently caused germline hyperplasia, which was, however, less severe
than in the rr91 or rr92 mutants (data not shown), probably owing to a weak
strd-1(RNAi) response.
rr111 was isolated from a similar additional screen that used a daf7(e1372); qIs56[lag-2p::GFP; unc-119(+)] background. Moderate linkage
to daf-7 was found and both aakb-2 and strd-1 were directly sequenced.
aakb-2 was found to be of wild-type sequence, but strd-1 carried a GC to AT
substitution at position 1 of the donor splice site of intron 1 that resulted in
cryptic mRNA splicing (see Fig. S1 in the supplementary material).
RNA interference
Double-stranded RNA was either synthesized from pJH573 (Fire et al.,
1998) and injected into animals or RNAi was induced by feeding animals
on individual recombinant bacterial clones following IPTG induction of
dsRNA (Fraser et al., 2000).
Antibody production and immunoprecipitation
To generate anti-STRD-1 antibodies, the strd-1(rr91) cDNA was cloned into
pGEX-6P-3 (GE Healthcare) to produce a GST fusion protein. Polyclonal
antibodies recognizing the GST::STRD-1 fusion protein were produced in
rabbits and the antisera were affinity purified and used at a dilution of 1:500
for immunoblotting or 1:25 for immunofluorescence.
To generate anti-PAR-4 antibodies, the full-length par-4 cDNA was
amplified by PCR, cloned into plasmids pDEST15 and pDEST17
(Invitrogen) to produce GST- and His-tagged proteins, respectively.
Polyclonal antibodies against GST-PAR-4 were produced in two rats
(Covance) and antisera were affinity purified using His-PAR-4. The
antibodies were used 1:25 in immunofluorescence.
Immunoprecipitation was performed as previously described (Liao et al.,
2004).
DEVELOPMENT
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strd-1 in cell quiescence and polarity
RESEARCH ARTICLE
663
Western blot analysis
Worm samples were mixed with 5⫻ sample buffer (250 mM Tris pH 6.8,
500 mM DTT, 10% SDS, 0.5% BPB, 50% glycerol) and lysed by five cycles
of –80°C freeze/100°C thaw. Lysates were electrophoresed on a 10% SDSpolyacrylamide gel and electroblotted onto a nitrocellulose membrane. The
membrane was incubated either with rabbit polyclonal anti-STRD-1, rabbit
polyclonal anti-PAR-1 (Guo and Kemphues, 1995), rabbit monoclonal antiP-AMPK (Cell signalling technology), mouse anti-a-tubulin (Sigma), rabbit
polyclonal anti-P-PAR-1 (Wang et al., 2007), or rabbit polyclonal anti-PMEX-5 (Tenlen et al., 2008) primary antibodies. Horseradish peroxidaseconjugated anti-rabbit or anti-mouse secondary antibodies were used.
Microscopy
For the visualization of early embryonic development in live specimens,
embryos were obtained from gravid hermaphrodites and mounted on a
coverslip coated with 1% poly-L-lysine in 2 mL of egg buffer (Edgar, 1995).
Time-lapse images were acquired by a Zeiss HRM camera mounted on a
Zeiss Axioimager Z1 microscope, and the acquisition system was controlled
by Zeiss Axiovision software (Carl Zeiss Canada). Images were acquired at
10-second intervals using a Plan Apochromat 63⫻/1.4 NA objective.
For immunofluorescence analysis, embryos were fixed in methanol and
stained as previously described (Labbé et al., 2004). Embryos were stained
for P granules (mouse OIC1D4 1:300, Developmental Studies Hybridoma
Bank, University of Iowa, USA), and PAR-6 protein [rabbit anti-PAR-6
1:150 (Hyenne et al., 2008)]. Secondary antibodies were Alexa546-coupled
goat-anti-mouse and Alexa488-coupled goat-anti-rabbit (1:500 each,
Invitrogen).
Fig. 1. Mutation of C. elegans LKB1/AMPK signalling components
causes dauer germline hyperplasia. (A-E)Representative images of
DAPI-stained hermaphrodite dauer larvae of the genotypes indicated. A
white line demarcates the germ line. All animals carry the daf-2(e1370)
allele. Scale bar: 50mm.
subunits cannot phosphorylate their relevant downstream effectors
to establish GSC quiescence in the absence of a functional b-subunit.
Furthermore, our data indicate that aakb-2 is the main b-subunit that
is required to inhibit GSC proliferation during dauer development,
whereas aakb-1 only has an accessory, yet non-negligible, role in
this regulation (Table 1).
par-4 requires strd-1 to establish GSC quiescence
In addition to aakb-2(rr88), we identified three other noncomplementing alleles also located on LGIII that disrupt the
establishment of GSC quiescence during dauer development.
