RESEARCH ARTICLE
867
Development 134, 867-879 (2007) doi:10.1242/dev.02790
The P-type ATPase CATP-1 is a novel regulator of C. elegans
developmental timing that acts independently of its
predicted pump function
Anne-Françoise Ruaud and Jean-Louis Bessereau*
During postembryonic stages, metazoans synchronize the development of a large number of cells, tissues and organs by
mechanisms that remain largely unknown. In Caenorhabditis elegans larvae, an invariant cell lineage is tightly coordinated with
four successive molts, thus defining a genetically tractable system to analyze the mechanisms underlying developmental
synchronization. Illegitimate activation of nicotinic acetylcholine receptors (nAChRs) by the nicotinic agonist
dimethylphenylpiperazinium (DMPP) during the second larval stage (L2) of C. elegans causes a lethal heterochronic phenotype.
DMPP exposure delays cell division and differentiation without affecting the molt cycle, hence resulting in deadly exposure of a
defective cuticle to the surrounding environment. In a screen for DMPP-resistant mutants, we identified catp-1 as a gene coding for
a predicted cation-transporting P-type ATPase expressed in the epidermis. Larval development was specifically slowed down at the
L2 stage in catp-1 mutants compared with wild-type animals and was not further delayed after exposure to DMPP. We demonstrate
that CATP-1 interacts with the insulin/IGF and Ras-MAPK pathways to control several postembryonic developmental events.
Interestingly, these developmental functions can be fulfilled independently of the predicted cation-transporter activity of CATP-1,
as pump-dead engineered variants of CATP-1 can rescue most catp-1-mutant defects. These results obtained in vivo provide further
evidence for the recently proposed pump-independent scaffolding functions of P-type ATPases in the modulation of intracellular
signaling.
INTRODUCTION
During embryonic and postembryonic stages, metazoans have to
synchronize the development of a large number of cells, tissues and
organs. As an example, the symmetrical organs of Bilateria usually
grow and develop at the same speed. Recent light has been shed on
the molecular mechanisms responsible for such regulation, with the
finding that retinoic acid is essential in vertebrates to synchronize
the somitogenesis clock between the left and right developing
mesoderm (reviewed by Brent, 2005). However, the mechanisms
responsible for synchronization during most steps of metazoan
development are still unknown (Rougvie, 2005).
The nematode Caenorhabditis elegans provides a genetically
tractable model system to understand how animals synchronize
postembryonic developmental events. After hatching, the C. elegans
larva proceeds to adulthood through a discontinuous development
with four larval stages (L1 to L4), each terminated by a molt. This
molt cycle is tightly coordinated with postembryonic cellular
divisions. Because the cell lineage is invariant in C. elegans, it is
possible to monitor developmental events at a single cell resolution
(Sulston and Horvitz, 1977). However, the mechanisms that control
and synchronize the timing of cell divisions (Kipreos, 2005) and
molts (Frand et al., 2005) are still poorly understood. We recently
described a novel paradigm to study developmental synchronization
in C. elegans by using the nicotinic agonist DMPP
(dimethylphenylpiperazinium) (Ruaud and Bessereau, 2006). We
demonstrated that illegitimate activation of nicotinic acetylcholine
receptors (nAChRs) by DMPP during the second larval stage
ENS, Biologie cellulaire de la synapse; INSERM, U789, Paris, F-75005 France.
*Author for correspondence (e-mail: [email protected])
Accepted 14 December 2006
induced a lethal heterochronic phenotype by disconnecting
developmental speed from the molting timer, hence resulting in
deadly exposure of a defective cuticle to the surrounding
environment at the subsequent molt. Environmental conditions that
delay the second to third larval molt suppress DMPP-induced
lethality by enabling resynchronization of the delayed development
with the molting time. The primary target of DMPP is likely
neuronal as loss of expression of the nAChR subunit UNC-63 in
neurons partially protects the animals from DMPP toxicity. Using a
forward genetic screen, we further demonstrated that the nuclear
hormone receptor DAF-12 is required to implement the
developmental effects of DMPP. These results uncovered two
independent pathways: one controlling the timing of C. elegans molt
and one regulating the timing of other developmental events.
Moreover, they likely define a previously undescribed
neuroendocrine pathway that is able to modulate the timing of
developmental events in response to environmental parameters.
Molecular players involved in this novel pathway as well as
environmental stimuli modulating DMPP sensitivity overlap with
key components of the dauer pathway, a genetic network
controlling entry into a facultative diapause stage. Under favorable
conditions, C. elegans goes from hatching to reproductive
adulthood in 3 days. If L1 larvae are exposed to adverse conditions
including limited food, high temperature and high population
density, animals can enter a facultative L3 diapause stage called the
dauer larva (Cassada and Russell, 1975). Dauer larvae survive for
several months without feeding and are able to resume development
to fertile adults when conditions are favorable again. A complex
neuroendocrine network involving a TGF␤, an insulin-like and a
nuclear hormone receptor (NHR) pathway controls dauer entry
(reviewed by Beckstead and Thummel, 2006; Riddle and Albert,
1997). Insulin/IGF and TGF␤ peptides are synthesized and released
from sensory neurons in response to favorable stimuli (Li et al.,
DEVELOPMENT
KEY WORDS: P-type ATPase, Developmental timing, Dauer formation, DAF-2/InsR, Ras-MAPK, Caenorhabditis elegans
RESEARCH ARTICLE
2003; Ren et al., 1996). Under these conditions, DAF-7/TGF␤
signals through its receptor to inactivate DAF-3/SMAD and DAF5/SNO (Patterson and Padgett, 2000), and insulin-like agonists
stimulate the DAF-2–insulin-like receptor, initiating a cascade that
inactivates DAF-16/FOXO by cytoplasmic segregation (Henderson
and Johnson, 2001; Kimura et al., 1997; Ogg et al., 1997), thus
allowing reproductive development. Under adverse conditions, a
DAF-3–DAF-5 complex and nuclear DAF-16 specify diapause. In
addition to their direct effect on specific transcription factors, the
DAF-7/TGF␤ and DAF-2/InsR pathways also act through
lipophilic hormone signaling, mostly by regulating the expression
of DAF-9, a cytochrome P450 involved in dafachronic acid
synthesis (Gerisch and Antebi, 2004; Gerisch et al., 2001; Jia et al.,
2002). Dafachronic acids are steroid hormones functioning as
ligands for the nuclear receptor DAF-12 (Antebi et al., 2000;
Motola et al., 2006). Under favorable conditions, DAF-7/TGF␤ and
DAF-2/InsR signaling pathways are active. This promotes DAF9/CYP expression and subsequent production of steroid hormones.
The DAF-12 NHR is mostly liganded and promotes reproductive
growth. Under adverse conditions, DAF-7/TGF␤ and DAF-2/InsR
signaling are poorly active and DAF-9 expression is low.
Consequently, dafachronic acid levels are low, and unliganded
DAF-12 triggers dauer diapause entry. We previously showed that
the liganded form of DAF-12 is specifically required to implement
nicotinic agonist toxicity during C. elegans larval development
(Ruaud and Bessereau, 2006).
In this study, we conducted a forward genetic screen to identify
additional molecular players involved in synchronizing the molt
cycle with other developmental events during the C. elegans L2
stage. We identified the cation-transporting ATPase CATP-1 as
being a novel factor required for DMPP sensitivity. catp-1 functions
in parallel with unc-63 and daf-12 for larval developmental timing
and interacts with the insulin/IGF and Ras-MAPK pathways to
control several postembryonic developmental events. Interestingly,
we demonstrated that CATP-1 mostly functions independently of its
predicted pump function to control C. elegans postembryonic
developmental timing by using ATPase-dead mutants. These results
are in line with previous data suggesting that Na+/K+-ATPases could
act as molecular scaffolds to modulate signaling pathways in
mammalian cells and provide the first in-vivo demonstration for a
transport-independent function of a P-type ATPase.
MATERIALS AND METHODS
General methods and strains
Unless noted otherwise, worms were cultured at 20°C on NG agar plates
inoculated with Escherichia coli OP50 (Sulston and Hodgkin, 1988).
