MAP kinase signaling specificity mediated by the LIN-1
Ets/LIN-31 WH transcription factor complex during C. elegans vulval induction
Patrick B. Tan, Mark R. Lackner*, and Stuart K. Kim
Department of Developmental Biology, Stanford University School of Medicine,
Stanford, CA, 94305
Running title: MAP kinase signaling mediated by LIN-1 and LIN-31
Key words: C. elegans, vulval induction, MAP kinase, mpk-1, lin-31,
winged-helix, lin-1, Ets
Tel: 415-725-7671
fax: 415-725-7739
email: kim@cmgm.stanford.edu
*present address: University of California, Department of Molecular and Cell Biology, 365 Life Science Addition, Berkeley, CA 94720
Summary
The let-23 receptor/let-60 ras/mpk-1 MAP kinase signaling pathway
induces the formation of the vulva in C. elegans. We show that MPK-1 acts
by directly regulating both the winged-helix transcription factor LIN-31
and the Ets transcription factor LIN-1 to specify the choice between vulval
and non-vulval cell fates. Both lin-31 and lin-1 act genetically downstream
of mpk-1, and both encode proteins that can be directly phosphorylated
by MAP kinase in vitro and in vivo. LIN-31 binds to LIN-1 and the LIN-1/LIN-31
complex inhibits vulval induction. Phosphorylation of LIN-31 by MAP kinase
disrupts the LIN-1/LIN-31 complex, which allows vulval induction to occur
by relieving vulval inhibition. In addition to disrupting the LIN-1/LIN-31
inhibitor complex, phosphorylation of LIN-31 may allow a LIN-31 trans-activation
domain to function, permitting phosphorylated LIN-31 to promote vulval
cell fates. We also show that LIN-31 acts as a vulval-specific effector
of mpk-1, while LIN-1 acts as a general effector of mpk-1 signaling. The
partnership of tissue-specific effectors with general effectors may be
one way to confer specificity onto generally-used signaling pathways, resulting
in the generation of distinct tissue-specific outcomes.
Introduction
The receptor tyrosine kinase (RTK)/Ras/MAP kinase signaling cascade
is an evolutionarily-conserved signaling pathway that controls key developmental
processes such as neuronal differentiation, fibroblast proliferation and
cell type specification (reviewed in Schlessinger and Ullrich, 1992; Perrimon,
1993; Dickson and Hafen, 1994). MAP kinases act at the end of this signaling
pathway - upon activation, MAP kinases translocate into the nucleus and
phosphorylate transcription factors (reviewed in Treisman, 1996). These
transcription factor targets form crucial links between the processes of
intercellular signaling and gene expression, and may directly mediate how
a cell responds to activation of the MAP kinase signaling pathway. Thus,
it is important to identify functional targets of MAP kinase, and determine
how phosphorylation of these substrates regulates cell fate specification.
Another key point to understand is the molecular basis underlying signaling
specificity (reviewed in Marshall, 1995; Hill and Treisman, 1995). The
MAP kinase signaling pathway acts in many cell types during development,
and yet is able to induce specific and distinct cellular responses in each
of these cell types. How can a generally-used signaling pathway elicit
different responses in distinct tissue types? One attractive hypothesis
is that there may exist tissue-specific targets of the MAP kinase signaling
cascade, and that these tissue-specific effectors may contribute to the
specificity of MAP kinase signaling.
In C. elegans, the MAP kinase signaling pathway is required for
the development of multiple tissues: the hermaphrodite vulva, the male
tail, the excretory system, the germline, the sex myoblasts, and possibly
the posterior ectoderm as well (Church et al., 1995; Sundaram et al., 1996;
Lackner and Kim, manuscript in prep.). Of these tissues, the function of
MAP kinase in the vulva has been the best characterized. During the L1
larval stage, six vulval precursor cells (P3.p - P8.p) are generated along
the ventral midline of the worm. LIN-3, a protein similar to epidermal
growth factor, is produced by the gonadal anchor cell and initiates vulval
development by activating the EGF receptor tyrosine kinase homolog LET-23
in the closest vulval precursor cell (P6.p). Activation of LET-23 RTK triggers
a conserved Grb2/Ras/Raf/MEK/MAP kinase cascade, the components of which
are encoded by the genes sem-5, let-60, lin-45, mek-2, and mpk-1/sur-1
respectively (reviewed in Kornfeld, 1997). Activation of this signaling
pathway causes P6.p to generate eight cells that ultimately form the inner
cells of the vulva, termed the 1° cell fate. In addition, activation
of LET-23 in P6.p also results in the production of a lateral signal that
induces the adjacent vulval precursor cells (P5.p and P7.p) to generate
seven cells that ultimately form the outer cells of the vulva, termed the
2° vulval fate. Besides the lateral signal, low levels of the anchor
cell signal may also contribute toward expression of the 2° cell fate.
The remaining vulval precursor cells (P3.p, P4.p, and P8.p) do not receive
either the anchor cell signal or the lateral signal and consequently express
the non-vulval, 3° cell fate.
In this report, we have focused upon two genes (lin-31 and lin-1)
that act downstream of mpk-1. lin-31 encodes a winged helix (WH) transcription
factor similar to mammalian HNF-3 and Drosophila forkhead (Miller et al.,
1993). lin-31 null mutants exhibit both a partially-penetrant vulvaless
(Vul) and multivulva (Muv) phenotype (Miller et al., 1993). Specifically,
in about 40% of lin-31 mutant animals, either P5.p, P6.p or P7.p adopts
the 3° non-vulval fate instead of the normal 1° or 2° vulval
fate, resulting in a vulvaless (Vul) phenotype. Conversely, in about 61%
of lin-31 mutant animals, either P3.p, P4.p or P8.p adopts the 1° or
2° vulval fate instead of the normal 3° non-vulval fate, resulting
in a multivulva (Muv) phenotype. This mutant phenotype suggests that LIN-31
may possess two activities: one that inhibits vulval induction (in P3.p,
P4.p, and P8.p) and another that promotes vulval induction (in P5.p, P6.p,
and P7.p).
lin-1 encodes an Ets-related transcription factor, and acts as
an inhibitor of vulval cell fates (Beitel et al., 1995). In lin-1 mutants,
most vulval precursor cells express 1° or 2° vulval fates, resulting
in a Muv phenotype (Horvitz and Sulston, 1980; Ferguson et al., 1987).
In wild-type animals, activation of the LET-23 RTK/MPK-1 signaling pathway
in P6.p may inactivate LIN-1 function, allowing this cell to express a
vulval fate. In addition to its role in vulval development, lin-1 is also
involved in the development of the excretory system and the male tail (Ferguson
and Horvitz, 1985; Han et al., 1990; H. Chamberlin, personal communication),
suggesting that lin-1 may act as a general downstream effector of MAP kinase
signaling in C. elegans.
In this paper, we have examined how LIN-31 WH and LIN-1 Ets control
vulval development in response to MAP kinase signaling. Our results suggest
that LIN-31 and LIN-1 are direct targets of MAP kinase, and that LIN-31
and LIN-1 physically interact with each other only when they are unphosphorylated.
We propose that when MAP kinase is inactive, unphosphorylated LIN-31 and
LIN-1 form a protein complex that inhibits vulval fates. Upon MAP kinase
phosphorylation, the LIN-31/LIN-1 complex is disrupted and phosphorylated
LIN-31 promotes vulval fates.
Furthermore, we show that LIN-31 mediates MAP kinase signaling
in the vulval precursor cells, and is not likely to do so in other tissues
where MAP kinase signaling is known to function. Thus, LIN-31 WH seems
to act as a tissue-specific effector of a generally-used signaling pathway.
The physical interaction of tissue-specific effectors (such as LIN-31 WH)
with general effectors (such as LIN-1 Ets) may allow different genes in
different tissues to be controlled by similar upstream signals, and may
permit common signaling pathways to trigger distinct developmental programs
and cell fate choices.
