1 Department of Genetics, University of Pennsylvania, Philadelphia, PA 19104,
USA
2 UCMP, Umea University, SE-901 87 Umeå, Sweden
3 Department of Biological Sciences, Simon Fraser University, Burnaby B.C. V5A
1S6, Canada
Author for correspondence (e-mail:
sundaram{at}mail.med.upenn.edu)
Accepted 4 November 2003
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SUMMARY |
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Key words: PP2A, PR55, Raf, PAR-1, Vulva, C. elegans
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Introduction |
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PP2A is a heterotrimeric serine/threonine phosphatase composed of invariant
catalytic (`C') and structural (`A') subunits and a variable regulatory
subunit (`B') that directs the AC core complex to different substrates
(Janssens and Goris, 2001).
PP2A both positively and negatively influences the Ras/MAP kinase pathway in
Drosophila and in mammalian cells, suggesting it may act on multiple
Ras pathway substrates (Sontag et al.,
1993
; Alessi et al.,
1995
; Wassarman et al.,
1996
; Maixner et al.,
1998
; Ugi et al.,
2002
; Strack,
2002
; Silverstein et al.,
2002
). Unfortunately, the pleiotropic defects caused by
interfering with PP2A activity in vivo and the very broad substrate
specificity of PP2A in vitro have hampered attempts to identify its most
functionally relevant substrates. The finding that partial loss-of-function
alleles of sur-6/B specifically reduce Ras signaling in C.
elegans (Sieburth et al.,
1999
) provides a potentially simpler genetic model system for
studying the effects of PP2A on the Ras pathway.
C. elegans vulval development is a well characterized model system
for studying the Ras signaling pathway
(Moghal and Sternberg, 2003).
The vulva is generated by a specialized subset of ventral ectodermal blast
cells called vulval precursor cells (VPCs)
(Fig. 1A). During larval
development, Ras signaling induces three of six equipotent VPCs to execute a
vulval lineage. The remaining three uninduced VPCs execute a non-vulval
hypodermal lineage. The EGF receptor/Ras/MAP kinase pathway
(Fig. 1B) is required for
vulval induction, as complete loss of pathway activity causes a Vulvaless
(Vul) phenotype in which no VPCs adopt vulval fates. Increased Ras activity
causes a Multivulva (Muv) phenotype in which greater than three VPCs adopt
vulval fates. Thus, the extent of vulval differentiation provides a sensitive
readout of Ras signaling levels. Other signaling pathways, including a
Wnt/ß-catenin pathway, independently influence vulval fate induction and
can also mutate to cause partial Vul or Muv phenotypes
(Eisenmann et al., 1998
;
Gleason et al., 2002
)
(Fig. 1B).
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The molecular identity of SUR-6 suggests a role for PP2A in modulating Raf
phosphorylation. Indeed, in mammalian cells, PP2A has been proposed to
activate Raf by removing inhibitory phosphates from the Raf N terminus
(Abraham et al., 2000;
Jaumot and Hancock, 2001
;
Dhillon et al., 2002
;
Kubicek et al., 2002
).
Phosphorylation of Raf-1 Ser259 by Akt and/or related serine/threonine kinases
inhibits Raf activity (Zimmermann and
Moelling, 1999
; Rommel et al.,
1999
; Guan et al.,
2000
; Zhang et al.,
2001
). PP2A may dephosphorylate Raf-1 on Ser259 as one step in Raf
activation. In support of this model, the PP2A catalytic subunit physically
associates with Raf-1 (Abraham et al.,
2000
), Raf-1 Serine 259 phosphorylation increases upon treatment
with the PP2A inhibitor okadaic acid
(Abraham et al., 2000
), and
mutation of Ser259 to Ala in Raf-1 increases Raf kinase activity above basal
levels (Michaud et al., 1995
;
Rommel et al., 1996
;
Clark et al., 1997
). B-Raf and
C. elegans LIN-45 RAF may be regulated in a similar manner to Raf-1
because each has multiple consensus Akt phosphorylation sites and mutation of
these sites elevates their activities
(Chong et al., 2001
). Although
the above experiments did not address which B regulatory subunit complexes
with PP2A to target Raf, the data are consistent with a model in which
SUR-6/PR55 and PP2A remove inhibitory phosphates from LIN-45 RAF. This model
predicts that the PP2A catalytic subunit should also promote Ras signaling in
C. elegans, and that mutating the candidate target sites on LIN-45
RAF should eliminate the requirement for SUR-6.