Synchronous developing populations were obtained by allowing young
gravid adults to lay eggs for 30 minutes. Eggs were then collected and
transferred onto new plates. The wild strain N2, the following mutant alleles
and transgenic markers were used: age-1(mg44) I, catp-1(kr17) I, daf2(e1368, e1370, e1391, m41, m577, m596) III, daf-7(e1372) III, daf-9(dh6)
X, daf-11(m47) V, daf-12(rh61rh411) X, daf-16(mgDf50) I, ksr-1(n2526) X,
let-60(n1046) IV, lin-15(n765ts) X, mek-2(n1989) I, mpk-1(ku1) I, sid-1(qt2)
V, unc-63(kr13) I, krEx36 and krEx49 [Pcatp-1::catp-1(C. briggsae); sur5-GFP], krEx45 and krEx46 [Pcatp-1(C. briggsae)::catp-1(C. elegans);
sur-5-GFP], krEx50 and krEx51 [Pcatp-1(C. briggsae)::GFP; lin-15(+)],
krEx251 and krEx252 [Pcatp-1(C. briggsae)::catp-1D409E; Pmyo3::GFP], krEx253 and krEx254 [Pcatp-1(C. briggsae)::catp-1R669Q;
Pmyo-3::GFP], krEx264 and krEx265 [Pdpy-7::catp-1(dsRNA); Prab3::GFP; Pmyo-3::GFP], ccIs4251
[Pmyo-3::Ngfp-lacZ; Pmyo3::Mtgfp].
Mutants of MAPK pathways that tested DMPP sensitive were: jkk-1(km2)
X, jnk-1(gk7) IV, mek-1(ks54) X, nsy-1(ag3) II, sek-1(ag1, km4) X.
Development 134 (5)
DMPP resistance assay and dauer pheromone
1,1-Dimethyl-4-phenylpiperazinium (DMPP) (Sigma) was dissolved in
water and added to 55°C-equilibrated NG agar at a concentration of 0.75
mM, unless noted otherwise, just before plates were poured. Gravid adult
worms were allowed to lay eggs for several hours on standard plates. Eggs
were then carefully transferred onto DMPP-containing plates and counted.
Surviving L4, adults and dauer larvae were scored after 3 days of
development (20°C). Dauer pheromone was purified as described (Golden
and Riddle, 1984) and added to streptomycin-containing (Sigma, 50 ␮g/mL)
plates when mentioned.
DMPP resistance screen and catp-1 allele identification
N2 worms were mutagenized by germline mobilization of the Drosophila
transposon Mos1 (Williams et al., 2005). Young-adult F1 worms were
transferred onto 0.75 mM DMPP plates and allowed to lay eggs for 1 day.
Three days later, plates were screened for healthy living adult animals. In
EN17 catp-1(kr17::Mos1), a mutagenic Mos1 insertion localizes at position
14,438,562 of chromosome I by inverse PCR (WormBase web site,
http://www.wormbase.org, release WS160, date 07/2006).
Plasmid constructions and PCR amplification
Pcatp-1(C. briggsae)::catp-1(C. briggsae)
An 8.1 kb genomic fragment corresponding to C. elegans Y105E8A.12 was
PCR amplified from C. briggsae AF16 genomic DNA using Expand Long
Range PCR (Roche) (primers 5⬘-CACATCATCGCATCATCGTC-3⬘ and 5⬘GATGAGTCGTCTTAGTAGTG-3⬘).
pAF29 Pcatp-1(C. briggsae)::GFP
A 2.9 kb C. briggsae catp-1 promoter fragment was PCR amplified from
C. briggsae genomic DNA using a Taq/Pfu mix and primers 5⬘-GCCTGCAGCACATCATCGCATCATCGTC-3⬘ and 5⬘-GCGGATCCCTTTCAGTGTATTCTTCTGTTTC-3⬘. This PCR fragment was cloned in
pPD115.62 using restriction sites PstI and BamHI.
pAF31 Pcatp-1(C. briggsae)::catp-1(C. elegans)
catp-1 cDNA was cloned by RT-PCR using primers 5⬘-GCGGTACCACCGGTTTACTGTATGACTCGGAAACC-3⬘ and 5⬘-GCGAATTCACCATTTGATAAGGCGAACA-3⬘ and inserted downstream of the catp-1 C.
briggsae promoter in pAF29.
pAF88 Pcatp-1(C. briggsae)::catp-1(D409E)
pAF31 was mutagenized using the QuikChange Site-Directed Mutagenesis
kit (Stratagene) using primer 5⬘-CCACCGTAATCTGCGCAGAGAAATCAGGCACTC-3⬘. A 1 kb-long region containing the mutagenized site
was sequenced and a pool of four independently mutagenized plasmids
was injected.
pAF90 Pcatp-1(C. briggsae)::catp-1(R669Q)
A 1637 bp EcoRV-EcoRI fragment from pAF31 was cloned in pBS SK and
mutagenized using primer 5⬘-GCTCGGCAACGAGGGTCGACAAGTGATCGCCTTTGC-3⬘. The mutagenized region was sequenced and
cloned back into pAF31.
pAF92 Pdpy-7::catp-1(dsRNA)
An 894 bp PstI-KpnI dpy-7 promoter fragment (Gilleard et al., 1997) was
subcloned into pPD115.62 (PstI-BamHI) to generate Pdpy-7::GFP. The 5⬘
fragment of catp-1 cDNA (5⬘-GCGGTACCACCGGTTTACTGTATGACTCGGAAACC-3⬘ to 5⬘-GACCTGGCAGCGACGGATTGA-3⬘) was
then inserted into Pdpy-7::GFP using AgeI and EcoRI. A 5⬘ fragment (5⬘GAATTCGCCCTTAAAGACTTCGTTCGTCGA-3⬘ to 5⬘-TATCGGTGTTCGACGCGTGGATCCCCCGGG-3⬘) of the cDNA was finally cloned
backward into the previous plasmid to create a 620 bp hairpin.
Germline transformation
Transformation was performed by microinjection of plasmid DNA into the
gonad (Mello et al., 1991). catp-1(kr17) worms were injected with a DNA
mixture containing a C. briggsae genomic fragment and pTG96 (sur-5GFP) (100 ng/␮L) or pAF31 (10 ng/␮L) and pTG96 (90 ng/␮L) for rescue
experiments. The pump-dead mutant plasmids pAF88 and pAF90 were
injected in catp-1(kr17) at 10 ng/␮L with pPD115.62 (Pmyo-3::GFP) (5
ng/␮L) as a co-injection marker and 1 kb+ DNA ladder (INVITROGEN)
DEVELOPMENT
868
(85 ng/␮L). For tissue-specific RNAi, pAF92 was injected in ccIs4251; sid1(qt2) hermaphrodites at 0.1 ng/␮L together with pHU4 (Prab-3::GFP,
20 ng/␮L), pPD115.62 (5 ng/␮L) and 1 kb+ DNA ladder (75 ng/␮L). pAF29
was injected in lin-15(n765ts) at 20 ng/␮L with EKL15 (lin-15(+))
(80 ng/␮L) as a co-injection marker.
Light microscopy
Light microscopy was performed as previously described (Ruaud and
Bessereau, 2006).
RESULTS
Mutation of catp-1 confers resistance to the
nicotinic agonist DMPP
Stimulation of nicotinic receptors by the agonist DMPP during the
second larval stage of C. elegans development causes a lethal
heterochronic phenotype by slowing cellular developmental events
but not molting. Consequently, animals die at the L2/L3 molt. To
identify the molecules required to implement the effect of DMPP,
we performed a forward genetic screen for mutants that can develop
RESEARCH ARTICLE
869
on the drug. An insertional mutagenesis technique based on the
mobilization of the drosophila transposon Mos1 in the C. elegans
germ line (Bessereau et al., 2001; Williams et al., 2005) was used to
facilitate rapid identification of the mutated genes. Among seven
resistant mutants (Ruaud and Bessereau, 2006), we identified one
strain containing the mutant allele kr17 that was strongly resistant
to DMPP compared with wild type (Fig. 1A). kr17 homozygous
mutants are healthy and do not display any gross behavioral nor
morphological abnormalities by DIC inspection.