Results
lin-31 WH acts Downstream of mpk-1
We analyzed the phenotype of a lin-31 mpk-1 double mutant to
determine if lin-31 WH acts downstream of mpk-1 (Figure 1). mpk-1(null)
mutants have a vulvaless (Vul) phenotype since P5.p, P6.p, and P7.p express
3° non-vulval cell fates instead of 1° or 2° vulval cell fates
(Lackner and Kim, manuscript in prep.). Conversely, lin-31(null) mutants
display a partially-penetrant multivulva (Muv) phenotype due to the ectopic
expression of vulval cell fates by P3.p, P4.p, and P8.p (Miller et al.,
1993). We constructed a lin-31(n1053); mpk-1(ga117) double mutant strain,
and observed that the double mutants exhibited a Muv phenotype similar
to that exhibited by lin-31 single mutants. These results show that lin-31
mutations can cause vulval induction independently of mpk-1 activity, suggesting
that lin-31 WH either acts downstream of mpk-1 or in a parallel pathway
to mpk-1. Recently, similar results have been obtained with lin-1 (Lackner
and Kim, manuscript in prep.), suggesting that lin-1 also acts downstream
or in a parallel pathway to mpk-1.
LIN-31 WH and LIN-1 Ets are Phosphorylated by MAP kinase
The results presented above and in Lackner and Kim (manuscript
in prep.) suggest that both LIN-31 WH and LIN-1 Ets may act as direct targets
of MPK-1 during vulval induction. Indeed, LIN-31 possesses four consensus
MAP kinase phosphorylation sites (S/T-P), and LIN-1 contains 18 consensus
MAP kinase sites (Clark-Lewis et al, 1991). To determine if MAP kinase
can directly phosphorylate LIN-31 or LIN-1, we performed in vitro phosphorylation
experiments by incubating activated rat ERK2 with purified GST-LIN-31 or
epitope-tagged FLAG-LIN-1. As rat ERK2 can functionally rescue the mpk-1
mutant phenotype in transformation experiments (Wu and Han, 1994), it is
likely that rat ERK2 can recognize the substrates that are normally phosphorylated
by C. elegans MPK-1. We found that both GST-LIN-31 and FLAG-LIN-1 were
efficiently phosphorylated by ERK2 in vitro (Figures 2A and B). We then
used site-directed mutagenesis to generate LIN-31(PhD) (for PHosphorylation
Defective), in which the threonines of all four LIN-31 consensus MAP kinase
phosphorylation sites were replaced by non-phosphorylatable amino acids
(see Experimental Procedures). We found that ERK2 was unable to phosphorylate
GST-LIN-31(PhD) (Figure 2A, lane 4), indicating that ERK2 phosphorylates
LIN-31(+) on some or all of the four consensus sites of LIN-31 in vitro.
We then determined if MAP kinase might phosphorylate LIN-31 and
LIN-1 in vivo, using a mammalian cell culture assay. NIH 3T3 fibroblasts
were transfected with vectors expressing either epitope-tagged versions
of LIN-31, non-phosphorylatable LIN-31(PhD), or LIN-1. These cells were
then stimulated by co-transfecting them with vectors expressing either
constitutively-activated H-Ras (G12V) or constitutively-activated MEK1
(*N3, S222D). Both activated proteins have previously been shown to stimulate
MAP kinase (ERK1 and ERK2) activity in vivo (Leevers and Marshall, 1992;
Mansour et al., 1994), and in particular, MEK1 is believed to act as a
highly-specific activator of ERK1 and ERK2. (Robbins et al., 1993; Derijard
et al., 1994; Robinson, et al., 1996). As shown in Figure 2C, stimulation
by activated H-Ras or activated MEK1 caused a substantial portion of LIN-31
protein to display a reduced electrophoretic mobility on SDS-PAGE gels.
This mobility shift was not seen in cells expressing LIN-31(PhD), suggesting
that it is most likely due to phosphorylation. Similarly, stimulation by
activated H-Ras or activated MEK1 caused LIN-1 to migrate as a slower and
more compact band on SDS-PAGE gels (Figure 2D), similar to the shift observed
when LIN-1 is phosphorylated by MAP kinase in vitro. These results indicate
that both LIN-31 and LIN-1 are likely to be phosphorylated by the MAP kinases
ERK1 or ERK2 in NIH 3T3 cells.
LIN-31 WH Binds to LIN-1 Ets
lin-31 and lin-1 behave similarly in a number of ways. Firstly,
mutations in both genes cause Muv phenotypes, indicating that both genes
act to inhibit vulval induction in P3.p, P4.p and P8.p (Horvitz and Sulston,
1980; Miller et al., 1993). Secondly, genetic epistasis experiments suggest
that both genes act at a similar point in the vulval signaling pathway,
i.e. downstream of mpk-1 (Kornfeld, 1997, Lackner and Kim, manuscript in
prep.). Thirdly, as shown above, LIN-31 WH and LIN-1 Ets are both likely
to be substrates for MAP kinase. Based upon these similarities, we decided
to test if LIN-31 WH and LIN-1 Ets might physically interact.
To determine if LIN-31 binds to LIN-1 in vivo, we co-infected
insect Sf9 cells with recombinant baculoviruses encoding GST-LIN-31 and
epitope-tagged FLAG-LIN-1. GST-LIN-31 protein was then purified from whole
cell lysates using glutathione beads, and the presence of FLAG-LIN-1 in
the GST-LIN-31 complex was determined using a-FLAG antibodies in Western
blotting experiments. This experiment revealed that FLAG-LIN-1 associated
with GST-LIN-31 (Figure 3A). As a negative control, a GST fusion protein
containing the Drosophila USP transcription factor (GST-USP) failed to
associate with FLAG-LIN-1 (Figure 3A), indicating that the association
of LIN-1 with LIN-31 is specific. We also performed this experiment in
reverse, and found that LIN-31 specifically co-immunprecipitated with LIN-1
(data not shown). These results show that LIN-1 specifically co-purifies
with LIN-31 when co-expressed in insect cells.
To determine if LIN-31 can directly bind to LIN-1, we performed
in vitro binding experiments using independently-purified GST-LIN-31 and
FLAG-LIN-1. As seen in Figure 3B, FLAG-LIN-1 bound to GST-LIN-31 in vitro.
We then defined the region of LIN-31 that contains the LIN-1 binding site
by expressing and purifying bacterial GST-LIN-31 fusion proteins containing
the LIN-31 DNA-binding domain, the middle region of LIN-31, and the C-terminus
of LIN-31. We tested each region for its ability to bind to FLAG-LIN-1
in vitro (Figure 3B), and found that the middle region (amino acids 39-169)
bound to FLAG-LIN-1 as well as full-length GST-LIN-31. However, the DNA-binding
domain, the C-terminus and the negative control GST-LIN-7 did not bind
to FLAG-LIN-1 under the same binding conditions. Interestingly, this 130
amino-acid middle region of LIN-31 that binds LIN-1 also contains a MAP
kinase phosphorylation site (Thr145) and may be a transcriptional activation
domain, since it is acidic and proline-rich.
MAP kinase Phosphorylation Disrupts Formation of the LIN-1/LIN-31
Complex
We considered the possibility that MAP kinase might regulate
the activity of LIN-31 WH and LIN-1 Ets by affecting their binding interaction.
To test this possibility, purified GST-LIN-31 and FLAG-LIN-1 proteins were
incubated together in the presence of activated MAP kinase and ATP. Phosphorylated
GST-LIN-31 was recovered using glutathione beads, and bound phosphorylated
FLAG-LIN-1 was detected using a-FLAG antibodies in Western Blotting experiments.
This experiment revealed that MAP kinase phosphorylation substantially
reduced the amount of FLAG-LIN-1 bound to GST-LIN-31 in vitro (Figure 3C).
We then determined if MAP kinase phosphorylation of either factor
alone was sufficient to prevent formation of the LIN-1/LIN-31 complex.
