1 Department of Biology, Queens College, and the Graduate School and University
Center, the City University of New York, 65-30 Kissena Boulevard, Flushing, NY
11367, USA
2 Department of Genetics and Developmental Biology, University of Connecticut
Health Center, 263 Farmington Avenue, Farmington, CT 06030, USA
* Author for correspondence (e-mail: csavage{at}qc1.qc.edu)
Accepted 6 June 2005
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SUMMARY |
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Key words: Body size, Pattern formation, Phosphorylation, Smad, TGF-ß, C. elegans
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Introduction |
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Two serines/threonines at the C terminus of R-Smad proteins (consensus
sequence SS*XS*) are the substrates of receptor phosphorylation
(Abdollah et al., 1997;
Souchelnytskyi et al., 1997
).
Phosphorylation is thought to relieve autoinhibition of the MH2 domain by the
MH1 domain, allowing Smad complexes to form and translocate to the nucleus
(Hata et al., 1997
). The
crystal structures of homomeric complexes containing Smad1 or phosphorylated
Smad2 (pSmad2), and of heteromeric complexes containing pSmad2:Smad4 or
pSmad3:Smad4, have provided insight into the structural basis of complex
formation (Qin et al., 2001
;
Wu et al., 2001b
;
Chacko et al., 2004
). The
crystal structures show that the phosphorylated C terminus of each R-Smad
subunit binds a positively charged pocket of another subunit
(Wu et al., 2001b
;
Chacko et al., 2004
). Other
studies have shown that pseudophosphorylation of R-Smads by the substitution
of negatively charged amino acids for C-terminal serines can result in their
activation independently of exogenous ligand
(Liu et al., 1997
;
Petritsch et al., 2000
;
Chacko et al., 2001
;
Qin et al., 2001
). Somewhat
paradoxically, in other contexts, pseudophosphorylated R-Smads (ppR-Smads)
were found to have dominant-negative effects
(Liu et al., 1997
;
Souchelnytskyi et al., 1997
;
Petrisch et al., 2000). Nevertheless, the functional consequences of Smad
phosphorylation have not been extensively addressed in any intact
organism.
The core TGF-ß signaling components have been identified in
Caenorhabditis elegans, providing a genetically tractable model
system for their study. There are multiple TGF-ß-related pathways that
control different events in C. elegans development. The Sma/Mab and
Dauer pathways regulate body size and dauer formation, respectively
(Patterson and Padgett, 2000;
Savage-Dunn, 2001
). These two
pathways share one type II receptor, DAF-4, but the type I receptors, SMA-6
and DAF-1, are pathway specific. When daf-4 is transformed into human
cell lines, the C. elegans receptor protein binds vertebrate BMP
ligands showing conservation between these pathway components in human and
C. elegans (Estevez et al.,
1993
). Loss of function of the Sma/Mab pathway results in a small
body size. Furthermore, in the male tail, some of the sensory rays become
fused and the spicules are crumpled
(Savage et al., 1996
). A model
for Sma/Mab pathway signaling has been developed on the basis of analogy with
biochemical experiments in other systems. The ligand DBL-1 interacts with the
type II receptor DAF-4 and the type I receptor SMA-6. The intracellular Smad
homologs SMA-2, SMA-3 (R-Smads), and SMA-4 (Co-Smad), propagate the signal,
presumably by forming complexes that accumulate in the nucleus. In the
nucleus, the Smads may interact with tissue-specific transcription co-factors,
such as LIN-31 (Baird and Ellazar,
1999
) and SMA-9 (Liang et al.,
2003
). We have previously shown that sma-3 is expressed
in the pharynx, intestine and hypodermis. Expression in the hypodermis is
necessary and sufficient to regulate body size. Also, pathway activity
activates a negative-feedback loop that reduces SMA-3 protein accumulation
(Wang et al., 2002
).