To identify the gene carrying the kr17 mutation, we performed
inverse PCR on genomic DNA of the mutant strain and detected a
Mos1 insertion in the predicted open reading frame Y105E8A.12
(Fig. 1B). This gene was named catp-1 because it codes for a cationtransporting ATPase of the P-type family (see below). The
identification of catp-1 as a novel DMPP resistance gene was
confirmed by three sets of experiments. First, the kr17 mutation
genetically mapped to the right end of chromosome I where
Y105E8A.12 is located. Second, RNAi against catp-1 phenocopied
the DMPP resistance of kr17 mutants (data not shown). Third, catp-
Fig. 1. Mutation of catp-1 confers resistance to the nicotinic agonist DMPP. (A) Dose-response sensitivity to DMPP of wild type (WT) and
catp-1(kr17) mutants. (B) catp-1 gene structure and constructs used for transgenesis experiments. Triangle, Mos1 insertion; dashes, direct repeats;
arrows, inverted repeats; *, STOP codon. (C) Survival on 0.75 mM DMPP. Error bars represent s.e.m. (n⭓4 independent experiments, N⭓96 total
individuals, two independent transgenic lines per construct tested). Pcatp-1(br)::catp-1(br): extrachromosomal array carrying the C. briggsae catp-1
genomic region; Pcatp-1(br)::catp-1(el): C. elegans catp-1 cDNA under the control of a C. briggsae catp-1 promoter. Both constructs rescued catp1(kr17) DMPP resistance. (D-G) catp-1 expression profile. Detection of GFP fluorescence by confocal (D) and epifluorescence microscopy (E) in
transgenic larvae expressing Pcatp-1(C. briggsae)::GFP. catp-1 is expressed in the hyp (white arrowheads) and Pn.p cells of the epidermis (D) and in
the duct cell of the excretory system (E-G). No GFP expression was detected in the lateral cells of the epidermis (seam cells: black arrowheads). In
(G), the GFP image (E) of the duct cell was overlayed on a Nomarski picture of the same field (F) where the excretory duct is visible (black arrow).
Scale bars: 20 ␮m (D); 5 ␮m (E-G). (H) Survival on 0.75 mM DMPP. Error bars represent s.e.m. (n⭓3 independent experiments, N⭓111 individuals,
two independent lines). Expression of a dsRNA targeting catp-1 in the epidermis induces partial DMPP resistance in a sid-1(qt2) background
(*P<0.05, Mann-Whitney test).
DEVELOPMENT
CATP-1 controls C. elegans postembryonic developmental timing
RESEARCH ARTICLE
1(kr17::Mos1) was rescued by expression of the CATP-1 protein.
Due to the presence of multiple repeated sequences in the
Y105E8A.12 genomic locus, we could not PCR amplify a rescuing
genomic DNA fragment from the wild-type N2 strain. However, in
the closely related nematode species C. briggsae, the genomic
region containing the catp-1 ortholog has a much simpler
organization (Fig. 1B). Based on the WABA software for crossspecies alignment (Kent and Zahler, 2000), the protein encoded by
the C. briggsae catp-1 ortholog was predicted to share 88% identity
and 92% similarity with C. elegans CATP-1 (see Fig. S1A in the
supplementary material). As cross-species rescue between C.
elegans and C. briggsae has already been achieved for several
Caenorhabditis genes (Aronoff et al., 2001; Maduro and Pilgrim,
1996; Thacker et al., 1999), we PCR amplified C. briggsae genomic
DNA to obtain a fragment spanning the entire catp-1 region. This
fragment rescued the DMPP resistance of catp-1(kr17) (Fig. 1C).
Next, we cloned C. elegans catp-1 cDNAs by RT-PCR and identified
16 exons, which encode a 1121-amino acid-long protein (CATP-1a)
with a start ATG codon at position 14,451,796 on chromosome I
(Fig. 1B). We also detected a short transcript lacking the 3⬘ part of
exon 16 (CATP-1b). It encodes a truncated product compared with
other proteins of the same family and was not used for further
analysis (see Fig. S1B in the supplementary material). The C.
elegans catp-1 cDNA expressed under the control of the C. briggsae
catp-1 promoter rescued the DMPP resistance of catp1(kr17::Mos1) (Fig. 1C). Taken together, these data demonstrate that
catp-1 is required to implement DMPP toxicity during C. elegans
larval development.
catp-1 encodes a cation-transporting ATPase of
the P-type family
Based on sequence homology, catp-1 was predicted to encode a Ptype ATPase (Fig. 2) (Kuhlbrandt, 2004; Okamura et al., 2003). Ptype ATPases form a large family of diverse membrane proteins that
actively transport charged substrates such as cations and
phospholipids across biological membranes. P-type ATPases
possess ten hydrophobic membrane-spanning helices (M1-M10),
and highly conserved cytoplasmic domains inserted between helices
M2 and M3 and between M4 and M5, an organization found in
CATP-1 (Fig. 2B, see Fig. S2 in the supplementary material). They
are biochemically characterized by the presence of an acid-stable
phosphorylated aspartate residue that forms during the pumping
cycle. This phosphorylable residue is easily identified in CATP-1 at
position 409 (Fig. 2C). In catp-1(kr17::Mos1) mutants, the
transposon insertion introduces a premature STOP codon before
helix M8 (see Fig. S1B in the supplementary material). As this
mutation is fully recessive and is phenocopied by catp-1 RNAi,
catp-1 truncation likely causes a loss of function of the protein.
Therefore kr17 is probably a null allele for CATP-1 enzymatic
activity. However, we cannot exclude that the protein is still being
made and retains the ability to interact with cellular partners.
Based on sequence similarity, the P-type ATPase family is divided
into five branches, referred to as types I-V, among which up to ten
different subtypes or classes can be distinguished, each subtype
being specific for a particular substrate ion (reviewed by Kuhlbrandt,
2004). Based on sequence analysis, CATP-1 can be assigned to the
␣-subunits of the type IIC ATPase subgroup, which contains the
Na+/K+- and H+/K+-ATPases (Fig. 2A). CATP-1 shares 32% identity
and 49% similarity with the human Na+/K+-ATPase NK1 and is
more distantly related to the human sarcoplasmic reticulum Ca2+ATPase (25% identity and 43% similarity). Na+/K+ and H+/K+ Ptype ATPases have been extensively characterized in vertebrates
Development 134 (5)
(Jorgensen et al., 2003; Kaplan, 2002). Both vertebrate Na+/K+- and
H+/K+-ATPases form functional heterodimers consisting of a larger
␣-subunit (110 kDa) and a highly glycosylated ␤-subunit (35 kDa
core molecular mass) (Moller et al., 1996). The ␣-subunits of both
enzymes probably incorporate all of the structural features required
for enzymatic activity, whereas the ␤-subunits are necessary to
ensure both the structural integrity of the dimeric protein complex
as well as its proper delivery to the plasma membrane (Geering,
1990; Gottardi and Caplan, 1993). Type IIC ATPases have been
subdivided into three subclasses based on their ionic specificity,
pharmacological properties and expression profile: (1) the Na+/K+ATPases, which are expressed in virtually all cells where they
extrude Na+ in exchange for K+ ions and are sensitive to the
antagonist ouabain, (2) the gastric H+/K+-ATPases, which are
expressed on the apical side of stomach cells where they exchange
H+ for K+ ions and are ouabain-insensitive, and (3) the so-called
nongastric H+/K+-ATPases can exchange both H+ and Na+ against
K+ and are ouabain-sensititive (Caplan, 1997; Jaisser and Beggah,
1999; Kuhlbrandt, 2004) (Fig. 2). The C. elegans genome encodes
five ␣ and three ␤ subunits of the type IIC ATPase family (Okamura
et al., 2003). The ␣-subunit EAT-6 clearly falls into the Na+/K+ATPases group by sequence homology and functional properties
(Davis et al., 1995). Other C. elegans class IIC ␣-subunits
(C01G12.8, C09H5.2, C02E7.1 and CATP-1) do not fall into any of
the vertebrate classes (Fig. 2). Out of these, C09H5.2, C02E7.1 and
CATP-1 lack the ouabain-binding site and the motifs correlated with
␣/␤ assembly, and show little conservation in the amino acids
associated with ion specificity (see Fig. S2 supplementary material)
(Okamura et al., 2003). These characteristics may reflect an
ancestral form of the Na+/K+, H+/K+-ATPase, and preclude a more
precise prediction of CATP-1 transport specificity.
catp-1 functions in the epidermis
To get further insights into catp-1 function, we analyzed its tissue
expression pattern. GFP was placed under the control of the
promoter sequence previously used to express CATP-1 for mutant
rescue. GFP was detected in epidermal cells including the head
epidermal cells hyp1 to hyp5, the hyp7 syncytium, the tail epidermal
cells hyp8 to hyp11, and the ventral Pn.p cells. GFP expression was
also detected in the excretory duct cell (Fig. 1D-F). To confirm that
catp-1 was required in the epidermal cells, we expressed CATP-1
under the control of the epidermal promoter Pdpy-7 (Gilleard et al.,
1997). Unfortunately, this construct was extremely toxic in vivo,
maybe due to improper expression during development or
overexpression levels, and was unable to rescue catp-1 mutants.