Phosphorylated GST-LIN-31 and FLAG-LIN-1 were generated independently
by pre-incubating each purified protein with MAP kinase. Phosphorylated
GST-LIN-31 was then bound to glutathione beads and phosphorylated FLAG-LIN-1
was bound to a-FLAG beads. Bound phosphorylated protein was mixed with
its unphosphorylated partner (phosphorylated GST-LIN-31 with unphosphorylated
FLAG-LIN-1, and vice versa), and binding between the two proteins was detected
using Western blots. We found that phosphorylation of LIN-31, but not LIN-1,
disrupted formation of the LIN-1/LIN-31 complex. Specifically, we found
that phosphorylated LIN-31 did not bind to unphosphorylated LIN-1 (Figure
3D), but that phosphorylated LIN-1 still bound to unphosphorylated LIN-31
(Figure 3E). Thus, phosphorylation of LIN-31 by MAP kinase is sufficient
to prevent the association of LIN-31 and LIN-1 in vitro.
These biochemical results suggest a model of how MAP kinase activity
could control the activity of LIN-31 WH and LIN-1 Ets in the vulval precursor
cells. In vulval precursor cells where MAP kinase is inactive (P3.p, P4.p,
and P8.p), unphosphorylated LIN-1 and LIN-31 form a complex that inhibits
the expression of vulval cell fates. Mutations in lin-31 or lin-1 would
lead to a failure of this inhibition, and as a result these cells would
ectopically express vulval cell fates. In vulval precursor cells where
MAP kinase is active (P6.p, and possibly P5.p and P7.p), MAP kinase phosphorylation
would disrupt this complex and alleviate vulval inhibition. In addition
to loss of the LIN-1/LIN-31 inhibitor complex, phosphorylated LIN-31 could
also actively promote the expression of vulval cell fates. This is because
lin-31 loss-of-function mutants also exhibit a partial Vul phenotype (indicating
a function in promoting vulval cell fates). In the following sections,
we use three different lin-31 constructs to test predictions of this model.
A LIN-1/LIN-31 Forced Heterodimer Inhibits Vulval Cell Fates
If the LIN-1/LIN-31 complex inhibits vulval induction, then maintaining
this complex in each of the vulval precursor cells should result in constitutive
vulval inhibition. To test this prediction, we constructed a LIN-1/LIN-31
forced heterodimer by engineering a transgene that uses the lin-31 promoter
to express a single polypeptide containing the entire lin-1 coding sequence
followed by the entire lin-31 coding sequence (termed LIN-1::LIN-31). Although
our biochemical studies indicate that phosphorylation greatly reduces LIN-1/LIN-31
binding, some binding is still observed at high protein concentrations
(P. Tan, unpublished observations). We reasoned that the close proximity
of LIN-1 and LIN-31 sequences in the forced heterodimer might permit binding
interactions to occur even when these proteins are phosphorylated. We injected
this construct into lin-31(+); lin-1(+) animals, obtained six transgenic
lines, and observed that 10%-50% of the transgenic animals exhibited a
dominant Vul phenotype (Figure 4B and Table 1). In contrast, transgenic
lines that expressed either lin-31 or lin-1 alone did not exhibit this
effect, indicating that the vulvaless phenotype of LIN-1::LIN-31 animals
is not likely to be due to overexpression of either lin-31 or lin-1 (Figure
4A and Table 1). Furthermore, we also determined that expression of a gfp::lin-31
gene did not exhibit a similar dominant Vul phenotype (Table 1), indicating
that the effects of the LIN-1::LIN-31 forced heterodimer cannot be explained
as the non-specific consequence of inserting a bulky polypeptide at the
N-terminus of the LIN-31 protein.
We then used Nomarski microscopy to determine the pattern of
vulval precursor cell division in animals expressing LIN-1::LIN-31. We
observed that P5.p, P6.p, and P7.p (which normally express 1° or 2°
vulval fates) often expressed non-vulval 3° cell fates (Figure 4B and
Table 2). These lineage patterns are similar to the phenotype caused by
mutations that diminish the activity of the vulval signaling pathway, such
as mutations in lin-3 EGF, let-23 receptor, let-60 ras, or mpk-1 (reviewed
in Kornfeld, 1997; Lackner and Kim, manuscript in prep.). Thus, these results
suggest that preventing dissociation of LIN-1 from LIN-31 blocks vulval
induction.
A Non-Phosphorylatable LIN-31 Protein Inhibits Vulval Cell Fates
Since phosphorylation of LIN-31 (but not LIN-1) is sufficient
to disrupt the LIN-1/LIN-31 complex, another prediction of the model is
that an unphosphorylatable form of LIN-31 (such as LIN-31(PhD)) should
remain complexed with LIN-1 and thus function as a constitutive inhibitor
of vulval induction. We microinjected lin-31(PhD) DNA into lin-31(+) animals,
obtained 3 transgenic lines, and found that an average of 42% of animals
exhibited a dominant Vul phenotype (Figure 4C and Table 1). We then directly
determined the pattern of cell fates expressed by P5.p, P6.p and P7.p in
these animals using Nomarski microscopy, and again found that these cells
often adopted uninduced or defective cell fates (Table 2). These results
suggest that LIN-31(PhD) can act to diminish the mpk-1 signaling pathway
in the vulva, perhaps by remaining complexed with LIN-1.
We also microinjected DNA containing lin-31(PhD) into lin-31(-)
worms and found that lin-31(PhD) could rescue the Muv but not the Vul phenotype
of lin-31 null mutants (out of 5 independent transgenic lines) (Table 1).
We also determined the cell lineages expressed by the vulval precursor
cells in one of the transgenic lines expressing LIN-31(PhD) (Table 2).
In these animals, P3.p, P4.p and P8.p expressed the non-vulval 3° cell
fate (as in wild-type), suggesting that lin-31(PhD) can inhibit vulval
induction. However, P5.p, P6.p, and P7.p often expressed partial vulval
cell fates or the 3° non-vulval cell fates (instead of the 1° and
2° cell fates), suggesting that lin-31(PhD) does not function to promote
vulval induction.
LIN-31(VP16) Promotes Vulval Cell Fates
In addition to inhibiting vulval cell fates in P3.p, P4.p and
P8.p, genetic analysis suggests that LIN-31 WH promotes the expression
of vulval fates in P5.p, P6.p, and P7.p. MPK-1 is active in P6.p (and possibly
P5.p and P7.p as well), suggesting that LIN-31 may be phosphorylated in
these cells. How might phosphorylation of LIN-31 allow it to promote vulval
induction? In many cases, the phosphorylation of a transcription factor
by a MAP kinase creates or reveals a potent trans-activation domain (Marais
et al., 1993; O'Neill et al, 1994; Kato et al., 1995; Wen et al., 1995).
We reasoned that this might also be the case for LIN-31 WH, especially
since the middle region of LIN-31 contains the LIN-1 binding site, a putative
LIN-31 trans-activation domain and a MPK-1 phosphorylation site. To pursue
this possibility, we replaced both the phosphorylation domain and the LIN-1
binding region of LIN-31 with a strong trans-activation domain (VP16),
reasoning that this might be functionally analogous to constitutive phosphorylation
by MPK-1. We injected DNA containing lin-31(VP16) into lin-31(+) animals,
generated 4 transgenic lines, and found that from 10% to 30% of the animals
from these transgenic lines exhibited a dominant Muv phenotype (Figure
4D). As a control, we also engineered a construct that expressed only the
DNA-binding region of LIN-31, introduced this construct into lin-31(+)
mutants, and generated 5 transgenic lines. We found that expression of
the LIN-31 DNA-binding region alone did not result in a dominant Muv phenotype
(Table 1), indicating that the effects of lin-31(VP16) are not due to the
DNA-binding domain of LIN-31 acting as a dominant-negative protein. In
addition, we also injected DNA containing lin-31(VP16) into lin-31(-) animals,
and obtained 3 transgenic lines. The penetrance of the Muv phenotype in
these transgenic lines is significantly higher than that of lin-31 null
mutants (Table 1), again suggesting that LIN-31(VP16) promotes the expression
of vulval cell fates.