We have noted that SMA-3 does not function well with GFP attached to the C
terminus, suggesting that the C terminus of SMA-3 is crucial for its activity
(Wang et al., 2002). As the C
terminus is likely to be the site of phosphorylation, we reasoned that
mutating C-terminal residues could provide an opportunity to investigate the
roles of Smad phosphorylation in vivo. We have therefore generated
C-terminally mutated sma-3 genes by site-directed mutagenesis. Our
results indicate that Smad phosphorylation at the C terminus is not required
for nuclear accumulation, but is probably required for Smad complex formation
and for interaction with transcription cofactors. We also find that different
developmental processes show a differential tolerance for Smad C-terminal
modification. These differences may be due in part to the need for interaction
with tissue-specific transcription co-factors that have different affinities
for unphosphorylated or pseudophosphorylated Smads. Finally, we have created
wild-type rescuing and pseudophosphorylated sma-2 constructs that
function differently from the sma-3 variants. These data, from both
sma-2 and sma-3, reveal some of the complex and
sophisticated signaling mechanisms by which the Sma/Mab pathway produces
tissue-specific developmental outcomes.
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Materials and methods |
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Cloning of genomic sma-2
The sma-2 ORF is contained within a large genomic region. One
large intron (3 kb) contains another gene. It is unknown whether this gene has
any relationship with sma-2. A second large intron (intron 7, 1 kb)
near the 3' end contains an interesting sequence. The first half is
complementary to the second half. During PCR, this intron creates aberrant
products. The final rescuing genomic clone, pCS309, does not include these two
introns. This clone was assembled from four PCR products using the following
primers (underlined bases indicate engineered restriction sites):
This strategy resulted in the insertion of six nucleotides encoding two amino acids between the 5' and 3' ends of the gene, due to the engineered restriction sites in the primers. The full predicted sequence of pCS309 is available upon request.
Site-directed mutagenesis
Using the MutaGene kit from BioRad, sma-3 C-terminal mutations
were generated by adding primers complementary to the uracil-containing
single-strand DNA purified from the CJ236 E. coli strain. After
extending by T7 DNA polymerase and ligating by T4 ligase, the DNA was
transformed and amplified in the DH5 E. coli strain. The
mutations were confirmed by DNA sequencing.
Primers for sma-3 mutagenesis:
By chance, we also generated a deletion of MT. The C-terminal mutations of sma-2 and sma-4 were generated by PCR using primers containing the mutations. The 5' primers were normal and the 3' primer sequences were:
Transformation and integration
All of the DNA constructs were amplified in the DH5 E. coli
strain and purified by mini-prep kit (Qiagen). The DNA concentration for
injection is 20 µg/ml using 100 µg/ml rol-6 marker gene
(Mello et al., 1991
). For each
transformation, at least three lines were observed and data are shown for one
representative line. Integration of extrachromosomal arrays was carried out by
exposure to 4000 Rad of gamma ray from a 137Cs source, as described
previously (Wang et al.,
2002
).
Body-length measurement and male tail analysis
Eggs were collected from gravid hermaphrodites by bleaching with
hypochlorite solution for 5 minutes. After the eggs were centrifuged and
cleaned by M9 buffer, they were spread onto new plates. At 96 hours after egg
collection, the photos were taken and body lengths were measured using
SigmaScan software. The males from non-starved plates were picked at young
adult stage. The male tail sensory rays were visualized by Nomarski DIC optics
using a Zeiss Axioplan and the defects were scored.
Yeast two-hybrid assay
The cDNAs of interaction partners were cloned into pPC86 and pPDLeu yeast
vectors. Competent cells of the yeast strain Mav203 were cultured at 30°C
until the OD value was about 0.5. After centrifugation, ice-cold 0.1 M LiCl
was added. The transformation was conducted by adding 5 µg boiled salmon
sperm DNA, 0.1 M lithium acetate, 10% PEG 3350. After adding the DNA and heat
shocking at 42°C for 5 minutes, the yeasts were incubated at 30°C for
30 minutes and spread onto plates without Trp or Leu. The interaction was
determined by spreading the transformed yeast onto the plates with 25 mM or 50
mM 3AT without His.
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Results |
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sma-3 C-terminal mutants are dominant negative in body size regulation
Interestingly, the integrated arrays with the mutated sma-3 genes
were dominant negative in body size regulation when crossed into a wild-type
background (Table 2). This
suggests that these constructs interfere with the function of the wild-type
sma-3 gene. During the cross, we observed that the heterozygotes of
the integrated array are a little longer than homozygotes (data not shown),
suggesting that the inhibition is dose dependent. The SMA-3 DME mutant, which
we expected to be constitutively active, is instead a strong inhibitor of
growth. The SMA-3 AMA is the weakest one among the dominant negatives. We can
postulate at least two different mechanisms for the inhibition. First, the
mutated SMA-3 proteins could compete with wild-type SMA-3 by binding to the
receptors or other Smad proteins. Alternatively, although these SMA-3 mutants
are not functional in body size regulation, they could form partially active
complexes with other Smad proteins and activate negative regulators in this
pathway. In this case, DME is more likely to be the strongest inhibitor
because it is similar to activated Smad and has the greatest tendency to form
a heteromeric complex and interact with transcription factors.