Therefore, we used RNA interference to selectively inhibit CATP-1
synthesis in epidermal cells by expressing a hairpin catp-1 dsRNA
driven by the Pdpy-7 promoter. Experiments were performed in a
sid-1 mutant background to prevent systemic spreading of RNAi
(Winston et al., 2002). Epidermal repression of catp-1 conferred
partial resistance to DMPP (Fig. 1H). Altogether, these data suggest
that the P-type ATPase CATP-1 functions in the epidermis to
implement DMPP toxicity.
CATP-1 has an ATPase-independent activity
Apart from regulating ion gradients through their pump activity,
recent reports suggest that Na+/K+-ATPases might function to
modulate intracellular signaling pathways. Specifically, reducing
Na+/K+-ATPase activity alters signaling through the Src, FAK and
MAPK pathways by direct protein-protein interactions between the
pump and the signaling kinase Src (Tian et al., 2006) (reviewed by
Xie and Askari, 2002). Signaling properties of the Na+/K+-ATPase
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CATP-1 controls C. elegans postembryonic developmental timing
RESEARCH ARTICLE
A
B
SCA-1
TM
human Na+/K +-ATPase α1
Ca2+-ATPases
PMR-1
871
yeast ATC1
P
human α2
N
P
rat α2
human α3
CATP-1
rat α3
Na+/K +-ATPases
bufo α1
P
N
P
human α1
rat α1
rat α4
100 aa
EAT-6
bufo α3
rat α2
C
non gastric H+/K +-ATPases
rabbit α6
P-domain part 1
human α4
400
xenopus α1
human α1
rabbit α1
NK
gastric H+/K +-ATPases
gastric
HK
mouse α1
non gastric
HK
C01G12.8
CATP-1
C02E7.1
C09H5.2
human α2
human α3
human α1
EAT-6
human α1
mouse α1
xenopus α1
rat α2
human α4
bufo α3
C01G12.8
C09H5.2
C02E7.1
CATP-1
410
NC LVK N LE A VE TLG S TS TI CS DKT GT L TQ N
NC LVK N LE A VE TLG S TS TI CS DKT GT L TQ N
NC LVK N LE A VE TLG S TS TI CS DKT GT L TQ N
NC LVK N LE A VE TLG S TS TI CS DKT GT L TQ N
NC VVK N LE A VE TLG S TS VI CS DKT GT L TQ N
NC VVK N LE A VE TLG S TS VI CS DKT GT L TQ N
NC VVK N LE A VE TLG S TS VI CS DKT GT L TQ N
NC LVK N LE A VE TLG S TS II CS DKT GT L TQ N
NC LVK N LE A VE TLG S TS II CS DKT GT L TQ N
NC LVK N LE A VE TLG S TS II CS DKT GT L TQ N
FC LVK K LQ A VE TLG S TS TI CS DKT GT L TQ N
NV FLK K LE K ID SVG A TT LI AS DKT GT L TK N
NV FLK K LE K ID SVG A TT LI AS DKT GT L TK N
NI LIK K LE L ID ELG A AT VI CA DKS GT L TM N
D409
D
N-domain
580
NK
gastric
HK
non gastric
HK
human α2
human α3
human α1
EAT-6
human α1
mouse α1
xenopus α1
rat α2
human α4
bufo α3
C01G12.8
C09H5.2
C02E7.1
CATP-1
600
620
640
660
680
N PK VA E IPF NS TNK Y Q LS I H ERE D S- --- -- --- - --- - -- PQS -H VLV MK GA P ERIL D RC STI LV QGK EI PL D KE MQ D AF QN A YMEL GGL GE R VL GF CQL NL P SG K
N KK VA E IPF NS TNK Y Q LS I H ETE D P- --- -- --- - --- - -- NDN RY LLV MK GA P ERIL D RC STI LL QGK EQ PL D EE MK E AF QN A YLEL GGL GE R VL GF CHY YL P EE Q
Y AK IV E IPF NS TNK Y Q LS I H KNP N T- --- -- --- - --- - -- SEP QH LLV MK GA P ERIL D RC SSI LL HGK EQ PL D EE LK D AF QN A YLEL GGL GE R VL GF CHL FL P DE Q
N KK IA E IPF NS TNK Y Q VS I H DNG D -- --- -- --- - --- - -- --- HY LLV MK GA P ERIL D VC STI FL NGK ES EL T DK LR E DF NT A YLEL GGM GE R VL GF CDF VL P AD K
F PK VC E IPF NS TNK F Q LS I H TLE D P- --- -- --- - --- - -- RDP RH LLV MK GA P ERVL E RC SSI LI KGQ EL PL D EQ WR E AF QT A YLSL GGL GE R VL GF CQL YL N EK D
F PK VC E IPF NS TNK F Q LS I H TLE D P- --- -- --- - --- - -- RDS RH LLV MK GA P ERVL E RC SSI LI KGQ EL PL D EQ WR E AF QT A YLSL GGL GE R VL GF CQL YL N EK D
F KK VT E VPF NS TNK F Q LS I H ELQ D P- --- -- --- - --- - -- LDL RY LMV MK GA P ERIL E RC STI MI KGQ EL PL D EQ WK E AF QT A YMDL GGL GE R VL GF CHL YL N EK E
N HK VA E IPF NS TNK F Q LS I H ETE D P- --- -- --- - --- - -- NNK RF LVV MK GA P ERIL E KC STI MI NGQ EQ PL D K S SA D SF HT A YMEL GGL GE R VL GF CHL YL P AE Q
N RK VA E IPF NS TNK F Q LS I H EMD D P- --- -- --- - --- - -- HGK RF LMV MK GA P ERIL E KC STI MI NGE EH PL D K S TA K TF HT A YMEL GGL GE R VL GF CHL YL P AD E
N RK VC E IPF NS TNK F Q LS I H ETD D P- --- -- --- - --- - -- QDQ RL LLV MK GA P ERIL E KC STI MI GGK EL PL D E S MK D SF QT A YMEL GGL GE R VL GF CHL YL P EE E
M PK IG E IPF NS TNK Y Q LS I H PMS K -- --- -- --- - --- - -- --K QN ILV MK GA P EKIL K LC STY YQ NGE TK NV S KK FE K EF QQ A YETL GSY GE R VL GF CDL EM S TT K
F HV VF E VPF NS VRK Y H LI L A TTE A -- --- TW AEI D DKK K VN ADV EF ILM IK GA P DVLI K SC STI NI DGV PM EL N GK RM D DF NE A YETF GDE GC R VI GF ACK KF R AP F QV VF E IPF NS VRK Y H LI L A TNK N -- --- TW NQV D K-- - -N DDV EF VVM IK GA P EVLI K NC STM NI NGE SK EL D LK RM E DF NE A YEAF GDE GC R VI GF AQK KF R AR Y QT VF E IPF NS IRR C Q VV V A RYL A SD FPM TS ELV D NPE - -E GQS RF MIF TK GA P EVIL G KC SNY RQ GKE LK TI D ET YR T EC QA A WEML GNE GR R VI AF AQK SF N AD -
R669
100
*
*
80
60
40
20
Fig. 2. CATP-1 encodes a cation-transporting ATPase of the P-type family with an ATPase-independent activity. (A) Phylogenetic tree of
C. elegans and vertebrate Ca2+-, H+/K+- and Na+/K+-ATPases determined using the ClustalW analysis on full-length sequences. SCA-1, PMR-1, EAT-6,
C01G12.8, CATP-1, C02E7.1 and C09H5.2 are C. elegans proteins. (B) Domain structure of human Na+/K+ P-type ATPase ␣1 and CATP-1. TM,
transmembrane domain (black); P, phosphorylable P-domain (light gray); N, ATP-binding N-domain (dark gray). (C-E) CATP-1 has an ATPaseindependent activity. (C,D) Amino-acid sequence comparison among the predicted H+/K+ (HK) and Na+/K+ (NK) P-type ATPase ␣ subunits. The aminoacid numbers are according to C. elegans CATP-1. Residues identical or similar in more than 50% of the proteins are shaded in black or gray,
respectively; residues similar to the identity consensus are also shaded in gray (BOXSHADE 3.21, http://www.ch.embnet.org/software/BOX_form.html).