These alleles affect the C. elegans orthologue of STRADa (strd1), a component of the heterotrimeric LKB1 kinase complex that
greatly enhances LKB1 activity in vitro (Baas et al., 2003). All of
these strd-1 alleles (rr91, rr92 and rr111) are recessive loss-offunction mutations that cause germline hyperplasia in dauer
larvae, a phenotype reminiscent of par-4/LKB1 mutants (Fig. 1;
Table 1). strd-1(rr92) is a typical EMS-induced (G727A)
transition that affects the region adjacent to the highly conserved
pseudokinase domain, whereas in strd-1(rr91), a similar transition
(C964T) introduces a premature stop codon that disrupts the two
last exons of the protein (see Fig. S1 in the supplementary
material). Finally, a third allele, strd-1(rr111), contains a mutation
at the donor splice site of intron 1 (GT to AT) (see Fig. S1 in the
supplementary material). This results in the production of
cryptically spliced mRNA variants, the most abundant of which
DEVELOPMENT
RESULTS
AMPK requirements for dauer-dependent GSC
quiescence
In a genetic screen designed to identify factors required for GSC
quiescence during dauer development in C. elegans, we previously
recovered a dominant-negative allele of the catalytic a2 subunit of
AMPK (aak-2) (Narbonne and Roy, 2006). RNAi of the other
AMPK catalytic a1 subunit aak-1 revealed that both subunits
function additively to regulate GSC quiescence. Similar dauer
germline hyperplasia phenotypes were also observed in mutants of
the C. elegans orthologues of the tumour suppressors PTEN (daf18) and LKB1 (par-4) (Narbonne and Roy, 2006). We extended the
screen to identify further loci that affect GSC quiescence. Two
independent loci were identified on linkage group (LG) III that gave
rise to par-4-like germline hyperplasia in dauer animals (Fig. 1).
One of these alleles (rr88) affects a C. elegans orthologue of an
AMPK regulatory b-subunit, which we hereafter refer to as aakb-2
(see Fig. S1 in the supplementary material). The mutant allele is
predicted to produce a truncated protein that lacks part of the
conserved glycogen-binding domain and the carboxyl-terminal
domain deemed critical for association with the other AMPK
subunits (Iseli et al., 2005; Polekhina et al., 2005). The molecular
nature and the determination of the cell/nuclei counts in the
hyperplastic germ lines of aakb-2(rr88) mutants indicate that this
mutation is a potential null allele. That is, aakb-2(RNAi) did not
enhance hyperplasia of the germ line of aakb-2(rr88) mutant dauers
(Table 1; data not shown).
A recently generated deletion allele of aakb-1(tm2658) that is
predicted to disrupt part of the glycogen-binding domain (see Fig.
S1 in the supplementary material) allowed us to analyze the
phenotype of aakb-1; aakb-2 double mutants, in which both of the
C. elegans AMPK b-subunits are impaired. These animals showed
a dauer germline hyperplasia phenotype that was slightly more
pronounced than that of aakb-2(rr88) animals and very similar
(P0.38) to that of aak-1; aak-2 double mutants that are catalytically
null for AMPK (Table 1). This suggests that the AMPK catalytic
664
RESEARCH ARTICLE
Development 137 (4)
Table 1. Success of embryonic development and germline proliferation during dauer formation
% maternal effect embryonic lethality† (n)
Genotype*
Control
aak-1(tm1944)
aak-2(ok524)
aak-1(tm1944); aak-2(ok524)
aakb-1(tm2658)
aakb-2(rr88)
aakb-2(rr88); aakb-1(tm2658)§
par-4(it57ts)
aak-1(tm1944); par-4(it57ts); aak-2(ok524)
par-1(zu310ts)
par-1(b274)¶
strd-1(rr91)**
strd-1(rr92)
strd-1(rr111)
strd-1(ok2275)††
strd-1(ok2283)**
strd-1(rr91); par-4(it57ts)
strd-1(rr92); par-4(it57ts)
strd-1(rr91); par-1(zu310ts)
strd-1(rr92); par-1(zu310ts)
15°C
25°C
Number of germ cells in dauer‡ (n)
0.0 (440)
0.3 (326)
0.8 (517)
0.4 (250)
0.2 (569)
1.3 (446)
1.0 (303)
4.1 (1076)
2.8 (252)
0.5 (418)
100 (307)
7.1 (434)
1.1 (523)
2.2 (504)
1.4 (214)
2.5 (395)
100 (807)
84.4 (334)
90.9 (419)
21.8 (591)
1.1 (375)
1.4 (219)
3.1 (260)
1.9 (212)
1.4 (222)
3.0 (328)
0.4 (283)
100 (414)
100 (191)
97.2 (318)
100 (181)
8.1 (211)
2.8 (529)
6.3 (398)
13.6 (656)
11.2 (660)
ND
100 (368)
100 (265)
100 (381)
31.4±4.3 (25)
61.4±9.2 (25)
60.2±7.2 (25)
182.1±18.5 (20)
36.5±4.2 (25)
150.0±29.9 (25)
183.9±18.4 (20)
128.1±13.0 (25)
240.0±15.0 (5)
32.3±4.3 (25)
22.0±3.2 (25)
148.0±24.7 (45)
86.4±13.4 (25)
139.3±20.4 (15)
153.6±20.1 (25)
153.5±26.8 (45)
ND
149.6±20.6 (20)
128.1±31.3 (7)
81.0±11.0 (25)
*Genotype includes daf-2(e1370ts) in all strains. †For data at 25°C, animals were upshifted at the L4 stage and eggs that were laid within the next 48 hours were scored.