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sma-3 C-terminal mutant forms are functional in male tail sensory ray morphogenesis
In addition to the small body size, the mutants of the Sma/Mab pathway have
male tail defects. Specific sensory rays become fused and the spicules are
crumpled. As most of the C-terminal mutations we generated were dominant
negative in body size control, we asked whether they are also dominant
negative in male tail development by crossing them into the
him-5(e1490) background. There was no defect in any of these
transgenic worms (Table 3). To
test the ability of sma-3 C-terminal mutants to rescue the male tail
defects of sma-3(wk30) animals, we crossed the integrated arrays into
sma-3;him-5. Although none of these mutant forms were functional in
body size regulation, all of them were at least partially functional in
specification of ray identity. Most surprisingly, the AMA mutation that
abolishes the phosphorylation site still can rescue the male tail sensory
rays. Thus, sensory ray development does not require pSMA-3. The decreased
activity of the deletion suggests that a full-length SMA-3 is preferred;
possibly only the backbone of the last amino acids is required.
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Pseudophosphorylated SMA-2 is functionally active
When the integrated sma-3 mutant arrays were crossed into
sma-6(wk7), none of the constructs rescued the male tail defects
(Table 3). Therefore, the
upstream signal from the SMA-6 type I receptor is still required for male tail
development, most likely to induce the phosphorylation of SMA-2. We wondered
whether there is an absolute requirement for SMA-2 phosphorylation, or whether
either SMA-2 or SMA-3 phosphorylation is sufficient for male tail development.
To address this question, we assessed the ability of ppSMA-2 DID (mutated from
SIS) to function in the male tail. We first needed to generate a rescuing
sma-2 clone. To test for rescue, we used the strong allele
sma-2(e297), as none of the sequenced sma-2 alleles are
predicted molecular nulls. A construct containing sma-2 upstream
sequences plus its cDNA failed to rescue the small body size defect of
sma-2(e297). The genomic DNA of sma-2 is long, including two
large introns that proved difficult to clone. We created a construct without
the two introns. After transforming into sma-2(e297), the
sma-2 clone shows a rescuing function in both body size and male tail
(Tables 2,
3). This indicates that the two
introns are not required for sma-2 function. A transcriptional fusion
of sma-2 promoter sequences with GFP shows that sma-2 is
expressed in pharynx, intestine and hypodermis, similar to the expression
pattern of sma-3 (Fig.
1B,C). This is consistent with sma-2 and sma-3
acting together in the signaling pathway. We generated a SMA-2 DID variant
(Table 1) by PCR and
transformed it into sma-2(e297). Interestingly, unlike SMA-3 DME,
ppSMA-2 could rescue body size (Table
2). So, SMA-2 DID is functionally active in body size control.
This difference in the behaviors of SMA-2 DID and SMA-3 DME suggests that
there are differential requirements for these Smad proteins in body size
regulation. In the Discussion, we present a model to reconcile this apparent
inconsistency.
Like ppSMA-3, ppSMA-2 can also rescue male tail sensory ray fusions and crumpled spicules (Table 3). As the pseudophosphorylation of either SMA-2 or SMA-3 could rescue male tail defects, it indicates these constructs could be independent from the receptor SMA-6. We hypothesized that the pseudophosphorylation of both SMA-2 and SMA-3 could control male tail patterning without upstream signaling. Thus, we integrated both of the constructs into him-5 and sma-6(wk7);him-5(e1490). In the wild-type background, the integrated array qcIs43 is dominant negative in body size (data not shown), but the male tail has no defects (Table 4). Although the integrated array has the ability to rescue partially the male tail sensory ray fusions and crumpled spicules of sma-2(e297) or sma-3(wk30), it provides no improvement in the male tail defects of sma-6(wk7) animals at all (Table 4). This result suggests that at least one of SMA-2 and SMA-3 must be truly phosphorylated, rather than pseudophosphorylated, for normal male tail morphogenesis. To test this hypothesis, and to exclude the possibility that male tail development also requires a Smad-independent but SMA-6-dependent signaling output, we crossed qcIs43 into a sma-3sma-2 double mutant. In this strain background, we were unable to homozygose the integrated array, so we scored sma-3sma-2 double-mutant animals carrying one copy of the array. Again, the combination of ppSMA-2 and ppSMA-3 was unable to rescue the male tail defects of sma-3sma-2 mutants (Table 4), thus confirming the difference in function between pseudophosphorylated and phosphorylated Smad.