(C) Part 1 of the phosphorylable P-domain. Arrow indicates the phosphorylable aspartate characteristic of P-type ATPases. (D) ATP-binding region of the
N-domain. Arrow indicates the arginine equivalent to R544 in pig kidney Na+/K+-ATPase, which is essential for ATP binding (Jacobsen et al., 2002).
(E) Survival on 0.75 mM DMPP. The D409E mutation disrupts the obligatory phosphorylation site conserved in all P-type ATPases. Error bars represent
s.e.m. (n⭓3 independent experiments, N⭓81 individuals, two independent lines). Two pump-dead mutants of CATP-1 partially rescue catp-1(kr17)
DMPP resistance (*P<0.05, Mann-Whitney test).
DEVELOPMENT
CA catp
TP -1(
-1( kr1
R6 7);
69
Q)
CA catp
TP -1(
-1( kr1
D4 7);
09
E)
1(k
CA r17)
TP ;
-1
ca
tp-
ca
tp-
1(k
r
17
)
0
WT
% survival (0.75 mM DMPP, 20°C)
E
872
RESEARCH ARTICLE
Development 134 (5)
are largely independent from its catalytic function as a pump-dead
mutant can rescue the signaling activity of Na+/K+-ATPase-depleted
cells (Liang et al., 2006). To test if CATP-1 might also function
independently of its pump activity during C. elegans postembryonic
development, we engineered two different mutations that had been
demonstrated to abolish the activity of the sodium pump. The
D409E mutation removes the conserved aspartate which is
phosphorylated during the catalytic cycle (Fig. 2C) (Liang et al.,
2006; Ohtsubo et al., 1990). The R669Q mutation disrupts ATP
binding and, consequently, the pump activity (Fig. 2D) (Jacobsen et
al., 2002). CATP-1 engineered point mutants were expressed in
catp-1(kr17). Each of these pump-dead proteins was able to partially
rescue DMPP resistance of the mutant animals (Fig. 2E). These
results indicate that CATP-1 functions, at least in part, independently
of its ATPase function during C. elegans postembryonic
development.
catp-1 modulates C. elegans developmental rate
at the second larval stage
Chronic exposure to DMPP is lethal by slowing development at the
second larval stage without affecting the molt timing (Ruaud and
Bessereau, 2006). Insensitivity to DMPP was achieved (1) when
DMPP was unable to induce a lineage delay, as demonstrated in the
daf-12(0) mutant, and (2) when development was slowed down at
the L2 larval stage, thus enabling resynchronization of molting with
a slowed development, as for animals entering a L2 diapause. To
identify which of these mechanisms might account for the DMPP
resistance of catp-1(kr17) mutants, we monitored the molt cycle and
L2 development. First, we observed that the duration of the L2 stage
was extended in mutants to 145% of wild type (Fig. 3A) and L2 cell
divisions were accordingly postponed (Fig. 3B). This developmental
delay was specific for the L2 stage, as the duration of the other larval
stages was not altered. Second, exposure of catp-1(kr17) mutants to
DMPP did not cause any further developmental delays (Fig. 3B).
Together, these results suggest that catp-1 functions to tune both the
developmental and molting timers during wild-type L2
development.
catp-1 functions in parallel with UNC-63containing nAChR and lipophilic hormone
signaling to implement DMPP toxicity
In a previous study, we showed that the nAChR subunit UNC-63
(Culetto et al., 2004) and the nuclear hormone receptor DAF-12
(Antebi et al., 1998; Antebi et al., 2000) are required to implement
DEVELOPMENT
Fig. 3. catp-1 modulates C. elegans L2
developmental rate. (A) catp-1(kr17)
specifically delays the timing of L2/L3 molt.
During the lethargus period that precedes
molting, rythmic contractions of the
pharynx (also called pharyngeal pumping)
ceases. Each dot represents the percentage
of worms pumping at a given time (n>25
individuals). (B) catp-1(kr17) is insensitive
to DMPP-induced developmental delay. L2
development was divided into five stages
based on seam cell (encircled nuclei)
divisions and anchor cell differentiation
(Ruaud and Bessereau, 2006).
Developmental stage was monitored using
DIC optics, which did not allow the
discrimination between classes 3 and 4.
The proportion of worms belonging to
each class was scored 32 hours after egg
laying (N⭓12 individuals). Error bar
represents s.e.m., n=3 independent
experiments.
CATP-1 controls C. elegans postembryonic developmental timing
DMPP toxicity. UNC-63 might be part of a DMPP receptor, whereas
DAF-12 is thought to provide a permissive activity to implement
DMPP signaling. These two genes interact in a non-linear pathway
(Ruaud and Bessereau, 2006). To further understand the function of
catp-1 during the developmental response triggered by exposure to
DMPP, we tested genetic interactions between catp-1(kr17::Mos1)
and null mutations of unc-63 and daf-12 that affect sensitivity to
DMPP. As both catp-1(kr17) and daf-12 null mutants show a strong
DMPP resistance (Ruaud and Bessereau, 2006) (Fig. 4), we used a
high drug concentration (1 mM) that kills a fraction of catp-1 and
daf-12 mutants in order to unmask possible synergistic effects. In
these conditions, we found that both double mutants [unc-63(kr13)
catp-1(kr17) and catp-1(kr17); daf-12(rh61rh411)] were more
resistant than any of the single mutants (Fig. 4), suggesting that catp1 acts in parallel to both daf-12 and unc-63.
**
100
*
80
*
**
60
873
2004). In these conditions, it is hypothesized that hormones
required for molting and for non-dauer development, derived from
non-methylated cholesterol, are not synthesized. Our result could
implicate CATP-1 in the control of the L2/L3d molt.
The decision to enter dauer diapause is controlled by a complex
genetic network. Schematically, signals from the DAF-2/InsR
(insulin receptor) and the DAF-7/TGF␤ pathways are integrated at
the level of DAF-12/NHR via the production of lipophilic hormones
(Fig. 5B). To place catp1 in this network, we performed epistasis
experiments between catp-1(kr17) and dauer constitutive mutants of
the different pathways. Among all mutant combinations tested, we
specifically detected genetic interactions between catp-1 and daf-2
(Fig. 5C). catp-1(kr17) fully suppressed the Daf-c phenotype of daf2(m41) mutants and caused abnormal dauer morphogenesis of daf2(m596) and daf-2(e1391) mutants in epidermal tissue (Fig. 5D and
data not shown): the dauer alae were abnormal and animals did not
elongate properly. Because the pharynx was constricted as in normal
daf-2 dauers, it appeared squeezed in the head. These results identify
catp-1 as a novel allele-specific suppressor of the daf-2/InsR dauer
constitutive phenotype and suggest that catp-1 could function
downstream or in parallel with daf-2/InsR to control dauer formation
and morphogenesis.
CATP-1 functions independently of DAF-16/FOXO
to modulate DAF-2/InsR signaling
To further investigate the interaction between catp-1 and daf-2/InsR
signaling, we examined the DMPP resistance of mutants of daf2/InsR and its main downstream effector, the transcription factor
daf-16/FOXO (Lin et al., 1997; Ogg et al., 1997). daf-2(e1370)
mutants are strongly DMPP resistant and this phenotype is fully
suppressed by a daf-16(mgDf50) mutant (Fig. 6). By contrast, a daf16(mgDf50) catp-1(kr17) double mutant is as resistant as catp1(kr17) alone (Fig. 6), suggesting that catp-1 functions downstream
or in parallel of daf-16 to mediate DMPP sensitivity and probably
dauer formation. Several genetic differences distinguish CATP-1
and DAF-16: (1) daf-16(0) suppresses constitutive dauer formation
and increased life span of all daf-2(lf) alleles (Kenyon et al., 1993;
Riddle et al., 1981) whereas catp-1(kr17) does not (Fig. 5C and see
Fig. S3 in the supplementary material); (2) daf-16(0) suppresses age1/PI3K whereas catp-1(kr17) does not (Fig. 5C); and (3) daf-16(0)
is DMPP sensitive whereas catp-1(kr17) is DMPP resistant (Fig. 6).
These data are not in favor of catp-1 acting downstream of daf-16
but rather support a model where catp-1 and daf-16 would act in
parallel to differentially modulate signaling through activated DAF2/InsR.