We analyze the effects of null mutations in sur-6 PR55/B and let-92 PP2A-C and provide support for the model that SUR-6 and PP2A cooperate to promote Raf activity. However, we find that mutating both consensus Akt phosphorylation sites in LIN-45 RAF does not eliminate the requirement for SUR-6. Therefore, SUR-6/PP2A does not act solely by dephosphorylating those inhibitory sites. We also provide genetic evidence that KSR activity is intact in sur-6 mutants, and that the kinase PAR-1 functions antagonistically to SUR-6 and KSR-1 during Ras-mediated vulval induction.
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Materials and methods |
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LGI: unc-13(e51), sur-6(ku123) and sur-6(cs24)
(Sieburth et al., 1999);
mek-2(h294), mek-2(ku114), pry-1(mu38), ksr-2(dx27)
(Ohmachi et al., 2002
) and
hT2[qIs48] (Wang and Kimble,
2001
).
LGIII: mpk-1(ku1) and unc-119(ed3). LGIV:
let-60(n1046gf), let-60(n2031dn), let-60 (sy100dn), lin-45(ku112)
(Rocheleau et al., 2002),
lin-45(oz166) and lin-45(dx19)
(Hsu et al., 2002
);
sur-8(ku167) (Sieburth et al.,
1998
), let-92(s504), let-92(s677), unc-22(s7), unc-22(e66),
dpy-20(e1282), dpy-20(e1362), him-8(e1489), lip-1(zh15)
(Berset et al., 2001
),
nT1[qIs5].
LGV: him-5 (e1490), let-341(cs41)
(Rocheleau et al., 2002),
par-1(b274), par-1(zu310ts), rol-4(sc8), unc-76(e911), gaIs36
(Lackner and Kim, 1998
).
LGX: lon-2(e678), ksr-1(n2526) and sem-5(n1779).
Isolation of the sur-6(sv30) deletion allele
A deletion library of N2 worms mutagenized with ethyl methane sulfonate
(Jansen et al., 1997) was
screened for deletions in the sur-6 gene. Pooled genomic samples
representing a total of 400,000 haploid genomes were used as templates in PCR
reactions with two primers with the sequences 5'-CGG AGG ACA GCT GAT AAG
TAA GAG GTT C-3' and 5'-GAT GTA GAG ATT GTT AGT GGC AGC AAG
AG-3'. A small amount of each reaction was used as template for a second
round of PCR with the primers 5'-GAA GTT CTT CTC TGC GTG ATC GCA
TAC-3' and 5'-GAA GTT GAT CAG ATG AAA GAT CCT CTT CG-3'. The
pool of worms containing the sur-6 deletion was thawed and used to
establish cultures from individual worms, from which individual heterozygous
animals were identified. Sequence analysis showed that the sv30
deletion removes 1.8 kb of the sur-6-coding region; it extends from
exon 2 to intron 8, eliminating at least five out of the seven WD40 repeats in
the SUR-6 protein. PCR experiments confirmed that a wild-type copy of the
sur-6 gene was not present elsewhere in the genome.
The sv30 strain was outcrossed by crossing six times with wild type and by selecting for recombinants on LGI. During this procedure, a second mutation that increased the penetrance of the Vul phenotype of the strain was identified and genetically removed.
Phenotypic characterizations
Vulval development was scored in early to mid fourth larval stage (L4)
animals using differential interference contrast (DIC) microscopy. Animals
with fewer than 22 vulval descendants (with losses in increments of three or
four cells) and greater than six non-vulval descendants were scored as
vulvaless (Vul). Animals with more than 22 vulval descendants (with gains in
increments of three or four cells) and fewer than six non-vulval descendants
were scored as multivulva (Muv). To calculate the number of induced vulval
precursor cells (VPCs) each normal lineage was given a value of 1.0 and each
partial lineage was given a value of 0.5, so that wild-type animals have a
value of 3.0, Vul animals have a value less than 3.0 and Muv animals had a
value greater than 3.0.