Taken together, these results indicate that LIN-31(VP16) causes
the vulval precursor cells to express vulval cell fates. The simplest interpretation
of these results is that LIN-31(VP16) is functionally analogous to phosphorylated
LIN-31 WH, since both proteins do not associate with LIN-1 and both may
act as transcriptional activators.
LIN-31 WH is Expressed in the Vulval Precursor Cells, but is Absent
from Many Other Tissues that Utilize the let-23 receptor/mpk-1 Signaling
Pathway
To test if LIN-31 WH is expressed at the appropriate time and
place to be a substrate for MPK-1, we performed immunocytochemistry experiments
to determine the LIN-31 expression pattern. LIN-31 is first expressed in
the nuclei of P1.p - P11.p (including the vulval precursor cells), from
the middle of the first larval (L1) stage until the early L3 stage (Figure
5A). Since activation of MPK-1 is thought to occur during this time (Kimble,
1981; Euling and Ambros, 1996), these results show that LIN-31 is present
in the vulval precursor cells at the appropriate time and place to respond
to activated MPK-1.
The mpk-1 signaling pathway is used in the vulva, the germline,
and the sex myoblasts (Church et al., 1995; Sundaram et al., 1996; Lackner
and Kim, manuscript in prep.). Additionally, when viewed with a dissecting
microscope, mpk-1 mutations cause L1 larval lethality (when maternal contribution
is removed) and a male spicule defect that appear to be identical to the
phenotypes caused by let-23 receptor and let-60 ras loss-of-function mutations
(Lackner and Kim, manuscript in prep.). These let-23 receptor and let-60
ras phenotypes are caused by defects in the excretory system and in the
male B cell lineage (Chamberlin and Sternberg, 1994; Koga and Ohshima,
1995; Yochem et al., 1996), suggesting that mpk-1 acts in these cells as
well. Finally, let-23 receptor, let-60 ras, and lin-45 raf also act to
establish the fate of P12 in the posterior ectoderm (Aroian et al., 1991,
Han et al., 1993; P. Sternberg, personal communication), and mpk-1 may
function in this cell as well.
We were interested in determining if lin-31 might interact with
the mpk-1 signaling pathway in tissues other than the vulva, and found
two pieces of evidence suggesting that lin-31 does not appear to function
in the mpk-1 signaling pathway in the germline, the excretory system, the
posterior ectoderm, or the sex myoblasts. Firstly, LIN-31 protein is expressed
primarily in the vulval precursor cells and not in these other tissues.
Specifically, we did not detect LIN-31 expression in the germ-line, P12
(the posterior ectoderm), and the sex myoblasts (Figure 5C-E, data not
shown). We also found that LIN-31 is not expressed in the excretory system
at the appropriate time to respond to mpk-1 signaling. mpk-1 signaling
in the excretory system probably occurs before or during the mid L1 larval
stage, as the larval lethality caused by null mutations in let-23 receptor,
let-60 ras, lin-45 raf, or mpk-1 occurs at this time (Aroian, 1991, Han
et al., 1993; Koga and Ohshima, 1995; Yochem et al., 1997; Lackner and
Kim, manuscript in prep.). We confirmed that the larval arrest caused by
a null let-23 mutation occurs at the L1 stage by picking arrested let-23
mutants and determining their precise developmental stage by staining them
with the MH27 monoclonal antibody (Figure 5F and Aroian et al., 1991).
We then determined the LIN-31 expression pattern in wild-type animals at
the same developmental stage, and found that LIN-31 is not expressed in
the excretory duct cell of wild-type animals at this time (Figure 5E).
Intriguingly, we did observe LIN-31 expression in this cell in the mid
L2 stage (Figure 5A).
Secondly, lin-31 and mpk-1 exhibit similar mutant phenotypes
only in vulval induction. Specifically, lin-31 mutants exhibit defects
in vulval cell fate specification, but do not exhibit apparent phenotypes
in the excretory system, sex myoblasts, germline, or posterior ectoderm
(Miller et al., 1993; Sundaram et al., 1996; P. Tan, unpublished observations).
Furthermore, lin-31 mutations do not suppress the larval lethality caused
by a let-23 mutation (L. Miller, personal communication) or suppress the
sterility caused by a mpk-1 mutation (M. Lackner, personal communication).
In addition to its role in vulval development, lin-31 functions
in the development of the male tail. However, the function of lin-31 in
this tissue is likely to be different from that of the mpk-1 signaling
pathway. As mentioned previously, the mpk-1 signaling pathway is thought
to act in the male tail to specify the fate of four cells that arise from
the B cell lineage (B.a(l or r)pp, B.alap, B.arap, and B.a(l or r)aa) (Chamberlin
and Sternberg, 1994). LIN-31 is expressed in three of these four cells
(all except B.a(l or r)aa) (Figure 5G), suggesting that LIN-31 WH might
be a target for the mpk-1 signaling pathway in these three cells in the
male tail. However, the lin-31 male tail phenotype is distinctly different
from the let-23 receptor and let-60 ras mutant phenotype (S. Baird, personal
communication). The defective mating spicules of lin-31 males result from
the abnormal migration of cells in the tail proctodeum (B.al/rappv and
B.al/rapapa), a process that occurs two (B.al/rappv) or three (B.al/rapapa)
cell divisions later than the cell fate specification defects of let-23
receptor or let-60 ras mutants. These results suggest that lin-31 may not
interact with the mpk-1 signaling pathway in the male tail to specify the
fates of the B cell progeny, despite being expressed in some of the appropriate
cells.
In summary, these genetic and expression studies suggest that
lin-31 does not interact with the mpk-1 signaling pathway in the germline,
the posterior ectoderm, the sex myoblasts, or the excretory system, and
most likely does not interact with this pathway in male tail development
to specify the fates of the B cell progeny. LIN-31 WH may thus act as a
vulval-specific effector of mpk-1 signaling, and contribute to the signaling
specificity of the MPK-1 signaling pathway.
Ectopic expression of LIN-31 Causes P12 to Express a Vulval-Specific Marker
We wanted to determine whether ectopic expression of LIN-31 in
another let-23 RTK-responsive cell type might partially induce that cell
to express vulval-specific markers in response to let-23 RTK signaling.
The 1° vulval cell fate consists of a specific cell division pattern
and the expression of specific molecular markers, such as increased LET-23
expression (for other markers see Burdine et al., 1998; Maloof and Kenyon,
1998). Staining with a-LET-23 antibodies shows that LET-23 RTK expression
sharply increases in P6.p (Figure 6a and Simske et al., 1996). This feedback
regulation is thought to be important for vulval patterning, as high LET-23
RTK levels may bind and sequester LIN-3, and thereby prevent LIN-3 from
inducing other vulval precursor cells (Hajnal et al., 1997). Increased
expression of LET-23 RTK is a vulval-specific event, because unlike the
1° vulval cell fate, LET-23 RTK expression does not increase in P12,
when the let-23 RTK/let-60 ras signaling pathway functions to specify the
P12 fate (Figure 6c).
We first found that lin-31 is required to increase LET-23 RTK
expression in P6.p. In lin-31 null mutants, we detected increased LET-23
RTK expression in P6.p in only 46% of lin-31 null mutants (n=50) versus
100% of wild-type animals (n=50) (Figure 6b). Thus, lin-31 is necessary
for the accurate and fidelitous execution of positive LET-23 RTK regulation
in P6.p.
Next, we tested if ectopic expression of lin-31 in P12 could
cause it to express this vulval specific marker. Using a heat-shock promoter,
we ectopically expressed lin-31 in the P12 posterior ectoblast during the
time of P12 specification. We detected increased LET-23 RTK expression
in P12 in 33% of heat-shocked animals (n=12), but no increased expression
in any wild-type (n=15) or heat-shocked control animals (n=20) (Figure
6d). These results demonstrate that lin-31 functions as a vulval-specific
effector of let-23 RTK signaling, as lin-31 misexpression causes a heterologous
let-23 RTK responsive cell to express a vulval specific marker (increased
LET-23 RTK expression).