20
3(k
c-6
un
c
f-1 atp2(r 1(k
h6
r
1rh 17);
41
1)
da
r13
)
r17
tp1(k
ca
)
ca
tpun 1(k
c-6 r17
3(k )
r13
da
)
f-1
2(r
h6
1rh
41
1)
0
Fig. 4. catp-1 functions in parallel with UNC-63–nAChR and
lipophilic hormone signaling. Survival of wild-type and mutant
animals developing on 1 mM DMPP. Error bars represent s.e.m. (n⭓3
independent experiments, N⭓287 individuals). catp-1(kr17) unc63(kr13) and catp-1(kr17); daf-12(rh61rh411) double mutants are
significantly more DMPP resistant than either single mutant alone
(*P<0.05, **P<0.005, Mann-Whitney test).
CATP-1 modulates Ras-MAPK signaling
Analysis of dauer formation and aging in C. elegans has defined
a linear DAF-2/InsR signaling pathway regulating DAF16/FOXO transcriptional activity. However, two recent studies
have unmasked functions of the Ras-MAPK pathway in DAF-2dependent regulation of development and aging in C. elegans
(Hopper, 2006; Nanji et al., 2005). An activated Ras mutation, let60(n1046gf), which affects the GTPase domain of Ras (Han and
Sternberg, 1990) and causes an extended life-time of LET-60 in
its active GTP-bound conformation (Barbacid, 1987; Beitel et al.,
1990; Polakis and McCormick, 1993) was demonstrated to
weakly suppress the constitutive dauer formation of some daf-2
mutants. However, the interaction between daf-2(m41) and let60(n1046gf) had not been analyzed previously. We observed that
dauer entry of daf-2(m41) was partially suppressed by let60(n1046gf), as opposed to daf-2(e1370) which was unaffected
DEVELOPMENT
40
WT
% survival (1 mM DMPP, 20°C)
catp-1 specifically interacts with the daf-2/InsR
branch of the dauer pathway to control dauer
formation and morphogenesis
Previously identified DMPP-resistant mutants such as daf-9 and
daf-12 do not properly take the decision to enter dauer, a
facultative L3 diapause stage chosen under adverse environmental
conditions (Antebi et al., 1998; Gerisch et al., 2001; Jia et al.,
2002). Does this property also apply to catp-1 mutants? Under
replete culture conditions, catp-1(kr17) animals never form dauer
larvae and are therefore not dauer formation constitutive (Daf-c).
We tested whether catp-1 mutants were dauer defective (Daf-d)
using partially purified dauer pheromone (Golden and Riddle,
1984). catp-1(kr17) mutants do not form morphologically typical
dauer larvae on high pheromone. Instead, we observed a high
proportion of short dauers that had not molted from their L2
cuticle (Fig. 5A). These animals were SDS-resistant, suggesting
that occlusion of the buccal cavity typical of dauer morphogenesis
had been completed. Interestingly, these abnormal dauer larvae
unable to molt are highly reminiscent of worms grown on
lophenol, a methylated derivative of cholesterol (Matyash et al.,
RESEARCH ARTICLE
874
RESEARCH ARTICLE
Development 134 (5)
by the activation of Ras (Fig. 7A). These results further support
the role of the Ras pathway in DAF-2/InsR signaling during larval
development.
Because catp-1 appeared to play a role in insulin signaling using
a branch parallel to daf-16, we speculated that it might function by
modulating a DAF-2-dependent Ras-MAPK pathway. To test this
hypothesis, we first assayed DMPP resistance of mutants in the RasMAPK pathway. A loss-of-function mutant of mek-2, the MAPKK
of the ERK-MAPK pathway (Wu et al., 1995), was partially DMPPresistant (Fig. 7B). We could not test stronger mutants of the
pathway because of their rod-like L1 lethality (Sundaram, 2006).
The weak viable mutant of mpk-1 (Lackner et al., 1994), which
encodes the C. elegans homolog of the ERK-MAPK, was not DMPP
resistant. However, this allele has been previously shown to induce
weaker phenotypes than the weakly resistant mek-2 allele (Nicholas
and Hodgkin, 2004). In addition, a null mutant of the adaptor
protein-encoding gene ksr-1 (Kornfeld et al., 1995; Sundaram and
Han, 1995) was also resistant to DMPP (Fig. 7B). The very weak
DMPP resistance of ksr-1(n2526) could result from redundancy
between KSR-1 and a second KSR protein, KSR-2, as previously
demonstrated for a subset of MAPK-controlled developmental
decisions (Ohmachi et al., 2002). By contrast with the ERK-MAPK
pathway, null mutants of the p38 and JNK MAPK pathways
(Sakaguchi et al., 2004) were sensitive to DMPP (data not shown).
Altogether, these results suggested that the C. elegans ERK-MAPK
pathway is required to implement DMPP toxicity.
DEVELOPMENT
Fig. 5. catp-1 specifically interacts
with the daf-2/InsR branch of the
dauer pathway to control dauer
formation and morphogenesis.
(A) Aberrant morphogenesis of catp1(kr17) dauer larvae induced by exposure
to dauer pheromone. Compared with
wild-type dauer larvae (upper row), catp1(kr17) dauer larvae are short (left), with a
constricted pharynx (middle), and are
trapped in their L2 cuticle (right,
arrowheads). Scale bar: dissecting scope,
50 ␮m (left); Nomarski microscopy, 10 ␮m
(middle and right). (B) Schematic
representation of the dauer pathway,
adapted from Beckstead and Thummel
(Beckstead and Thummel, 2006), Gerisch
and Antebi (Gerisch and Antebi, 2004)
and Riddle (Riddle and Albert, 1997).
(C) Genetic interactions between catp1(kr17) and mutants of the dauer
pathway: dauer formation at 25°C scored
by visual inspection. age-1(mg44), daf7(e1372) and daf-9(dh6) are null alleles.
The arrow points to the suppression of
constitutive dauer formation in catp1(kr17); daf-2(m41). * defective dauer
morphogenesis. Error bars represent
s.e.m. (n⭓3 independent experiments,
N⭓89 individuals). (D) Aberrant
morphogenesis of catp-1(kr17); daf2(m596) dauer larvae. catp-1(kr17); daf2(m596) dauer larvae (bottom row) are
short with a constricted pharynx which
appears squeezed in the head (left),
abnormal alae (middle) and normal small
gonads (right). Scale bar: 10 ␮m.
80
60
40
20
da
f-2
(e1
37
0)
da
f-1
6(m
da gD
f-2 f50
(e1 );
37
0)
ca
tp1(k
r17
)
da
f-1
6(m
ca
g
tp- Df5
1(k 0)
r17
)
da
f-1
6(m
gD
f
50
)
0
Fig. 6. CATP-1 functions independently of DAF-16/FOXO to
modulate DAF-2/InsR signaling. Survival on 0.75 mM DMPP. No
dauer larvae were observed in daf-2(e1370) under these experimental
conditions. Error bars represent s.e.m. (n⭓3 independent experiments,
N⭓215 individuals) (*P<0.05, Mann-Whitney test).
To test if CATP-1 was interacting with the MAPK pathway, we
used the activated Ras mutation. let-60(n1046gf) individuals were
DMPP sensitive. The activated Ras partially suppressed catp1(kr17) DMPP resistance (Fig. 7B), hence suggesting that catp-1
and let-60 might act in the same pathway to implement DMPP
toxicity. To test if Ras signaling is indeed involved in DAF-2dependent sensitivity to DMPP, we introduced the let-60(n1046gf)
in daf-2 mutants. Interestingly, activation of Ras only weakly
suppresses the DMPP resistance of the daf-2(e1370) mutants
whereas it fully suppresses the resistance of daf-2(m41) mutants
(Fig. 7B). Altogether, these results suggest that both Ras-dependent
and Ras-independent pathways are involved in DAF-2/InsR
signaling during development modulation by DMPP-stimulation of
AchRs. Moreover, CATP-1 would mostly interact with DAF-2/InsR
signaling by modulating a Ras-dependent pathway.
DISCUSSION
In a forward genetic screen for C. elegans mutants able to develop
in the presence of the nicotinic agonist DMPP, we identified catp-1,
a gene coding for an epidermal cation-transporting ATPase. catp-1
is involved in the control of postembryonic developmental timing
and in the decision to enter the dauer diapause stage. These features
likely result from interactions with daf-2/InsR signaling, most
probably through the interaction between CATP-1 and the RasMAPK pathway. By rescuing catp-1(kr17) mutants with ATPasedead versions of CATP-1, we could demonstrate that the
developmental functions of CATP-1 are largely independent of its
putative cation transport function but might rely on scaffolding
properties provided by this transmembrane protein, as recently
proposed for other members of the P-type ATPase family.