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The C terminus of SMA-4 is not important for its activity
The C-terminal amino acid residues of SMA-4 are two serines (SS), whereas
those of its homolog Smad4 are leucine and aspartic acid (LD). To test whether
these C-terminal serines have any significant function, we produced several
mutations of SMA-4. The SS was changed into SSIS (more like an R-Smad), DD
(negatively charged) and AA (remove the hydroxyl groups)
(Table 1). These constructs
were introduced into sma-4(e729) mutants, which carry a null allele
with a premature termination codon in sma-4
(Wang et al., 2002). All of
the three constructs function as well as wild-type SMA-4 in body size
regulation (Table 2). The
construct with SSIS could not substitute for any other Smads, as it failed to
rescue the small body size of sma-2 or sma-3 mutants (data
not shown). This suggests, not surprisingly, that the identity of Smads is not
determined by the most C-terminal amino acids, but by the internal sequences
that are necessary for receptor activation and subtype-specific functions. The
results also tell us that the C terminus of SMA-4 is different from that of
SMA-2 or SMA-3, in that it is dispensable for SMA-4 function. In the crystal
structure of the Smad4 MH2 domain, the C terminus is randomly located
(Shi et al., 1997
), unlike
that of pSmad2 (Wu et al.,
2001b
). It is likely that the C terminus of Co-Smad is not
involved in complex formation or other intermolecular interactions.
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Discussion |
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Our results with SMA-2 DID and SMA-3 DME indicate that SMA-2 DID can rescue
the body size defect of a sma-2 mutant, whereas SMA-3 DME cannot
rescue the body size defect of a sma-3 mutant
(Table 5). This difference
suggests that the complex containing SMA-2 DID and pSMA-3 is functionally
different from the one containing pSMA-2 and SMA-3 DME. One model that can
reconcile all of our data is presented in
Fig. 4. In a normal
heterotrimer, there are two possible configurations of the subunits. In one,
the phosphorylated C terminus of SMA-2 interacts with SMA-4, that of SMA-3
interacts with SMA-2, and the unphosphorylated C terminus of SMA-4 interacts
with SMA-3. In the other configuration, the phosphorylated C terminus of SMA-3
interacts with SMA-4, that of SMA-2 with SMA-3, and the SMA-4 C terminus
interacts with SMA-2. Chacko et al. (Chacko
et al., 2004) have designated the two R-Smad subunits in a
heterotrimer as subunits A and C, and the Co-Smad as subunit B. The AB
interface is that of the pR-Smad C terminus with Co-Smad, and it forms more
favorable interactions than that of the CA interface between R-Smads. It is
possible that the interaction between a pR-Smad (A subunit) and SMA-4 (B
subunit) drives complex formation, leading to the recruitment of a second
R-Smad (C subunit). According to this model, the C subunit R-Smad need not be
phosphorylated. Thus, in the SMA-2 DID background, there would be an excess of
pSMA-3:SMA-4:ppSMA-2 complexes relative to ppSMA-2:SMA-4:pSMA-3 complexes.
Conversely, in the SMA-3 DME background, there would be an excess of
pSMA-2:SMA-4:ppSMA-3 complexes. For some target gene(s) involved in body size
regulation, we hypothesize that the configuration of SMA-3:SMA-4:SMA-2 is
required for normal function. By contrast, the SMA-2:SMA-4:SMA-3 configuration
may be sufficient for the feedback loop, which can be triggered by SMA-3 DME.
In male tail development, both kinds of trimers are functional. Thus,
different branches of the pathway may depend on different complexes.
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ACKNOWLEDGMENTS |
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