Discussion
We have examined how the LIN-31 winged helix transcription factor
and the LIN-1 Ets transcription factor function as direct targets of MAP
kinase to control vulval induction. Genetic epistasis experiments indicate
that both lin-31 and lin-1 function downstream of mpk-1 (this report, Lackner
and Kim, manuscript in prep.), and LIN-31 is expressed in the vulval precursor
cells at the appropriate time and place to respond to activated MPK-1.
Both factors can be phosphorylated by MAP kinase in vitro and in vivo in
NIH 3T3 cells. Furthermore, unphosphorylated LIN-31 WH and LIN-1 Ets directly
bind to each other in vitro and in vivo, and that this interaction is prevented
by MAP kinase phosphorylation of LIN-31 in vitro.
These genetic and biochemical observations suggest the following
model for how MAP kinase might regulate LIN-31 and LIN-1 during vulval
development (Figure 6A). In vulval precursor cells where MAP kinase is
inactive (P3.p, P4.p and P8.p), LIN-31 and LIN-1 are associated as a complex
that inhibits vulval induction and causes these vulval precursor cells
to express the non-vulval 3° cell fate. Since the LIN-1 binding site
on LIN-31 overlaps two possible transcriptional activation domains (a proline-rich
and highly-acidic region), LIN-1 binding to LIN-31 may mask the LIN-31
trans-activation domain and prevent LIN-31 from functioning as a transcriptional
activator. Activation of MAP kinase in P6.p (and possibly P5.p and P7.p)
results in the phosphorylation of LIN-31 and LIN-1, leading to the dissociation
of the LIN-1/LIN-31 complex. In addition to preventing LIN-1 from binding
to LIN-31, phosphorylation of LIN-31 at threonine 145 may also potentiate
the ability of the proline-rich or the acidic region of LIN-31 to function
as a trans-activation domain. Once dissociated from LIN-1, phosphorylated
LIN-31 may then act as a transcriptional activator to promote the expression
of vulval fates.
We tested this model in three ways. Firstly, if LIN-1 and LIN-31
form a complex in vivo that functions to inhibit vulval induction, then
a strain in which LIN-1 is always associated with LIN-31 should be Vul.
We engineered a gene that expresses a LIN-1::LIN-31 forced heterodimer,
similar to that reported for the bHLH factors MyoD and E47 (Neuhold and
Wold, 1993). We reasoned that such a LIN-1::LIN-31 fusion protein might
maintain high local concentrations of both the LIN-31 and LIN-1 fragments
and associate intramolecularly even when phosphorylated by MPK-1. We found
that the lin-1::lin-31 transgene caused a partial dominant vulvaless phenotype,
indicating that LIN-1 and LIN-31 most likely associate as a vulval inhibitor
complex in vivo.
Secondly, if phosphorylation of LIN-31 dissociates the LIN-1/LIN-31
complex, then preventing LIN-31 phosphorylation might cause the LIN-l/LIN-31
heterodimer to persist in vivo resulting in constitutive vulval inhibition.
We engineered a non-phosphorylatable form of LIN-31 (LIN-31(PhD)), and
showed that a lin-31(PhD) transgene caused a partial dominant vulvaless
phenotype. We also determined that lin-31(PhD) could rescue the Muv phenotype
of a lin-31 null strain, indicating that LIN-31(PhD) can function to inhibit
vulval induction in P3.p, P4.p and P8.p. In these cells in wild-type animals,
MAP kinase is most likely inactive and LIN-31(+) is most likely unphosphorylated.
Thus, LIN-31(PhD) retains the function associated with unphosphorylated
LIN-31(+). However, LIN-31(PhD) does not rescue the Vul phenotype of a
lin-31 mutant, indicating that LIN-31(PhD) does not retain the function
associated with phosphorylated LIN-31 (in P6.p, and possibly P5.p and P7.p).
These results suggest that LIN-31 is phosphorylated in vivo, and that this
phosphorylation determines whether vulval precursor cells express vulval
or non-vulval cell fates.
Thirdly, if MAP kinase phosphorylation results in dissociation
of the LIN-1/LIN-31 inhibitor complex and activates a LIN-31 trans-activation
domain, then replacing the LIN-1 binding region of LIN-31 with the VP16
trans-activation domain should be functionally analogous to constitutive
LIN-31 phosphorylation. This is because the VP16 trans-activation domain
should not bind to LIN-1 and should function as a trans-activation domain
independently of MAP kinase phosphorylation. As predicted, we found that
a transgene expressing a lin-31(VP16) chimeric activator caused a dominant
Muv phenotype. This result suggests that the activation of LIN-31 target
genes is sufficient to cause vulval precursor cells to ectopically express
vulval cell fates.
Our work does not address the functional consequences of MAP
kinase phosphorylation of LIN-1. lin-1 loss-of-function mutations do not
obviously affect the specification of vulval cell fates by P5.p, P6.p,
and P7.p (Beitel et al., 1995), unlike mutations in lin-31. This suggests
that phosphorylated LIN-1 may be functionally inactive. Phosphorylation
of LIN-1 may inactivate its inhibitory function either by inhibiting the
DNA binding ability of LIN-1, by preventing LIN-1 from acting as a trans-repressor,
by causing LIN-1 to be rapidly degraded or by altering the subcellular
localization of LIN-1. Alternatively, LIN-1 phosphorylation may not be
critical for vulval induction, and phosphorylation of LIN-31 alone may
be sufficient to inactivate LIN-1 by disrupting the LIN-1/LIN-31 inhibitor
complex.
Positive and Negative Regulation of Vulval Cell Fates
How does this proposed model explain the deregulated vulval phenotype
of lin-31 null mutants? The simplest explanation would be that in lin-31(null)
animals, downstream vulval specification genes normally regulated by lin-31(+)
would fail to be either strongly activated or inhibited. Instead, these
genes would be transcribed at an intermediate or basal level, which might
also be near the threshold for vulval induction. Consequently, stochastic
variation in the expression levels of these LIN-31 target genes would eventually
cause some vulval precursor cells to express vulval cell fates.
In lin-31 mutants, the vulval precursor cells do not adopt a
uniform hybrid fate that might result from the uniform basal transcription
of downstream specification genes (the LIN-31 targets). Instead, the vulval
precursor cells typically express discrete cell fates (1°, 2° or
3°). This observation suggests that initial small stochastic differences
in the transcription of these LIN-31 target genes from cell to cell and
from animal to animal in lin-31 mutants are somehow ultimately translated
into large differences in the expression of terminal cell fates (i. e.
1°, 2° or 3°). We speculate that this might occur through positive
and negative autoregulatory feedbacks; specifically, the products of these
LIN-31 target genes might positively regulate their own expression and
also negatively cross-regulate genes promoting the alternative cell fate
(Miller et al., 1993). In this model, if these postulated LIN-31 target
genes were initially expressed above a certain threshold, then these feedback
loops could amplify their own expression and cause the vulval precursor
cell to adopt a discrete 1° or 2° cell fate. If the initial level
were lower than a certain threshold, then these feedback loops would not
be maintained and such a vulval precursor cell would express the 3°
cell fate. Similar mechanisms have been proposed to explain how small qualitative
differences can be amplified to yield distinct biological responses in
the life cycle of phage l and in the process of Drosophila sex determination
(Ptashne et al., 1980; Bell et al., 1991)
A key feature of this model is that in wild-type animals, LIN-31
target genes are either strongly activated or repressed. Recent reports
suggest that many diverse transcription factors (such as Mad, CREB, c-Jun,
NF-KB, and the various nuclear hormone receptors) can both activate and
repress transcription, depending upon their association with co-repressors
(such as SMRT or mSin3a) (Ayer et al., 1995; Chen and Evans, 1995; Schreiber-Agus
et al., 1995) or co-activators (such as SRC-1, ACTR, and CBP) (Onate et
al., 1995; Chakravarti et al., 1996; Kamei et al., 1996; Chen et al., 1997).