CATP-1 regulates L2 developmental timing
We previously demonstrated that illegitimate activation of nAChRs
by DMPP during the second larval stage induced a lethal
heterochronic phenotype by slowing developmental speed without
affecting the molting timer, hence resulting in deadly exposure of a
defective cuticle to the surrounding environment at the subsequent
molt (Ruaud and Bessereau, 2006). Most probably, the defective
RESEARCH ARTICLE
875
cuticle exposed at the L2/L3 molt does not fulfill its diffusion barrier
function and animals dissolve rapidly in a way reminiscent of an
osmotic shock. Different parameters can be modified by
environmental conditions or genetic mutations to cause DMPP
resistance. Some mutants, such as daf-12(0), do not slow
development in the presence of DMPP and thus do not
desynchronize. Alternatively, animals reared on restricted amounts
of food or mutants such as eat-6(lf) possess extended intermolt
periods, hence enabling the compensation of developmental delay.
Finally, some osmotic-stress-resistant mutants like osm-7 (Wheeler
and Thomas, 2006) might survive even with a defective cuticle
(A. F. Ruaud and J. L. Bessereau, unpublished). At first glance, catp1 might function in osmoregulation, osmotic stress sensing or
response to osmotic stress as CATP-1 is expressed in the epidermis
and has a predicted ion transport function. However, we do not favor
this hypothesis. First, catp-1 mutants are as sensitive as wild-type
animals to high osmolarity on 800 mM sodium acetate, in contrast
to osm-7(n1515) (data not shown). Second, catp-1 mutants do not
delay their development in the presence of DMPP. Under
physiological conditions, catp-1(kr17) animals develop at normal
rate during all larval stages, except at the L2 stage which is
considerably extended compared with wild type. L2 lengthening
could then account for the DMPP insensitivity of catp-1 mutants by
occluding the reduction of development speed normally caused by
activation of nicotinic receptors. Interestingly, the two daf-2 alleles
that tested DMPP resistant are also slow growing: at 20°C, daf2(m41) and daf-2(e1370) need one additional day to reach adulthood
compared with wild type (Gems et al., 1998) (A.F.R. and J.L.B.,
unpublished). Part of this delay is due to an extended L2 stage,
which is likely to be different from L2d as dauer formation was
marginal in these conditions (A.F.R. and J.L.B., unpublished). As in
catp-1 mutants, L2 lengthening in daf-2 might account for the
resistance to DMPP.
Signal bifurcation downstream of the C. elegans
DAF-2/InsR
Signaling downstream of the insulin and IGF receptors has been
extensively studied in vertebrates at the cellular and molecular
levels. When activated, these receptor tyrosine kinases (RTKs) are
able to phosphorylate multiple intracellular substrates, including the
insulin receptor substrate proteins (IRS), Shc, Gab-1, Cbl and APS.
Upon tyrosine phosphorylation, each of these substrates can recruit
a distinct subset of signaling proteins containing Src homology 2
(SH2) domains and initiate different signaling pathways, among
which are the Akt/PKB and the MAPK pathways (Lizcano and
Alessi, 2002; Saltiel and Pessin, 2002). Each of these pathways plays
a separate role in the different cellular effects of insulin and IGF-1.
Most of the insulin and IGF receptors transduction machinery
described in vertebrates is conserved in C. elegans. However, the
genetics of dauer formation and aging have defined a linear pathway
for insulin signaling, which consists of elements both necessary and
sufficient for dauer formation and aging under the control of daf-2.
Ultimately, DAF-2/InsR activation leads to the segregation of DAF16/FOXO in the cytoplasm, preventing its interaction with
transcriptional targets. This pathway is required to implement
DMPP toxicity as daf-16(0) suppresses daf-2(e1370) DMPP
resistance (Fig. 8A).
Despite the functional importance of the DAF-16/FOXOdependent pathway for DAF-2/InsR signal transduction,
increasing evidence substantiates the existence of DAF-16independent DAF-2/InsR pathways (Gerisch and Antebi, 2004;
Yu and Larsen, 2001). Our results, together with two recent
DEVELOPMENT
100
WT
% survival (0.75 mM DMPP, 20°C)
CATP-1 controls C. elegans postembryonic developmental timing
876
RESEARCH ARTICLE
B
*
n.s.
(n1
let
-60
tp1
ca
n2
r-1
(
ks
(kr
1
52
6
7)
)
1)
(ku
k-1
mp
04
6g
f)
ca
t
p
le t
-60 -1(kr
(n1 17)
04 ;
6g
f)
da
f-2
(m
41
)
da
let f-2(
-60 m
(n1 41)
04 ;
6)
da
f-2
(e1
37
0)
da
f-2
let
-6 0 (e 1 3
(n 1 7 0 )
04 ;
6)
0
9)
m4
1)
da
f
le t
-60 2(m
(n1 41)
04 ;
6)
da
f-2
(e1
37
0)
da
f-2
let
(
-6 0 e 1 3
(n 1 7 0 )
04 ;
6)
f-2
(
da
le t
-60
(n1
04
6)
0
*
20
98
20
40
(n 1
40
*
60
WT
60
**
80
k-2
80
100
me
% dauer formation (25°C)
100
% survival (0.75 mM DMPP, 20°C)
A
Development 134 (5)
Fig. 7. DAF-2 and CATP-1 interact with the Ras-MAPK signaling pathway. (A) Dauer formation at 25°C scored by visual inspection. Error bars
represent s.e.m. (n⭓3 independent experiments, N⭓72 individuals). let-60(n1046) partially suppresses daf-2(m41) but not daf-2(e1370) constitutive
dauer formation (*P<0.05, Mann-Whitney test). (B) Survival on 0.75 mM DMPP. Error bars represent s.e.m. (n⭓3 independent experiments, N⭓136
individuals). mek-2(n1989), and ksr-1(n2526) are significantly more DMPP resistant than N2. let-60(n1046) strongly suppresses catp-1(kr17) and
daf-2(m41) DMPP resistance (*P<0.05, **P<0.005, Mann-Whitney test).
Dual interaction between catp-1 and a Ras-MAPKdependent DAF-2/InsR pathway
Genetic interaction data indicate that CATP-1 participates in two
processes regulated by DAF-2/InsR signaling during C. elegans
larval development: catp-1 positively interacts with daf-2 in the
control of L2 developmental timing and negatively interacts in the
decision to form dauer larvae. In both cases, CATP-1 function seems
to interact with the Ras-MAPK pathway discussed above. Like daf2 mutants, the DMPP resistance of catp-1 mutants is suppressed by
let-60(gf). However, catp-1(kr17) DMPP resistance was
independent of DAF-16/FOXO, thus indicating an interaction of
CATP-1 with a DAF-16/FOXO-independent DAF-2 pathway (Fig.
8A). Opposite to their interaction in the control of L2 developmental
timing, CATP-1 negatively interacts with LET-60/Ras signaling to
control entry into the dauer diapause, as both catp-1(kr17) and let60(gf) suppress constitutive dauer formation in a daf-2(m41) mutant.
Such dual interaction illustrates the complexity of the network that
integrates insulin/IGF-1 signaling into developmental decision at the
organism level.
One striking feature of the interactions between catp-1, let-60/ras
and daf-2 is the high degree of allele specificity, as previously
reported for many phenotypic traits of the daf-2 mutants. Despite
extensive analysis of multiple daf-2 mutant alleles, the relationship
between the molecular lesions of the DAF-2 receptor and mutant
phenotypes remains poorly understood. In our study, we observed
the strongest interaction with m41, which corresponds to a G383E
mutation in the Cys-rich region of the ectodomain (Yu and Larsen,
2001), outside of the ligand-binding interface (McKern et al., 2006).
Three other mutations in the ectodomain (Kimura et al., 1997; Scott
et al., 2002) show weak (m596) or no (e1368 and m577) genetic
interaction with catp-1(kr17). Similarly, mutants of the kinase
domain display either weak (e1391) or no (e1370) genetic
interaction with catp-1(kr17). If differences in relative levels of
disruption of LET-60/Ras and PI3-kinase signaling account for
phenotypic differences based on genetic data (Nanji et al., 2005), the
cellular and molecular mechanisms at work are unknown. For
example, the m41 mutation in the ectodomain might affect the
binding of one or a group of the many C. elegans insulin-like
peptides that preferentially activate the Ras-MAPK pathway.