Embryonic lethality was assessed by allowing hermaphrodites to lay eggs for 12-18 hours and then counting unhatched eggs 24 hours later.
Soaking RNAi
Embryos were allowed to develop on plates until most of them were at a
point just before vulval development starts. The larvae were washed and
200 larvae were mixed in an Eppendorf tube with 1 mg/ml of the
appropriate dsRNA and OP50 bacteria at OD595 of 1.0 in a 40 µl
volume followed by incubation at 20°C with gentle rotation for 24 hours.
The larvae were pipetted onto seeded plates and examined for vulval
development by differential interference contrast microscopy.
Immunostaining
Embryo immunostaining was performed by the freeze/crack method followed by
methanol/acetone fixation (Miller and
Shakes, 1995). Fixed embryos were incubated at 37°C for 1 hour
with 1:100 dilution of YL1/2 rat anti-
-tubulin (Accurate Chemical &
Scientific). Samples were washed twice with PBS+2% Tween-20 and incubated at
37°C for 30 minutes with 1:100 dilution of Cy3 conjugated donkey-anti rat
IgG (Jackson Immuno Research) and washed three times as before. DAPI
(4',6-diamidino-2-phenylindole) was added to the penultimate wash at 0.5
mg/ml. Excess liquid was wiped off and a coverslip containing 5 µl of
mowiol mounting medium was placed over the slide.
Western blotting
Worm lysates from 25-100 L4 animals were separated on 7.5% SDS-PAGE gels
and transferred onto Hybond nitrocellulose (Amersham). Blots were probed with
antibodies against di-phosphorylated MAPK (MAPK-YT, Sigma, 1:2500 dilution) or
total MAPK (K23, Santa Cruz, 1:200 dilution) overnight at 4°C before
incubation with horseradish peroxidase-conjugated secondary antibodies
(Jackson Immuno Research) for 1 hour at room temperature and exposure to West
Pico chemiluminescent substrate (Pierce). Membranes were stripped before
reprobing with the second primary antibody.
Rescue of let-92 larval lethality
A 5.2 kb genomic fragment from the F38H4.9 locus containing 3 kb of
promoter sequence and 1 kb of predicted 3'UTR was amplified from N2
genomic DNA and cloned into pBluescript II (SK+) as a
NotI/KpnI fragment to generate pGK182. Transgenic lines were
generated in wild-type animals by co-injecting pGK182 at 10 ng/µl along
with pTG96_2 (sur-5::GFP) (Yochem
et al., 1998) at 30 ng/µl. Transgenic males were crossed with
let-92(s504) unc-22 (s7)/DnT1 hermaphrodites. All GFP (+) Unc-22
progeny grew up to adulthood indicating rescue of the let-92 (s504)
larval arrest phenotype. Rescued animals were sterile indicating a role for
let-92 in the germline that could not be rescued as most C.
elegans transgenes are transcriptionally silent in the germline
(Kelly et al., 1997
). The
sterile phenotype of the rescued animals was similar to the sterile phenotype
of wild-type larvae soaked in let-92 dsRNA. Vulva development was
normal in the rescued animals.
Site directed mutagenesis of lin-45
Point mutations were introduced by PCR into the lin-45 cDNA and
the mutated versions were cloned into pPD49.83 (kindly provided by A. Fire) as
NheI/NcoI fragments to put them under control of the
hsp16-41 promoter (Stringham et
al., 1992). Mutagenic primers oMS103 (5'-GAT CGG AGC TCT GCT
GCT CCG AAT ATC-3') and oMS104 (5'-GAT ATT CGG AGC AGC AGA GCT CCG
ATC-3') were used on the lin-45 cDNA clone pRaf107a to change
the codon for Ser312 to Ala in pGK167. Mutagenic primers oGK90 (5'-CGT
AGT CGA GCG CCA GGC GAA CG-3') and oGK91 (5'-CGT TCG CCT GGC GCT
CGA CTA CG-3') were then used to change Ser453 to Ala in pGK209. The
lin-45 inserts were completely sequenced to verify that the only
changes were the desired ones. lin-45(+) cDNA was cloned into
pPD49.83 to generate pGK170.