The results of this work show the importance of such an activation/repression
mechanism in a developmental patterning context. LIN-31 can both positively
and negatively regulate vulval induction, and both functions are required
for the proper spatial specification of vulval cell fates. Vulval precursor
cells are not fully activated if the positive function is defective (the
Vul phenotype), and they are not fully inhibited if the negative function
is defective (the Muv phenotype). The ability of transcription factors
to both activate and repress gene expression may contribute to the precision
and fidelity of gene transcription that characterizes many developmental
programs.
Signaling Specificity by the MAP kinase Pathway
Our results also address the problem of signaling specificity,
in which the activation of common upstream signaling components (such as
Ras and MAP kinase) can elicit different responses in different cell types
(Figure 6B). Our results suggest that C. elegans LIN-31 WH is a tissue-specific
effector of MAP kinase signaling. lin-31 mutations cause defects in only
one of the six processes in which mpk-1 signaling is known to act (Miller
et al., 1993; P. Tan, unpublished observations), and the highly-restricted
expression pattern of LIN-31 WH indicates that LIN-31 can transduce the
MAP kinase signal only in the vulval precursor cells and possibly in the
B cell progeny in the male tail. Furthermore, lin-31 is required to faithfully
induce a vulval-specific response (increased LET-23 RTK expression), and
lin-31 misexpression in a heterologous Ras responsive cell (the P12 posterior
ectoblast) partially causes that cell to respond like a vulval precursor
cell by expressing a vulval specific marker.
We did not observe any dramatic P12 lineage alterations in our
lin-31 misexpression experiments, indicating that lin-31 alone is not sufficient
to fully transform P12 into a vulval precursor cell. One explanation is
that lin-31 is not the only vulval-specific effector of MAP kinase, and
that other vulval-specific effectors also mediate vulval signaling specificity.
Expression of the full vulval fate by a heterologous cell would thus require
the simultaneous expression of all these multiple effectors.
In contrast to the tissue specific function of LIN-31 WH, LIN-1
Ets may function as a more general component of the mpk-1 signaling pathway,
since loss-of-function lin-1 mutations affect at least four (and possibly
five) of the six cell types that utilize Ras/MAP kinase signaling in C.
elegans. One aspect of MAP kinase signaling specificity may thus involve
the interaction between a general Ras/MAP kinase effector (LIN-1 Ets) and
a tissue-specific effector (LIN-31 WH, in the case of the vulval precursor
cells). Differences in the DNA-binding specificity of tissue-specific MAP
kinase effectors might allow different genes in different tissues to be
targeted for either activation or repression.
In flies and mammals, MAP kinase phosphorylation of general factors
are well known, but only recently have tissue specific effectors been found.
In mammals, the bHLH transcription factor Microphthalmia and the PPARg
nuclear receptor are tissue-specific effectors of MAP kinase signaling
in melanocyte and adipocyte differentiation, respectively (Hu et al., 1996;
Hemesath et al., 1998). These tissue specific effectors may act with general
effectors (eg ELK-1, Ets-1, Ets-2) to initiate new programs of gene expression.
In Drosophila, rolled MAP kinase may directly regulate three transcription
factors: Aop/Yan, PointedP2, and dJun. However, none of these transcription
factors are tissue specific. Thus, the mechanism of signaling specificity
in Drosophila is currenly unknown. Tissue-specific effectors of MAP kinase
signaling may form an important class of proteins that confer specificity
onto generally used signaling pathways, so that diverse cellular outcomes
can ultimately be generated.
Experimental Procedures
Further details concerning the experimental procedures, including the construction of expression vectors, are available at http://cmgm.Stanford.edu/~kimlab.
General Methods and Strains
Standard methods were used to handle C. elegans (variety Bristol,
strain N2) at 20°C (Wood, 1988). All mutations are described in Wood
(1988) except when otherwise noted. Linkage group I (LG I): unc-29(e1072).
LG II: lin-31(n1053, ga37) (Miller et al., 1993). LG III: mpk-1(ga117)
(Lackner and Kim, manuscript in prep.), dpy17(e164), sDp3, LG V; him-5(e1490).
Not yet assigned to a linkage group is gaIs50[hs-lin-31; unc-29(+)].
Pn.p and other cells were analyzed as described in Sulston and Horvitz
(1977), using the criteria for the assignment of 1°, 2°, and 3°
cell fates described in Sternberg and Horvitz (1986). Hybrid fates (e.g.
LTN) and undivided fates (e.g. U) were scored as underinduced or non-vulval,
as these fates can be associated with mutations that reduce the activity
of the anchor cell signaling pathway (e.g. see Aroian et al., 1991, Hajnal
et al., 1997).
Molecular Biology
Standard techniques were used, with site-directed mutagenesis
performed using the Unique Site Elimination procedure (Pharmacia). All
introduced mutations were verified by sequencing. All lin-31 genomic constructs
are derived from PB8, a 8.5 kb Spe I - Eco RI genomic fragment that spans
the lin-31 promoter, coding region, and 3' UTR. To create LIN-31(PhD),
Thr145 was replaced with Met, while Thr 200, 218 and 220 were replaced
with Ala. As expression from the lin-31 promoter is dependent upon an enhancer
located in the third intron of lin-31 (P. Tan, unpublished observations),
experiments involving LIN-31(VP16) were performed using PB255, a modified
lin-31 promoter in which this enhancer was inserted directly upstream of
the endogenous lin-31 promoter, followed by multiple restriction enzyme
sites for cloning and a 3' UTR derived from the unc-54 gene (Fire et al.,
1990). This construct allows cDNA sequences to be expressed in the vulval
precursor cells specifically. The lin-1::lin-31 forced heterodimer was
made by first inserting a Sal I site into PB8 just after the start ATG
of the lin-31 coding sequence, and then inserting a full length lin-1 cDNA
(gift of G. Beitel, Stanford University) into the Sal I site. To make the
heat-shock lin-31 transgene, a full-length lin-31 cDNA was inserted into
pPD49.83 (bearing the hsp16-41 promoter (Stringham et al., 1992)
Germline Transformation
Germline transformants were obtained by standard DNA microinjection
(Mello et al., 1991). Concentrations of DNAs used: all lin-31-derived constructs
(100 µg/ml), unc-29(+) (F35D3, 100 µg/ml), rol-6 (pRF4, 80
µg/ml). All results described in this work are based on the average
of at least three or more transmitting lines. In the case of experiments
employing lin-31(VP16), the dominant Muv phenotype was only seen when rol-6
was used as a co-transformation marker (except for lin-31(VP16) and gfp::lin-31,
all other experiments in this paper used unc-29(+) as a co-transformation
marker). One explanation is that GFP reporter studies indicate that transgenes
are expressed at higher levels using rol-6 as a co-transformation marker
than unc-29 (P. Tan, unpublished observations). Chromosomal integration
of the heat-shock lin-31 transgene was induced by standard g-irradiation
and integrated strains were subsequently backcrossed twice. For heat-shock
treatment, synchronized eggs were isolated by allowing gravid adults to
lay eggs on a single plate for 5 hours before removing the parents. Newly
hatched worms (2-3 hours after hatching) were heat-shocked (33°C, 30
min) and fixed for immunostaining 3-4 hours later.
Generation of Antibodies and Immunoflouresence
The bacterial MBP-LIN-31 fusion protein construct contains a
full length lin-31 cDNA subcloned into an MBP (Maltose Binding Protein)
expression vector (NEB). Rabbit a-LIN-31 antibodies were generated (Josman
Labs) using MBP-LIN-31 as an immunogen, followed by affinity-purification
using a GST-LIN-31 column (containing full length lin-31 cDNA). Generation
and purification of MBP-LIN-31 and GST-LIN-31 proteins were performed according
to standard protocols (NEB, Pharmacia). Antibodies were used at dilutions
of 1:200 for immunofluoresence and 1:1000 for Western blotting. Generation
of a-LET-23 antibodies will be described elsewhere (Candia et al., manuscript
in prep.)