Alternatively, the different mutations might cause subtle changes of
the overall receptor activity, and phenotypic differences might arise
from differential coupling with intracellular signaling pathways
among the cells executing insulin/IGF-1-dependent programs. The
multiplicity of the functions of DAF-2 in C. elegans and the
prominence of cell non-autonomous processes has hampered such
analysis so far.
A scaffolding function of CATP-1 for signal
transduction?
Sequence analysis unambiguously identifies CATP-1 as an ␣subunit of the Na+/K+- and H+/K+-pump P-type ATPase family. Four
additional C. elegans genes are predicted to encode closely related
proteins, including eat-6 which codes for a bona fide Na+/K+ATPase ␣-subunit required for proper excitability of pharyngeal
muscle cells (Davis et al., 1995). The three other predicted genes
DEVELOPMENT
studies (Hopper, 2006; Nanji et al., 2005), support the role of the
Ras pathway in DAF-2/InsR signaling during larval development.
First, we demonstrated that activated Ras efficiently suppressed
the constitutive dauer formation of daf-2(m41) mutants but not of
daf-2(e1370), as previously reported. Second, activated Ras was
fully suppressing the DMPP resistance of daf-2(m41) but weakly
suppressed the resistance of daf-2(e1370). Such genetic
interaction data must be interpreted cautiously because we could
not test the interactions between the null alleles of daf-2 and let60/Ras which are both lethal early during development. However,
these results raise the possibility that two signaling branches
bifurcate downstream to DAF-2/InsR, one independent of LET60/Ras and one involving LET-60/Ras. According to this simple
model, the LET-60/Ras-dependent pathway would be prominently
reduced in daf-2(m41) while both pathways would be depressed
in daf-2(e1370) mutants. As suggested by Nanji et al. (Nanji et al.,
2005), such differential alteration of the coupling between DAF2/InsR and the Ras-MAPK pathway might participate in the
phenotypic differences observed between daf-2 mutant alleles as
DAF-2/InsR probably positively regulates LET-60/Ras activity
to control both L2 developmental timing and dauer formation
(Fig. 8).
A
DMPP
CATP-1
?
nAChRs
?
DAF-2
(InsR)
LET-60
(Ras)
[UNC-63+X]
MEK-2
(MEK)
DAF-12
liganded
DAF-16
(FOXO)
L2 cell division
L2/L3 molt
lethal heterochrony
B
CATP-1
?
?
DAF-2
(InsR)
LET-60
(Ras)
DAF-9
(CYP450)
MEK-2
(MEK)
dafachronic
acids
DAF-16
(FOXO)
DAF-12
(NHR)
DAF-12
+
dafachronic
acids
dauer formation
Fig. 8. A genetic model for CATP-1 action in L2 developmental
timing and dauer formation. (A) Control of L2 developmental
timing. CATP-1 speeds up both a cell division and a molting timer
independently of the UNC-63/nAChR and DAF-12/NHR pathways
described in Ruaud and Bessereau (Ruaud and Bessereau, 2006). daf-2
mutant DMPP resistance probably results from a similar developmental
delay and involves both the DAF-16/FOXO and Ras-MAPK pathways.
CATP-1 effect on developmental timing likely involves a Ras-MAPK
branch of the daf-2 pathway. Whether catp-1 directly interferes with
daf-2/InsR, modulates Ras/MAPK activity and/or functions through a
third unidentified parallel pathway remains equally possible at this
stage. Dashed lines: hypothetical interactions. (B) Dauer formation. In
addition to the DAF-16/FOXO and DAF-12/NHR pathways, DAF-2/InsR
controls dauer formation through a Ras-MAPK pathway. CATP-1 is
likely to modulate dauer formation by negatively interacting with this
Ras-MAPK branch, but could also directly alter daf-2/InsR signaling or
work through an uncharacterized parallel pathway (see the text for a
full discussion).
RESEARCH ARTICLE
877
have not been analyzed. The primary function of these ATPases is
to maintain ionic gradients across plasma membranes and
intracellular homeostasis. By sequence comparison, CATP-1 is
equally distant from Na+/K+- and H+/K+-ATPases, thus precluding
a prediction of which ions might be transported by this protein.
However, our results suggest that the identified functions of CATP1 are mostly independent from its transport function, as recently
proposed for some signaling functions of the vertebrate Na+/K+ATPase in response to cardiac glycosides (Schoner, 2002).
Cardiac glycosides are the most widely employed therapy for
cardiac insufficiency. They block the Na+/K+-ATPase, which affects
a wide range of cellular phenomena (Kaplan, 2002; Xie and Askari,
2002). Those effects have long been thought to result from the
change in cell Na+ content, which can lead to changes in intracellular
pH (via the Na+/H+ exchange system), or intracellular Ca2+ (via the
Na+/Ca2+ exchange system) (Kaplan, 2002; Scheiner-Bobis and
Schoner, 2001). Such ionic changes would subsequently initiate
various signaling responses. However, disrupting Na+/K+-ATPase
function has also been reported to modulate several classical
intracellular signaling pathways such as ERK and Src pathways in
different lines of cultured cells with no obvious alteration of
intracellular cation concentrations (reviewed by Xie and Askari,
2002). Recent evidence substantiates a direct interaction of the
Na+/K+-ATPase with intracellular signaling machineries,
independently of its pump function. The cytoplasmic region of the
Na+/K+-ATPase was shown to interact in vitro and in vivo with the
soluble tyrosine kinase Src. This interaction would maintain Src in
an inactive form. Ouabain, an inhibitor of the Na+/K+-ATPase, is
proposed to cause Src release and activation of Src and downstream
effectors such as ERK and FAK. Interestingly, the inhibition of Src
activity could be achieved with a pump-dead mutant as efficiently as
with the wild-type protein (Liang et al., 2006; Tian et al., 2006). In
our study, we mutated a CATP-1 residue which is essential for the
catalytic cycle, similar to the Liang study, or a residue absolutely
required for ATP binding, hence catalytic activity. Both mutant
proteins were able to partially rescue catp-1(kr17) defects. Partial
rescue could be explained by some contribution of the pump
function. Alternatively, it might result from inappropriate expression
in transgenic animals or from additional modifications of CATP-1
properties in the mutant forms. In any case, our data provide
evidence that CATP-1 can function independently of its pump
function to regulate L2 developmental timing.
Although a scaffolding function has been postulated for the
Na+/K+-ATPase at septate junctions in the Drosophila tracheal
system (Genova and Fehon, 2003; Paul et al., 2003), this is to our
knowledge the first in vivo demonstration of a signaling function of
a P-type ATPase that is independent of its pump function. Whether
CATP-1 interacts directly of indirectly with the Ras-MAPK and
DAF-2/InsR machinery remains equally possible at this stage.
However, it is tempting to speculate that the cytoplasmic region of
CATP-1 could serve as a membrane-bound docking platform to
recruit components or modulators of the Ras-MAPK and/or DAF2/InsR signaling pathways. The multiple developmental phenotypes
of catp-1 mutants offer an interesting opportunity to dissect the
mechanisms underlying signaling pathway modulation by cationtransporting ATPases in vivo.
We thank Ian Johnstone for the gift of a Pdpy-7 plasmid. Some nematode
strains used in this work were provided by the Caenorhabdidtis Genetics
Center, which is funded by the NIH National Center for Research Resources
(NCRR). B. Matthieu is thanked for help with confocal microscopy, H. Gendrot
for technical help, M.A. Félix, M. Labouesse, I. Katic and V. Robert for critical
reading of the manuscript. A.F.R. was supported by a fellowship from the
DEVELOPMENT
CATP-1 controls C. elegans postembryonic developmental timing
RESEARCH ARTICLE
Ministère de la Recherche and by the Association pour la Recherche contre le
Cancer. This work was funded by an AVENIR grant from the Institut National
de la Santé et de la Recherche Médicale and by the ACI BDPI from the
Ministère de la Recherche.
Note added in proof
After this manuscript was accepted, Paul et al. reported an in vivo
pump-independent function of the Na,K-ATPase for epithelial
junction function in Drosophila (Paul et al., 2007).
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/134/5/867/
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DEVELOPMENT
CATP-1 controls C. elegans postembryonic developmental timing