‡
Mean values ± s.d. Except for par-1(zu310ts) (P0.2), statistical significance was found using the one-tailed t-test with unequal variance (P≤0.0005) versus control. §Dauer
formation was ~100% for all strains at 25°C, except for this strain, which was 82.2% Daf-c (n437). ¶Complete genotype includes sqt-3(sc8). **The maternal effect
embryonic lethality of strd-1(rr91) and strd-1(ok2283) was also verified in a daf-2(+) background: 7.0% (n329) and 2.9% (n105) at 15°C versus 9.5% (n654) and 22.7%
(n440) at 25°C, respectively. ††Complete genotype includes qIs56. ts, temperature-sensitive allele; n, sample size; ND, not determined.
par-4-dependent phosphorylation of AAK-2 at
Thr243 requires strd-1
PAR-4 and AAK-2 are both required to survive oxidative stress,
during which AAK-2 becomes highly phosphorylated in the
activation loop at Thr243 (equivalent to Thr172 of the AMPK asubunit in humans), whereas PAR-4 is absolutely required for this
activating phosphorylation of AAK-2 (Lee et al., 2008). To evaluate
the requirement for STRD-1 in PAR-4-dependent AAK-2
phosphorylation, we examined the phosphorylation status of AAK2 in strd-1 mutant dauer larvae by western blot analysis. We
observed a marked increase in AAK-2 Thr243 phosphorylation in
dauer larvae compared with a mixed-stage growing culture,
confirming that reduced insulin-like signalling and/or dauer
development induces AAK-2 activation. Phosphorylation of AAK2 was, however, undetectable in both par-4 and strd-1 mutant dauer
larvae (Fig. 2A). These results therefore suggest that PAR-4 requires
STRD-1 to phosphorylate AAK-2 to effectively inhibit GSC
proliferation during dauer development.
par-4 does not regulate embryonic polarity
through aak-1/2
In mouse and Drosophila, the complete removal of LKB1, or of both
of the AMPK catalytic subunits together, equally results in fully
penetrant embryonic lethality, owing to diverse polarity defects (Lee
et al., 2007; Mirouse et al., 2007). In C. elegans, the LKB1
orthologue par-4 is similarly required for proper embryonic
development, in which it functions to set up the early polarity axis.
par-4-associated polarity defects include a failure to localize P
granules to the posterior of the embryo at the one-cell stage and the
abnormal synchronization of blastomere divisions at the two-cell
stage, resulting in fully penetrant maternal effect embryonic lethality
(Kemphues et al., 1988; Morton et al., 1992). Moreover, mutant
larvae that carry a temperature-sensitive par-4 allele develop
germline hyperplasia as they enter dauer diapause when shifted and
maintained at the restrictive temperature following embryogenesis
DEVELOPMENT
lacks the last 13 base pairs of exon 1, causing a frameshift and a
premature stop codon, thus disrupting the production of STRD-1
protein (see Fig. S1 in the supplementary material; data not
shown).
Based on the degree of hyperplasia observed in each of the mutants,
strd-1(rr91) and strd-1(rr111) are similar and considerably more
severe than strd-1(rr92) (Fig. 1; Table 1). Two recently generated strd1 deletion alleles, (ok2275) and (ok2283), allowed us to confirm the
null phenotype of strd-1. Both of these deletions are predicted to
produce similarly truncated proteins that end after exon 3, in which
most of the conserved pseudokinase domain is deleted (see Fig. S1 in
the supplementary material). Correspondingly, the dauer germline
hyperplasia of strd-1(ok2275) and strd-1(ok2283) is similar and also
statistically indistinguishable from that of strd-1(rr91) or strd1(rr111), thus suggesting that strd-1(rr91, rr111) mutations are very
strongly hypomorphic and potentially null (Table 1).
If par-4 requires strd-1 to induce GSC quiescence, it would be
expected that par-4- and strd-1-null mutants should show a
similar degree of hyperplasia, and that reducing par-4 activity in
a strd-1-null mutant background would not further enhance the
observed dauer germline hyperplasia. Consistent with this, the
germline hyperplasia typical of strd-1-null mutant dauer larvae is
similar and slightly more severe than that of par-4(it57) strong
hypomorphic mutant animals, and is also comparable to that of
strd-1(rr92); par-4(it57) double mutants (Table 1). strd-1(rr91);
par-4(it57) double mutants die during embryogenesis, even at the
permissive temperature (15°C) for par-4(it57), and therefore we
did not evaluate germline hyperplasia in these animals. Our data
nonetheless suggest that the presumptive null germline
phenotypes of strd-1 and par-4 are similar, and are likely to
resemble that of strd-1(rr91, rr111, ok2275, ok2283) or strd1(rr92); par-4(it57) animals. strd-1 and par-4 therefore
participate in the same genetic pathway, wherein PAR-4 requires
the presence of STRD-1 to appropriately establish GSC
quiescence during dauer development.