Generation of transgenic animals
A mixture of pGK167 (100 ng/µl) and pPD#MM016 (unc-119+)
(Maduro and Pilgrim, 1995) (30
ng/µl) was injected into unc-119(ed3) animals. Stable
extrachromosomal array lines that gave a robust Muv phenotype after heat-shock
were kept. One such line was irradiated with 1800 rads of X-rays to integrate
the array into the genome. One line carrying the insertion csIs34 on
the X chromosome was outcrossed four times with wild type before use in strain
construction. pGK167 was injected at 20 ng/µl along with
punc-119:GFP at 100 ng/µl to yield csEx2.
pGK209 was injected into N2 animals at 20 ng/µl along with pTG96_2
(sur-5::GFP) at 30 ng/µl and pBluescript at 50 ng/µl to yield
transgenic lines csEx52 and csEx53. pMS88 containing
hsp16-41::torso4021-Draf
(Sieburth et al., 1998) was
similarly injected to yield csEx64. Stable transgenes were crossed
into the desired genetic backgrounds using standard genetic methods.
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Results |
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To further characterize sur-6, we generated a deletion mutation, sv30 (Materials and methods). As predicted for a null allele, sv30 causes maternal effect embryonic lethality (Table 1). This embryonic lethality could not be rescued by mating sv30 hermaphrodites with wild-type males (data not shown). The deficiency qDf8, which removes sur-6, fails to complement sv30 for the maternal effect lethal phenotype (Table 1). In addition, the sv30/qDf8 phenotype was not more severe than that of sv30/sv30 homozygous animals (Table 1) supporting the notion that sv30 is a genetic null. The sur-6(cs24) allele complements sv30 for the maternal effect lethal phenotype (Table 1), indicating that cs24 does not significantly perturb the essential function of sur-6 (see Discussion).
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let-92(RNAi) embryos showed catastrophic failures in cytokinesis
during the earliest cell divisions (data not shown). These defects are more
severe than those seen in sur-6 mutant embryos, consistent with
studies in yeast and mammals that suggest the catalytic subunit acts in
concert with multiple regulatory subunits to participate in distinct cellular
and developmental events (Janssens and
Goris, 2001). Although the sur-6 and let-92
embryonic arrest phenotypes are distinct, a let-92 loss-of-function
mutation shows strong dominant synthetic lethal interactions with both viable
sur-6 missense alleles (Table
3), suggesting that sur-6 and let-92 do function
together during embryogenesis.
|
Our previous experiments suggested a positive role for let-92
because, like sur-6(RNAi), let-92(RNAi) could partially suppress the
let-60(gf) Muv phenotype
(Sieburth et al., 1999). We
also found that let-92(RNAi) and sur-6(RNAi) caused similar
weak synthetic Vul phenotypes in sur-8 mutant larvae
(Table 4), further supporting a
positive role for let-92.
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Based on these experiments, we conclude that let-92, like sur-6, plays a positive role in vulval fate induction. We find no evidence for a negative role. Thus, sur-6 most probably promotes PP2A activity toward a Ras pathway substrate. If so, SUR-6/PP2A either removes inhibitory phosphates from a positively acting substrate or removes stimulatory phosphates from a negatively acting substrate.
sur-6functions independently of inhibitory phosphorylation sites on LIN-45 RAF
We tested whether SUR-6/PP2A acts by removing inhibitory phosphates from
LIN-45 RAF. Mammalian Raf-1 has a single consensus Akt phosphorylation site
(Serine 259) that is subject to inhibitory phosphorylation and that PP2A has
been proposed to dephosphorylate (Chong et
al., 2003). C. elegans LIN-45 RAF has two consensus Akt
phosphorylation sites (Ser312 and Ser453) that appear to function analogously
to the single Raf-1 Ser259 site (Chong et
al., 2001
). We mutated both serines to alanine and expressed the
mutant LIN-45 proteins under the control of the heat shock promoter
hsp16-41 (Stringham et al.,
1992
). When overexpressed in this manner, LIN-45+ had
no apparent effect (data not shown). However, LIN-45S312A caused a
moderate Muv phenotype and LIN-45S312A S453A caused a somewhat
stronger Muv phenotype (Table
2C, Fig. 3E). These
results are similar to those reported previously
(Chong et al., 2001
), except
that those authors saw a Muv phenotype with LIN-45S312A S453A but
not LIN-45S312A, possibly because of lower expression levels. Taken
together, our results are consistent with the model that both Ser312 and
Ser453 in LIN-45 RAF are sites of inhibitory phosphorylation.