For immunostaining, populations of animals were first synchronized
by bleaching gravid adults to isolate unhatched eggs (Wood, 1988). Animals
were then fixed and stained at various time points using the method of
Finney and Ruvkun (1990). The junctional antibody MH27 (gift of Bob Waterston)
was used to determine the precise developmental stage of stained worms
(at 1:1000 dilution) (Austin and Kenyon, 1994; Podbilewicz and White, 1994).
DAPI was used at 1µg/ml (Molecular Probes, Inc.). a-LET-23 antibodies
were used at 1:1000 dilution.
In vitro Kinase Assays
The test substrate GST-LIN-31 was created by subcloning a full
length lin-31 cDNA into vector pAC-GHLT A (Pharmingen), and FLAG-LIN-1
was created by introducing the FLAG epitope sequence just after the start
Met residue of a full length lin-1 cDNA subcloned into vector pAC-HLT B
(Pharmingen). Recombinant baculoviruses using these constructs were then
produced and amplified in Sf9 cells using Baculogold, according to the
directions of the manufacturer (Pharmingen). Affinity purification was
performed using glutathione beads (for GST-LIN-31) or a-FLAG-beads (Kodak).
Briefly, after gentle centrifugation, insect cells were resuspended in
kinase/washing buffer (250 mM NaCl, 1 mM MgCl2, 0.5% NP-40, 1 mM DTT, 10%
glycerol and 1x protease inhibitor cocktail (Pharmingen)) to a final concentration
of 1 x 107 cells/ml. Cells were then lysed using a microsonicator, and
the insoluble fraction was removed by centrifugation at 30,000 rpm for
30 min. Affinity purification was then performed according to standard
protocols (Pharmacia, Kodak). All proteins were stored at -80 °C. Kinase
assays were performed using 0.1 µg of test protein, 1 unit activated
rat ERK2 (NEB), and 10 µCi 32P-ATP (for GST-LIN-31) or 0.5 mM ATP
(for FLAG-LIN-1) in kinase buffer. Reactions were incubated at 30°C
for 30 min. Phosphorylated complexes were resolved on 9% SDS-PAGE gels,
transferred to nitrocellulose filters, and analyzed using phosphoimaging
or Western Blotting. All, or nearly all, of GST-LIN-31 and FLAG-LIN-1 are
phosphorylated under these conditions (Figures 2B and 3D).
Cell Culture and DNA Transfections
Transfections into NIH 3T3 cells were performed in 60 mm dishes
with 5 µg of total DNA using Superfect (Qiagen), according to the
directions of the manufacturer. Cells were harvested 48 hr post-transfection
for analysis on SDS-PAGE (12% for LIN-31 or LIN-31(PhD), and 9% for LIN-1).
Vectors expressing HA-epitope-tagged LIN-31 and LIN-31(PhD) were constructed
by inserting full-length lin-31 or lin-31(PhD) cDNAs containing a single
C-terminal HA epitope into the mammalian expression vector pcDNA 3.1(+)
(Invitrogen). The lin-1 expression vector was created by inserting a full-length
lin-1 cDNA containing an N-terminal FLAG epitope into the mammalian expression
vector pCB7 (gift of R. Coffey, Vanderbilt University). Vectors expressing
H-Ras(V12) (pEF-Ras) and activated MEK1(*N3, S222D) were the gifts of G.
Crabtree (Stanford University) and M. Schwartz (Scripps Research Institute,
La Jolla), respectively.
Binding assays
For co-infection experiments, Sf9 cells were co-infected with
recombinant baculoviruses expressing FLAG-LIN-1 and either GST-LIN-31 or
GST-USP (gift from M. Arbeitman, Stanford University) at an M.O.I. of 3.
Cells (5 x 106 cells in 1 ml) were lysed 48 hr post-infection in binding
buffer (50 mM Tris-Cl 7.5, 250 mM NaCl, 1 mM DTT, 0.2% NP-40, 1x protease
inhibitor cocktail (Pharmingen)); and centrifuged at 30,000 rpm for 30
min. GST fusion proteins were purified at 4°C using glutathione beads,
washed with 4 x 10 bed volumes of binding buffer, and resolved on 10% SDS-PAGE
gels. FLAG-LIN-1 was detected using M2 monoclonal antibodies (Kodak) in
Western blotting experiments.
In vitro binding assays were performed using independently-purified
GST-LIN-31 or FLAG-LIN-1. GST-LIN-31 (A, B, C) and the negative control
GST-LIN-7 (gift of S. Kaech) were expressed and purified in E. coli under
standard conditions (Pharmacia). GST-proteins and FLAG-LIN-1 (1 µg
each) were co-incubated in 200 µl binding buffer for 1 hr at 25°C.
GST fusion proteins were then anchored using glutathione beads, and washed
in 4 x 10 bead volumes of binding buffer. Proteins that remained bound
to GST-LIN-31 were resolved on 10% SDS-PAGE gels and analyzed by Western
blotting. In experiments where MAP kinase was also used, binding reactions
also included 2 mM MgCl2 and 0.5 mM ATP, and 0.1 µg of each protein
was used. In experiments involving MAP kinase pre-incubations, phosphorylated
proteins were extensively washed with binding buffer including 10 mM EDTA
and no ATP to inactivate MAP kinase before testing their protein-binding
ability.
Acknowledgments
We thank members of the Kim lab for discussions and critical comments. We thank M. Arbeitman, R. Coffey, G. Beitel, G. Crabtree, S. Kaech, Martin Schwartz, and B. Waterston for reagents. Some of the strains used in this work were supplied by the C. elegans Genetics Center. This work was supported by a Fairchild Foundation grant to P.T., a NIH training grant to M.L., and a NIH grant to S. K. K.
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Figure 1 : lin-31 acts Genetically Downstream of mpk-1 MAP kinase
Vulval induction at the L4 stage in (A) wild-type, (B) mpk-1(ga117), (C) lin-31(n1053), and (D) lin-31(1053); mpk-1(ga117) animals. mpk-1(ga117) animals are 100% Vul (M. Lackner, manuscript in prep.), lin-31(n1053) animals are 40% Vul and 61% Muv, and lin-31(n1053); mpk-1(ga117) animals are 35% Muv. The percentage of lin-31; mpk-1 animals that are Vul was not determined, as the Vul phenotype (scored as defective egg-laying) was not scored due to sterility caused by the mpk-1(ga117) mutation. Hypodermal cells derived from the 3° fate are shown as white lines, and vulval cells derived from 1° and 2° cell fates are shown as arrows. Scale bar is 20 µm for A, C, D, and 10 µm for B.
Figure 2 : LIN-31 and LIN-1 are Phosphorylated by MAP kinase in vitro and in vivo
(A) In vitro phosphorylation of LIN-31 by MAP kinase. GST-LIN-31 proteins
possessing all 4 MAP kinase phosphorylation sites (lane 2), 3 sites, T145M
(lane 3), or no sites, T145M, T200A, T218A, T220A (lane 4) were incubated
with 32P g-ATP and activated rat ERK2. The top figure indicates the relative
amount of 32P g-ATP incorporated into the test substrate. The bottom figure
is a loading control, showing the same blot stained with a-LIN-31 antibodies.
Quantitative analysis using a phosphoimager indicates that the amount of
incorporation in lane 3 is approximately 50% that of lane 2. LIN-31 is
phosphorylated 25% as well as the reference substrate Myelin Basic Protein
(P.T., data not shown).
(B) In vitro phosphorylation of FLAG-LIN-1 by MAP kinase. Epitope-tagged
FLAG-LIN-1 protein was incubated with ATP in the absence or presence of
activated ERK2. Proteins were separated on a 9% SDS-PAGE gel, blotted,
and probed with a-FLAG antibodies. Phosphorylated FLAG-LIN-1 appears as
a slower migrating band on SDS-PAGE (left). Treatment with l phosphatase
(NEB) confirms that the slower migration is due to phosphorylation (right).