strd-1 in cell quiescence and polarity
RESEARCH ARTICLE
665
Fig. 2. PAR-4 requires STRD-1 to phosphorylate AAK-2, but not PAR-1. (A)Phosphorylation of AAK-2 at Thr243 was detected using a
monoclonal anti-phospho-AMPK antibody. All strains carried the daf-2(e1370) mutation. Alleles are as follows: aak-1(tm1944), aak-2(ok524), par4(it57), strd-1(ok2283). Mixed-stage larvae were cultured at 15°C and dauers at 25°C. (B)STRD-1 protein was detected using rabbit polyclonal antiSTRD-1 antibodies. A specific band was apparent at ~44 kDa, the expected molecular weight for STRD-1. Animals from lane 1 are unc-119(ed3);
rrEx211[HS::STRD-1; unc-119(+)] and were heat-shocked at 30°C for 1 hour and kept at 20°C for 90 minutes before they were lysed. Lanes 2-6 were
cultured at 15°C. Animals from lanes 7-9 were cultured at 25°C and the alleles used are daf-2(e1370), par-4(it57) and strd-1(rr91). (C)Coimmunoprecipitation of PAR-4 with STRD-1. Whole worm lysates prepared from wild-type and strd-1(ok2275) animals were immunoprecipitated with
anti-STRD-1 antibodies and subsequently immunoblotted with anti-PAR-4. (D)Phosphorylation of PAR-1 at Thr325 was detected using rabbit
polyclonal anti-phospho-PAR-1 antibodies (Wang et al., 2007). par-1(b274) has a nonsense (Q814>STOP) mutation downstream of the kinase
domain (E. Griffin and G. Seydoux, personal communication). PAR-1 was used as a loading control in a parallel blot. One hundred gravid adults were
loaded per lane in each blot. (E)Phosphorylation of MEX-5 at Ser458 was detected using polyclonal anti-phospho-MEX-5 antibodies (Tenlen et al.,
2008). mex-5(rr38) carries a mutation in the donor splice site of the last intron (g1352a), resulting in aberrant splicing (M. Hebeisen, personal
communication), whereas Ser458 is within the last exon. Recapitulating previous results (Tenlen et al., 2008), MEX-5 is phosphorylated in par-1(b274)
animals, which retain kinase activity. Fifty gravid adults were loaded per lane. (C,D)The asterisk (*) indicates a non-specific band. (D,E)par-1(b274)
animals are also homozygous for sqt-3(sc8), and are the progeny of sqt-3(sc8) par-1(b274)/nT1 hermaphrodites. par-4(it33) animals are also
homozygous for dpy-21(e428), and are the progeny of dpy-21(e428) par-4(it33)/ozDf2 hermaphrodites. (A,B,E) TBA-1 was used as a loading control.
(Table 1). In C. elegans, there are two catalytic AMPK subunits
(aak-1 and aak-2) that act additively to inhibit GSC proliferation
during dauer development downstream of par-4 (Narbonne and Roy,
2006) (Fig. 1; Table 1). A deletion allele of the aak-1 gene (tm1944)
allowed us to study the effect of the removal of all AMPK catalytic
activity during C. elegans early embryonic development. Unlike in
mammals or Drosophila, aak-1; aak-2 AMPK catalytic null (as well
as aakb-1; aakb-2 double mutants) animals are viable and fertile,
even in conditions of reduced insulin-like signalling, or following
starvation (Table 1; data not shown). Furthermore, none of the
AMPK mutant embryos, or any combinations thereof, showed
defects in the segregation of P granules (data not shown). Moreover,
mutations in the AMPK a catalytic subunits, which have highly
penetrant effects on GSC quiescence, do not enhance the embryonic
lethality of a temperature-sensitive par-4 loss of function at the
permissive temperature (Table 1). We therefore conclude that par4/LKB1 does not establish embryonic polarity through aak/AMPK
in C. elegans.
par-4 does not require strd-1 to establish
embryonic polarity
Because STRAD was shown to enhance the kinase activity of LKB1
in tissue culture (Baas et al., 2003) and because our findings suggest
that PAR-4 requires STRD-1 to phosphorylate its downstream target
(AAK-2) and establish GSC quiescence, we wondered whether strd1 is required for all par-4 functions. Thus, we were curious whether
Table 2. Early phenotypes of C. elegans embryos*
Wild-type
par-4(it57ts)
strd-1(rr91)
strd-1(rr92); par-4(it57)
par-1(zu310)
strd-1(rr91); par-1(zu310ts)
Posterior P granule localization
15°C
25°C
15°C
25°C
5% (20)
88% (17)
24% (25)
100% (21)
17% (18)
100% (24)
5% (20)
100% (13)
100% (20)
ND
13% (8)
ND
100% (16)
67% (18)
100% (16)
53% (19)
100% (14)
96% (24)
100% (34)
0% (24)
100% (17)
ND
ND
ND
*Phenotypes were determined either by time-lapse analysis of developing embryos (blebbing) or by immunofluorescence on fixed specimens (P granule localization). The
values correspond to the percentage of embryos displaying the given phenotype. The number in parentheses corresponds to the number of embryos scored for each
phenotype. ND, not determined; ts, temperature-sensitive allele.
DEVELOPMENT
Blebbing at 1st cytokinesis
Genotype
RESEARCH ARTICLE
Fig. 3. STRD-1 and PAR-4 co-localize in the early C. elegans
embryo. (A-D)STRD-1 (left, green) co-localizes with PAR-4 (middle,
red) at the cell cortex (arrows) during early embryonic development.