If Ser312 and Ser453 in LIN-45 RAF are the relevant targets of SUR-6/PP2A during vulval development, then mutation of those serines to alanine should eliminate the need for sur-6. However, the Muv phenotypes of LIN-45S312A and LIN-45S312A S453A still required sur-6 (Table 2C, Fig. 4E,F). This requirement for sur-6 cannot be explained by an effect on endogenous LIN-45+ as LIN-45S312A S453A produced a potent Muv phenotype even in a strong loss-of-function lin-45(dx19lf) mutant background (Table 2C). Because removing both inhibitory sites did not eliminate the requirement for sur-6, SUR-6 must promote LIN-45 RAF activity via a mechanism distinct from dephosphorylating those sites. SUR-6/PP2A may regulate LIN-45 RAF through as yet unidentified phosphorylation sites, or it may regulate LIN-45 RAF indirectly by targeting other Raf regulatory proteins.
sur-6functions independently of ksr-1or ksr-2
The putative scaffold protein KSR is a positive Raf regulator whose
function can be inhibited by phosphorylation
(Muller et al., 2001), making
it another candidate PP2A substrate. Furthermore, KSR and SUR-6 appear to act
at a similar step of Raf activation, as ksr-1 also suppresses the Muv
phenotype caused by LIN-45S312A S453A but not
Torso4021-Draf (Table
2B,C) (Sieburth et al.,
1999
). C. elegans has two ksr genes,
ksr-1 and ksr-2, that are redundantly required for viability
and vulval development (Ohmachi et al.,
2002
). Although ksr-1 and ksr-2 single mutants
are viable and have normal vulvae, the mutants are very sensitive to further
reductions in KSR activity (Ohmachi et
al., 2002
). Therefore, if sur-6 mutations reduce KSR
activity, we would expect to see a strong genetic interaction between a
sur-6 null mutation and ksr-1 or ksr-2. Contrary to
this prediction, ksr-1 and ksr-2 mutations each failed to
enhance sur-6(sv30) Vul defects
(Table 2A). These results
suggests that KSR activity is relatively intact in sur-6 mutants.
Therefore, we do not favor the model that KSR is a key substrate of
SUR-6/PP2A.
The PAR-1 kinase acts antagonistically to SUR-6 and KSR during vulval development
The serine/threonine kinase C-TAK1/PAR-1 has been identified biochemically
as an inhibitor of mammalian KSR (Muller
et al., 2001). Most C. elegans par-1 mutants have
wild-type vulval fate specification (Hurd
and Kemphues, 2003
). However, we find that
par-1(b274lf)/par-1(zu310ts) transheterozygotes (in which both
maternal and zygotic par-1 activities are reduced) are weakly Muv
(Table 5). Additionally we find
that reducing par-1 function strongly reverts the suppressed
(non-Muv) phenotype of sur-6(ku123);let-60(n1046gf) and
let-60(n1046gf);ksr-1(n2526) double mutants
(Table 5). By contrast,
reducing par-1 function does not revert the suppressed (non-Muv)
phenotype of lin-45(ku112) let-60(n1046gf). Therefore par-1
has an inhibitory role in vulval development, probably acts upstream or
parallel to lin-45 raf, and functions antagonistically to
sur-6/PP2A and ksr-1.
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Discussion |
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Positive versus negative roles for PR55/PP2A in Ras signaling
In other systems, PR55/B and PP2A have been found to have both positive and
negative effects on Ras signaling. For example, in Drosophila a
positive role for PR55/PP2A is supported by findings that mutations in
tws/PR55 suppress the lethality caused by activated sevenless
receptor and activated Ras (Maixner et
al., 1998), and mutations in the PP2A catalytic subunit enhance
photoreceptor defects caused by a hypomorphic Draf allele
(Wassarman et al., 1996
).