The right-most column shows a merged image with DNA (blue) stained
with DAPI. P granules (punctate pattern) are non-specifically labelled by
our polyclonal anti-STRD-1 antibodies (see Fig. S3 in the supplementary
material). A, early one-cell; B, late one-cell; C, two-cell; D, four-cell
stage. (E-J)Cortical STRD-1 is absent in (E) strd-1(rr91), (G) strd1(rr111), (H) strd-1(ok2283) [strd-1(ok2275) were identical; not shown],
and is reduced in (F) strd-1(rr92) embryos. Cortical localization of STRD1 is lost in (I) daf-2(e1370); par-4(it57) embryos raised at 25°C (note the
uniform repartition of the non-specific P granule staining, a
characteristic of par-4 mutants). Cortical localization of PAR-4 is
maintained in (J) strd-1(ok2283) embryos. Four-cell stage embryos are
shown. Arrows indicate the cell cortex. Scale bar: 20mm.
strd-1 mutants would share some of the early embryonic polarity
defects that are reminiscent of par-4 mutants. strd-1(rr91, rr92, rr111,
ok2275, ok2283) mutant fertilized embryos that lack both maternal
and zygotic strd-1, however, all hatch at a relatively high frequency,
even if insulin signalling is reduced or if the temperature is raised to
25°C (Table 1). Indeed, time-lapse images of developing embryos
revealed that strd-1(rr91) mutant embryos execute the invariant events
typical of early embryonic development similar to wild type and
undergo early development relatively normally (see Movie 1 in the
supplementary material). We did not observe gross abnormalities
during the first or the second cell division of these embryos.
Furthermore, we found that the PAR-6 protein and P granules, which
segregate to the presumptive anterior and posterior regions of wildtype embryos respectively (Kemphues and Strome, 1997), were
properly localized in strd-1-null mutant embryos (Table 2; Fig. 3E-H;
data not shown). This indicates that strd-1 mutants do not display
Development 137 (4)
strong defects in embryonic polarity and infer that, in contrast to GSC
quiescence, par-4 functions largely independently of strd-1 to ensure
the correct asymmetric segregation of developmental determinants
during the early phases of C. elegans embryonic development.
The PAR-4 protein is uniformly enriched at the cortex of the onecell embryo, as well as at the cortex of every cell that arises
following cell division (Watts et al., 2000) (Fig. 3A-D). To
determine the expression pattern and localization of STRD-1 during
C. elegans embryonic development, we raised antibodies against
STRD-1. Our antibodies recognize an ~44 kDa protein in wild-type,
mixed-stage C. elegans extracts that is lost in strd-1 mutants rr91,
rr111, ok2275 and ok2283 (Fig. 2B). Using immunofluorescence,
we found that STRD-1 is enriched at the cell cortex in the early
embryo (Fig. 3A-D). The STRD-1 signal is reduced in strd-1(rr92)
and lost in strd-1 mutants rr91, rr111, ok2275 and ok2283 (Fig. 3EH). To independently confirm the endogenous localization of
STRD-1, we generated a transgenic line expressing a STRD-1::GFP
fusion driven by the strd-1 operon promoter using the Mos1mediated single-copy insertion strategy (Frokjaer-Jensen et al.,
2008). STRD-1::GFP is enriched at the cortex of every cell in early
transgenic embryos as well as at the cortex in the germ line of
developing larvae and adults (see Fig. S2 in the supplementary
material). Overall, STRD-1 localization seemed very similar to the
localization pattern that has been described for PAR-4 (Watts et al.,
2000), and double staining confirmed that the two proteins colocalize at the cell cortex in early C. elegans embryos (Fig. 3A-D).
To determine whether the two proteins interact, we
immunoprecipitated STRD-1 from wild-type and strd-1(ok2275)
lysates using rabbit anti-STRD-1 antibodies and probed the
precipitates on a western blot with rat anti-PAR-4 antibodies. PAR4 was detected specifically in the wild-type precipitate (Fig. 2C),
indicating that PAR-4 and STRD-1 associate in a complex in vivo.
Interestingly, we find that strd-1 is not required for the cortical
localization of PAR-4 during embryonic development, whereas par4 is required for the cortical localization of STRD-1 (Fig. 3I,J).
Moreover, STRD-1 levels are reduced in par-4 mutant animals (Fig.
2B), suggesting that the interaction with PAR-4 stabilises STRD-1.
Therefore, although STRD-1 co-localizes with PAR-4 in the early
embryo and could thus contribute to cortical PAR-4 functions, PAR4 is still present at the cortex in the absence of STRD-1 and this is
sufficient to ensure the establishment of the early polarity axis.
strd-1 and par-4 mutants share non-essential
functions during early embryonic divisions
Although par-4 does not require strd-1 to establish the polarity axis in
the early C. elegans embryo, we wondered whether strd-1 contributes,
even weakly, to the early events of embryonic development, which
could explain the low percentage of embryonic lethality observed in
these mutants (Table 1). Although strd-1 single mutants are largely
viable, detailed examination of strd-1 mutant embryos did reveal some
phenotypic abnormalities that are shared with par-4 mutant embryos.
In addition to the essential polarity defects attributed to par-4
impairment, a reduction in par-4 activity notably causes cortical
‘blebbing’ in the anterior blastomere during the first cytokinesis, and
this defect is also seen at high penetrance in strd-1 mutants (see Movie
1 in the supplementary material). Indeed, this blebbing phenotype is
rarely observed in wild-type embryos, but occurs in 100% of par4(it57) or strd-1(rr91) embryos grown at 25°C (Fig. 4; Table 2).
Furthermore, this defect was also observed in the temperaturesensitive par-4(it57) embryos grown at permissive temperature, albeit
at a reduced frequency and intensity, suggesting that this defect is
linked to a reduction of par-4 activity. Blebbing was proposed to result
DEVELOPMENT
666
Fig. 4. strd-1 mutant embryos display weak par-4-like
phenotypes. (A-F)Time-lapse images of wild-type (A,B), par-4(it57)
(C,D) and strd-1(rr91) (E,F) developing embryos undergoing the first
cytokinesis. The frames correspond to two consecutive images of the
same embryo captured at 10-second intervals and the insets are 2-fold
magnifications of the upper (⬘) or lower (⬙) cortex for better
visualization of membrane blebs. In each frame, anterior is to the left
and embryo length is ~50mm. (G)Difference in cell division timing (in
seconds) between AB and P1 in embryos of various genotypes.
Between 12 and 20 embryos were scored for each genotype and error
bars correspond to the s.d. strd-1(rr92); par-4(it57) double mutant
embryos divide significantly faster than par-4(it57) embryos at the
permissive temperature of 15°C (P4⫻10–4, t-test), and strd-1(rr91);
par-1(zu310) double mutant embryos divide significantly faster than
par-1(zu310) embryos at 15°C (P3⫻10–7, t-test).
from an uncoupling of the cytoplasmic membrane and the underlying
actin meshwork (reviewed by Sheetz et al., 2006). Thus, the data
suggest that par-4 regulates the integrity of the early embryonic cortex
in a strd-1-dependent manner.
Temperature-sensitive par-1 mutants, conversely, also show a
blebbing phenotype like strd-1 and par-4 mutant embryos, but at a
much lower frequency, and this is not affected by temperature,
suggesting that this phenotype is not related to the essential function
of par-1 (Table 2). Consistent with this, only 25% of par-1(RNAi)
(n12) embryos showed a weak blebbing phenotype at 25°C (data
not shown). Thus, it seems that par-4 and strd-1 function together to
prevent these defects in cortical integrity during cytokinesis,
whereas par-1 does not have a predominant role in this process.
To address the importance of the mechanism that regulates
cortical integrity during early embryonic development when
polarity is weakly compromised, we assessed whether weak par4 or par-1 mutations could enhance the embryonic lethality
phenotype of strd-1 mutant animals. When maintained at the
RESEARCH ARTICLE
667
Fig. 5. Model for differential STRD-1 requirements in distinct
PAR-4 functions. (A)Under reduced insulin-like signalling during dauer
development, PAR-4 requires STRD-1 to phosphorylate and activate
AAK-1 and AAK-2, both in association with AAKB-2, to inhibit GSC
proliferation during dauer. AAKB-1 also significantly contributes to
AAK-1/2-dependent inhibition of GSC proliferation, albeit to a much
lesser extent (not shown). (B)During early embryonic development,
PAR-4 does not require STRD-1 to induce phosphorylation of PAR-1 and
MEX-5 (and potentially other proteins) to asymmetrically distribute early
fate determinants (left), whereas PAR-4 requires STRD-1 to ensure
proper cortical integrity (right). Arrows indicate direct or indirect
phosphorylation events and/or positive regulation. Bars indicate
negative regulation.
permissive temperature (15°C), par-4(it57) or par-1(zu310), as
well as strd-1(rr91, rr92) animals hatch at a high frequency.
However under the same conditions strd-1; par-4(it57) and strd1; par-1(zu310) double mutants show substantially decreased
embryonic viability, whereby strd-1(rr91) is more severe than
strd-1(rr92), resulting in fully penetrant maternal effect
embryonic lethality in a par-4(it57) background (Table 1). Thus,
strd-1 function becomes essential when polarity is weakly
compromised. Consistent with this, time-lapse analysis of
developing embryos revealed that the blebbing defect observed in
strd-1 mutant embryos was enhanced when the activity of either
par-1 or par-4 was weakly compromised (Table 2). Furthermore,
disruption of strd-1 enhanced the abnormalities in cell division
timing between the AB and P1 blastomeres in par-1 or par-4
mutants (Fig. 4G). Other phenotypes, such as spindle positioning
and orientation, as well as the localization of PAR-6 protein and
P granules were unaffected in double mutants (Table 2; data not
shown). These data suggest that the integrity of the early
embryonic cortex is regulated by a pathway that requires both
strd-1 and par-4, whereas the essential asymmetric segregation of
embryonic determinants occurs in a par-4- and par-1-dependent
manner. Therefore, although these different processes that take
place during early embryonic development seem to be regulated
through divergent pathways, they are interrelated and together
clearly affect the viability of the embryo.
strd-1 is not required for par-4-dependent
phosphorylation of PAR-1
In human cells in culture, as well as in Drosophila, LKB1
phosphorylates and activates the MARK kinase PAR-1 at a
conserved site in the activation loop (equivalent to Thr325 in C.
DEVELOPMENT
strd-1 in cell quiescence and polarity
RESEARCH ARTICLE
elegans PAR-1) (Lizcano et al., 2004; Wang et al., 2007). We
therefore hypothesized that in the early C. elegans embryo, cortical
PAR-4 might be required to phosphorylate and activate PAR-1,
which is enriched at the posterior cortex (Guo and Kemphues,
1995), to asymmetrically segregate early fate determinants.