However, a negative role for PR55/PP2A is supported by findings that RNAi
against tws/PR55 elevates the level of phospho-ERK in cultured S2
cells (Silverstein et al.,
2002
), and mutations in the PP2A catalytic subunit enhance
photoreceptor defects caused by activated Ras
(Wassarman et al., 1996
).
Thus, in Drosophila the role of PR55/PP2A appears complex, and PP2A
may act on multiple substrates within the Ras pathway. Similarly, in mammalian
cells PP2A has been suggested to positively regulate Ras signaling by removing
inhibitory phosphates from Raf (Abraham et
al., 2000
; Jaumot and Hancock,
2001
; Dhillon et al.,
2002
; Kubicek et al.,
2002
) and to negatively regulate Ras signaling by removing
activating phosphates from MEK or ERK
(Alessi et al., 1995
). By
contrast, we find no evidence for a negative role of SUR-6/PR55 or LET-92/PP2A
in C. elegans, despite having tested sur-6 and
let-92 mutations in numerous genetic backgrounds. Therefore either
PP2A lacks a negative role in C. elegans, or its negative role is
masked by its stronger positive role.
SUR-6/PP2A as a positive regulator of LIN-45 RAF activity
Prior studies of SUR-6 and its genetic placement between (or in parallel
to) Ras and Raf relied on partial loss-of-function sur-6 alleles
(Sieburth et al., 1999). Our
characterization of a sur-6 null mutation, sv30, is
consistent with the prior studies but clarifies several previously unresolved
points. First, our data suggest that SUR-6 promotes Ras signaling but is not
absolutely essential for Ras signaling under normal circumstances. Therefore,
SUR-6/PP2A may dephosphorylate a site that has modest effects on substrate
activity, or SUR-6/PP2A function may be partly redundant with that of another
phosphatase complex. Second, our finding that sur-6(sv30) reduces the
levels of activated MPK-1 ERK in vivo argues that SUR-6 acts upstream of
rather than in parallel to MEK and ERK. SUR-6 could still act either upstream
or in parallel to LIN-45 RAF. Finally, our data dispute two of the prevailing
models for SUR-6 function.
Like mammalian Raf proteins, LIN-45 RAF appears to be inhibited by
phosphorylation on sites that match the consensus sequence for the Akt kinase
(Chong et al., 2001). To date
we have not seen effects of akt-1 or akt-2 RNAi on Ras
signaling (G.K. and M.V.S., unpublished), so it is unclear whether Akt itself
or some other kinase(s) normally phosphorylates these LIN-45 RAF inhibitory
sites. Although a simple and attractive model was that SUR-6 and PP2A
dephosphorylate these LIN-45 RAF inhibitory sites, our data are inconsistent
with that being their sole mechanism of action. We found that
sur-6(sv30) suppresses the Muv phenotype caused by LIN-45S312A
S453A, which lacks both presumptive inhibitory Akt phosphorylation
sites, indicating that SUR-6 must promote LIN-45 RAF activity independently of
those sites. One possibility is that SUR-6/PR55 and PP2A dephosphorylate
LIN-45 RAF on other inhibitory sites; however, no such sites have been
identified as yet. An alternative possibility is that SUR-6 and PP2A
indirectly influence LIN-45 RAF by dephosphorylating some other Raf regulatory
protein(s).
A second proposed model was that SUR-6 and PP2A regulate the scaffold
protein KSR (Sieburth et al.,
1999; Ory et al.,
2003
). KSR may regulate Raf at a similar step as SUR-6, as
ksr-1 mutations also suppress the Muv phenotype caused by
LIN-45S312A S453A but not Torso4021-Draf. However, as
discussed above, the failure of sur-6 mutations to genetically
interact with ksr-1 or ksr-2 suggests that KSR activity is
relatively intact in sur-6 mutants. Therefore, KSR is unlikely to be
the sole SUR-6/PP2A substrate. It remains possible that SUR-6/PP2A has
multiple substrates, and that LIN-45 RAF and/or KSR are among these, but our
data argue that another important substrate(s) remains to be identified.