Consistent with this, we found that par-4 is required for the
phosphorylation of PAR-1 at Thr325 in vivo, as phospho-PAR-1 is
absent in par-4(it33) catalytic null animals (Fig. 2D). Surprisingly
however, PAR-1 remains highly phosphorylated in strd-1-null
mutants, as well as at the restrictive temperature in two par-4
temperature-sensitive mutants (Fig. 2D). Thus, PAR-4-dependent
phosphorylation of PAR-1 is robust and, unlike AAK-2
phosphorylation, it does not require strd-1.
strd-1 is not required for par-4- and par-1dependent phosphorylation of MEX-5
Following the initial asymmetric segregation of the PAR proteins,
the zinc finger protein MEX-5 accumulates in the anterior cytoplasm
of the early embryo, such that embryos depleted of PAR proteins fail
to localize MEX-5 (Schubert et al., 2000; Tenenhaus et al., 1998).
Recent findings have demonstrated that phosphorylation of MEX-5
at a crucial residue (Ser458) within its C-terminal domain is essential
for its asymmetric distribution and interestingly, PAR-4 and PAR-1
are both required for this phosphorylation (Tenlen et al., 2008). As
both PAR-1 and PAR-4 activities are required for MEX-5
phosphorylation, we reasoned that if PAR-4 does not require STRD1 to phosphorylate and activate PAR-1, strd-1 should equally be
dispensable for the phosphorylation of MEX-5 at Ser458. Western
blot analysis using anti-phospho-MEX-5 antibodies revealed that
MEX-5 is still highly phosphorylated at Ser458 in strd-1 mutants,
even at 25°C (Fig. 2E). This result confirms that both par-4 and par1 have strd-1-independent functions, further supporting the
hypothesis that PAR-4 directly phosphorylates and activates PAR-1
independently of STRD-1.
DISCUSSION
Using genetic analysis we have identified and characterised new
alleles of genes that function with PAR-4/LKB1 in establishing
GSC quiescence during dauer formation in C. elegans. We have
shown that the germline hyperplasia phenotype of strd-1-null
mutants is comparable to that of strong par-4 alleles, and that
these genes do not act in an additive manner, suggesting that they
function in a single linear pathway, potentially as a single
molecular complex. In addition, we provide biochemical evidence
that PAR-4 interacts with and requires STRD-1 to phosphorylate
AAK-2 at its activation loop during dauer development. Thus, we
propose that PAR-4 forms a complex with STRD-1 that
phosphorylates and activates AAK-2 to induce GSC quiescence
during dauer development. AAK-1 is likely to be activated during
dauer development through a similar mechanism. We have further
demonstrated that each of the catalytic AMPK subunits also
require the b-subunit aakb-2 or, to a lesser extent, aakb-1, to
inhibit GSC proliferation during dauer formation (Fig. 5). A
second function of aak-2, that is not shared with aak-1, is to
ensure the long-term survival of dauer larvae by inhibiting
lipolysis in the hypodermis. In this case, aak-2 requires either one
of the two b-subunits (Narbonne and Roy, 2009). Therefore,
multiple AMPK complexes seem to carry partially overlapping
roles in different tissues. Whether this is due to differential
expression patterns of the AMPK subunits, or to functional
differences between the possible complexes, however, remains
unclear.
Development 137 (4)
We have further shown that despite the fact that they are relevant
STRD-1-dependent PAR-4 targets, the C. elegans orthologues of
AMPK a- and b-subunits, are not involved in regulating cell
polarity, at least not in the early embryo. This contrasts with
observations made both in mouse and Drosophila reporting that
catalytically active AMPK is required for embryonic development
and also to maintain epithelial polarity under energetic stress (Lee
et al., 2007; Mirouse et al., 2007). Nonetheless, a more thorough set
of assays might reveal subtle polarity defects in C. elegans AMPK
mutants. Consistent with this, aak-2 mutants show modest motility
and behavioural response defects (Lee et al., 2008). Yet, it seems
most probable that AMPK did not originally evolve as a regulator
of polarity, but might have progressively been co-opted for this
novel function in increasingly complex organisms.
In early C. elegans embryos, mutations in par-4 or par-1 similarly
result in the loss of the asymmetric distribution of the early fate
determinants, suggesting that they function in the same process (Guo
and Kemphues, 1995; Kemphues et al., 1988; Watts et al., 2000).
PAR-4 is present in the cytoplasm and is enriched and evenly
distributed at the cortex of all the cells of the germ line and of the
early embryo (Watts et al., 2000). PAR-1, conversely, is first
detected evenly distributed at the periphery of the germ cells during
meiosis, becoming asymmetrically localized to the posterior cortex
of embryos upon fertilization (Guo and Kemphues, 1995). In
Drosophila and mammals, LKB1 was shown to directly
phosphorylate and activate several kinases of the AMPK family,
including the microtubule affinity regulating kinase (MARK) PAR1/Par1b/EMK1 (Lizcano et al., 2004). Based on genetic and cell
biological data in C. elegans, as well as on biochemical data from
other systems, the most widely accepted hypothesis is that PAR-4
directly phosphorylates and activates PAR-1 at the posterior cortex
in the early embryo, where their expression overlaps, and this would
be necessary for the segregation of the germline determinants to the
posterior pole. We have shown that par-4 is indeed required for the
activating phosphorylation on PAR-1. This phosphorylation might,
however, not be the sole essential function of PAR-4 as par-4(ts)
mutants maintain significant phosphorylation of PAR-1, despite the
observed complete embryonic lethality at the restrictive temperature.