The kinase PAR-1 acts in opposition to PP2A
Murine C-TAK/PAR-1 has been suggested to phosphorylate KSR to modulate KSR
localization (Muller et al.,
2001). The relevant phosphorylation site is not conserved in
C. elegans KSR-1 or KSR-2, although related sites are present
elsewhere. PAR-1 can also phosphorylate Raf as well as other substrates
(Benton et al., 2002
) and
therefore could have broader roles in Ras signaling. C. elegans par-1
has been previously found to play a role in vulval morphogenesis
(Hurd and Kemphues, 2003
). We
found that par-1 also plays an inhibitory role in vulval fate
specification. This inhibitory role of par-1 is partly masked by
perdurance of maternally provided gene product, but could be seen in animals
in which both maternal and zygotic par-1 contributions were
diminished, as well as in some sensitized genetic backgrounds. We found that
zygotic removal of par-1 reverts the suppressor of ras (gf)
phenotypes of sur-6 and ksr-1 but not lin-45 raf,
consistent with models where PAR-1 acts on KSR, LIN-45 RAF or both to inhibit
vulval development. Our results also raise the possibility that SUR-6/PP2A
inhibits PAR-1 to indirectly affect LIN-45 RAF activity.
Genetically separable functions for SUR-6/PR55 in Ras signaling and mitotic progression
The sur-6 maternal effect lethal phenotype reveals that in
addition to Ras signaling, sur-6 is required for mitotic progression.
sur-6(sv30) and sur-6(RNAi) embryos display a variety of
mitotic defects such as ectopic and aberrant cytokinesis, the collapse and
re-elaboration of well-extended anaphase spindles, abnormally shaped spindles
and chromatin bridges during anaphase. Similar mitotic defects have been
observed in Drosophila tws/PR55 mutants
(Gomes et al., 1993;
Mayer-Jaekel et al., 1993
).
Premature sister chromatid separation and cytokinesis defects have also been
observed in S. cerevisiae cdc55/PR55 mutants
(Minshull et al., 1996
;
Wang and Burke, 1997
). Thus,
the mitotic role of PR55 appears to be evolutionarily conserved. The early
C. elegans embryo is a particularly tractable system for further
study of this poorly understood mitotic role of PR55.
Interestingly, the original two missense alleles of sur-6, ku123
and cs24, behave similarly to the sur-6 null allele
sv30 with respect to Ras signaling but do not cause mitotic defects
or embryonic lethality even when placed in trans to sur-6(sv30) or a
deficiency of the sur-6 locus as shown here and previously
(Sieburth et al., 1999). Thus,
the functions of sur-6 in Ras signaling and mitotic progression are
genetically separable. The cs24 allele causes an E to K change at an
absolutely conserved site of the third WD repeat of SUR-6/PR55
(Sieburth et al., 1999
).
Equivalent mutations in the human PR55 protein have been shown to severely
compromise binding of PR55 to the A subunit of PP2A, preventing PR55
association with the catalytic core
(Strack et al., 2002
). Thus,
cs24 would be predicted to similarly compromise the interaction of
SUR-6 with the PP2A core. As cs24 is nearly wild type for mitotic
function but nearly null for Ras signaling function, one possible model is
that SUR-6 acts independently of the A and C subunits during mitosis but acts
with the A and C subunits during Ras signaling. An alternative model is that
SUR-6 acts with the A and C subunits during both processes, but that only very
low levels of SUR-6/PP2A are required for mitotic function. The latter model
is supported by the fact that maternally rescued sur-6(sv30) mutants
show few or no mitotic defects during larval development (presumably owing to
perdurance of low levels of maternal product), and by the fact that reducing
the let-92/PP2A dose by half causes synthetic embryonic lethality in
both sur-6 missense backgrounds. Thus, there appear to be different
threshold requirements for the various roles of SUR-6-associated PP2A, with
Raf activation being most sensitive to reductions in sur-6
activity.
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Conclusions |
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ACKNOWLEDGMENTS |
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Footnotes |
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REFERENCES |
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