It is thus possible that PAR-4 carries out several essential functions
during the establishment of the A/P axis. Most importantly, we have
shown that strd-1 is not required for the PAR-4-dependent activating
phosphorylation on PAR-1. Consistent with this, PAR-4 remains
enriched at the cell cortex even in the absence of STRD-1 and, most
convincingly, strd-1-null mutants do not share the fully penetrant
maternal effect embryonic lethality typical of par-4 and par-1
mutants. Furthermore, we provide evidence that there is sustained
par-4 and par-1 activity in the absence of strd-1 because MEX-5
Ser458 is still highly phosphorylated in these mutant embryos. Thus,
in contrast to the case in the dauer germline, PAR-4 does not require
association with STRD-1 to set up the polarity axis in the early
embryo, at least in part owing to its ability to phosphorylate and
hence activate or induce the phosphorylation and activation of PAR1 and MEX-5 (Fig. 5).
Nonetheless, weak par-4-like early embryonic phenotypes were
observed in strd-1 mutants. Notably, strd-1 mutant embryos showed
membrane blebbing at the anterior blastomere during the first
cytokinesis, and strd-1 also enhanced the AB and P1 blastomere
division timing defects of weak par-4 and par-1 mutants. It is not
well defined how defects in these gene products result in the
observed defects in cortical integrity. As par-1 is not necessary to
prevent the blebbing phenotype downstream of par-4, the data
suggest that PAR-4 requires STRD-1 to phosphorylate additional
DEVELOPMENT
668
substrate(s) in the early embryo, to ensure proper cortical integrity
(Fig. 5). Further elucidation of how PAR-4 and STRD-1 affect this
process will be facilitated by the identification of the target
substrates that mediate these events.
The mechanisms through which cells elicit change often impinge
on key effector molecules that commonly include protein kinases.
Yet still unresolved is how individual protein kinases can affect
numerous distinct cellular processes in a manner that is both highly
specific and robust. LKB1 is one such kinase that plays pivotal roles
in the appropriate regulation of cell polarity and cell/tissue growth
during development. Taken together, our findings indicate that
STRD-1 and PAR-4 work as a unit in many, but not all, cellular
contexts. The defective germline quiescence phenotype of strd-1
mutants corresponds to a strong loss of par-4 function, whereas the
embryonic phenotypes correspond to a weak, or partial loss of par4 activity. One explanation for this finding is that the function of
STRD-1 could be to maintain high-levels of PAR-4 kinase activity,
whereas the PAR-4 effector(s) that polarizes the early embryo (i.e.
PAR-1) might be a better substrate for PAR-4 phosphorylation, such
that it is phosphorylated even when par-4 kinase activity is low.
This, however, seems unlikely because we did not observe a marked
reduction in PAR-1 and MEX-5 phosphorylation in the absence of
STRD-1. Alternatively, the association of PAR-4 with STRD-1
could induce conformational changes such that the complex would
acquire novel properties including an enhanced affinity for a given
set of substrates. It is also possible that a second adapter protein
activates PAR-4 and compensates for STRD-1 during
embryogenesis but, in its absence, this adapter becomes limiting
when par-4 function is compromised. Nonetheless, our data suggest
that PAR-4 is capable of phosphorylating specific substrates
independently of STRD-1 (i.e. PAR-1) but acquires the ability to
phosphorylate additional targets upon association with STRD-1 (i.e.
AAK-1,2).
That STRAD or AMPK have not been directly implicated in PJS
or in any form of cancer suggests that the most robust LKB1dependent processes, those that require only minimal LKB1 activity,
might indeed be the crucial hinge pins that underlie the aetiology of
PJS. Our data hint that these might include LKB1-mediated events
in polarity establishment and further emphasizes the importance of
uncovering the identities of these important targets. How other genes
associated with hamartoma-producing syndromes that are similar to
PJS might converge on common substrates to regulate such
decisions will be of considerable interest to better understand how
their impairment translates to tumour growth.
Acknowledgements
We thank Ken Kemphues, Jim Priess and Bingwei Lu for sharing antibodies
and Paulina Salazar for generating the rat anti-PAR-4 antibodies. Some of the
strains used were obtained from the Caenorhabditis Genetic Center, the C.
elegans Gene Knockout Consortium and the Japan National Bioresource
Project. This work was supported by research grants from the Canadian
Cancer Society and CIHR to R.R., and from the Terry Fox Foundation (#16392)
and CIHR (#158715) to J.-C.L. P.N. holds a NSERC studentship, V.H. a Terry Fox
Foundation fellowship. J.-C.L. holds the Canada Research Chair in Cell Division
and Differentiation and R.R. is a CIHR New Investigator. IRIC is funded in part
by the Canadian CECR, the CFI and the FRSQ.
Competing interests statement
The authors declare no competing financial interests.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/lookup/suppl/doi:10.1242/dev.042044/-/DC1
RESEARCH ARTICLE
669
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