(C) Stimulation of NIH 3T3 cells by activated H-Ras (lanes 2, 4) or
activated MEK1 (lanes 6, 8) causes a retardation in electrotrophoretic
mobility (LIN-31-P) of HA epitope-tagged LIN-31, but not LIN-31(PhD). The
lowermost band in lanes 6 and 8 (AM*) is a proteolytic product of the activated
MEK1 construct (which also possesses a HA epitope tag), and is seen in
cells transfected with activated MEK1 alone. Phosphorylation causes HA-LIN-31
(C) but not GST-LIN-31(A) to migrate more slowly on SDS-PAGE gels, possibly
because the former is smaller (26 kD) than the latter (56 kD).
(D) Stimulation of NIH 3T3 cells by activated H-Ras (lane 2) or activated
MEK1 (lane 4) causes a mobility retardation (LIN-1-P) in epitope-tagged
FLAG-LIN-1.
Figure 3 : LIN-1 binding to LIN-31 is Inhibited by MAP kinase
(A) LIN-1 binds to LIN-31 in vivo. GST-LIN-31 was purified from Sf9
cells co-expressing GST-LIN-31 and FLAG-LIN-1 using glutathione beads.
FLAG-LIN-1 (arrow) associated with GST-LIN-31, and not with another transcription
factor, GST-USP. Equal amounts of FLAG-LIN-1 and GST-fusion protein were
produced in both lysates (left and data not shown).
(B) LIN-1 and LIN-31 directly interact in vitro. Overlapping regions
of LIN-31 (Regions A (aa 1-39), B (aa 39-169), and C (aa 169-237)) were
made as bacterial GST fusion proteins, purified using glutathione beads
and tested for their ability to bind FLAG-LIN-1 using a-FLAG antibodies
in Western blotting experiments (right gel). Only full length GST-LIN-31
protein and GST-B (aa 39-169) bound to FLAG-LIN-1. FLAG-LIN-1 did not bind
to GST-A, GST-C, or the negative control GST-LIN-7.
(C) Inhibition of LIN-1 and LIN-31 interaction by MAP kinase. GST-LIN-31
and FLAG-LIN-1 were independently purified and then co-incubated in the
presence or absence of rat ERK2 and ATP. Both proteins were mixed, and
GST-LIN-31 was subsequently isolated using glutathione beads. Bound FLAG-LIN-1
was detected using a-FLAG antibodies on Western blots, and was only seen
to associate with GST-LIN-31 when MAP kinase was absent from the reaction
mix.
(D and E) Phosphorylation of LIN-31 inhibits binding to LIN-1. (D)
Unphosphorylated FLAG-LIN-1 binds to unphosphorylated GST-LIN-31, but not
pre-phosphorylated GST-LIN-31-P (prepared by pre-incubation with ERK2).
(E) In the reciprocal experiment, unphosphorylated GST-LIN-31 binds to
both pre-phosphorylated and unphosphorylated FLAG-LIN-1.
(A-E) Gels on the left are fusion protein loading controls, showing
that equivalent levels of bait protein were used. 10% inp. indicates 10%
of the total float protein present in the extract before binding.
Figure 4 : Vulval Phenotypes Caused by LIN-1::LIN-31, LIN-31(PhD) and
LIN-31(VP16)
(A-D) Vulval induction is normal in animals that express lin-31(+)
(A) or lin-1(+) transgenes(Table 1). Expression of the lin-1::lin-31 forced
heterodimer (B) or lin-31(PhD) (C) blocks vulval induction. Expression
of a lin-31(VP16) promotes vulval induction (D). White lines indicate hypodermal
cells derived from Pn.p cells that have adopted non-vulval 3° or U
cell fates rather than vulval 1° or 2° cell fates. White arrows
indicate cells derived from Pn.p cells that have expressed vulval 1°
or 2° cell fates. The exact vulval cell lineages of the animals in
(B) and (C) are shown in Table 2 (animal B3 and C5). Scale bar is 10 µm
for (A-C), and 25 µm for (D).
Figure 5 : LIN-31 expression pattern
a-LIN-31 staining is shown in green, MH27 staining is shown in red, and DAPI staining in blue. (A) Expression pattern of LIN-31 in wild-type animals at the L2 stage. LIN-31 is localized in the nuclei of the Pn.p cells (P1.p-P11.p), including the vulval precursor cells (P3.p-P8.p). MH27 stains the cell junctions of P3.p - P8.p at this stage. (B) LIN-31 expression is not observed in lin-31(ga37) or in lin-31(n1053) animals (P. Tan, unpublished observations). (C) DAPI staining at the L4 stage shows the gonad (large arrows). (D) LIN-31 is expressed in the vulval precursor cell descendants (arrows), but not in the gonad or sex myoblasts, which flank the P6.p progeny during this time. (E) At the L1 larval stage, LIN-31 is not expressed in the excretory duct cell (exc) or in P12.p, but is expressed in P1.p to P11.p. L1 larval stage worms were identified by the presence of MH27 staining in P9.p - P12.p, indicating that these cells have not yet fused with the syncitial hypodermis (which normally occurs at the mid-L1 stage). (F) At this same stage, let-23(mn23) (shown) and let-60(null) mutants (Yochem et al., 1997; and P.T. unpublished observations) arrest in development indicating prior function of this signaling pathway in cells comprising the excretory system. (G) In males 25 hr after hatching, LIN-31 is expressed in 3 of the 4 B cell progeny (B.a(l or r)pp, B.alap, B.arap, but not B.a(l or r)aa) that utilize LET-23 RTK signaling. Green indicates nuclei expressing LIN-31 located in the sagital mid-line (same plane as MH27 staining), while blue indicates nuclei expressing LIN-31 located laterally left of the mid-line. Other cells expressing LIN-31 during the L2/L3 stage in the hermaphrodite include the excretory duct cell (exc) and the neuronal cell T.ppa (known as PVWL/R). Scale bar for (A-G) is 10 µm.
Figure 6 : Ectopic expression of lin-31 in P12 Causes Induction of a Vulval-specific Marker
(A) LET-23 staining of a wild-type animal at the mid-L2 larval stage reveals that P6.p exhibits increased LET-23 RTK expression (100% of animals, n=50). Staging of worms and identification of Pn.p cells was achieved by co-staining animals with MH27 (data not shown). (B) In a lin-31(n1053) mutant, increased expression of LET-23 RTK is observed in only 46% of animals (n=50). (C) Increased LET-23 RTK expression is not observed in P12 in a wild-type L1 animal (5 hrs after hatching, n=15). (D) Ectopic expression of LIN-31 in P12 from a heat-shock promoter results in increased expression of LET-23 RTK in P12 in 33% of observed animals (n=12, see Experimental Procedures). Increased LET-23 RTK expression was not observed in heat-shocked controls that did not carry the hs-lin-31 transgene (n=20, data not shown). Scale bar is 10 mm.
Figure 7 : Models for LIN-31 Function and as a Tissue-Specific Effector of MAP kinase Signaling
(A) LIN-31 function in Vulval Development. See text for details. In
cells where MAP kinase is inactive (P3.p, P4.p and P8.p), the LIN-1/LIN-31
complex inhibits vulval induction. Conversely, in P6.p (and possibly P5.p
and P7.p) where MAP kinase is active, LIN-1 and LIN-31 dissociate and phosphorylated
LIN-31 promotes vulval induction. In a lin-31 loss-of-function mutant,
the absence of LIN-31 results in a failure of LIN-31 target genes to be
either actively repressed or activated. Arrows and inhibitory bars reflect
genetic activities, and may not necessarily indicate direct transcriptional
activation or repression. For example, the LIN-31/LIN-1 complex may act
as a vulval inhibitor complex by transcriptionally activating the expression
of a vulval repressor gene.
(B) LIN-31 is a Tissue-Specific Effector of MAP kinase Signaling. The
Ras/MAP kinase signaling pathway is used in the development of at least
six distinct tissues in the nematode. LIN-31 may transduce the mpk-1 signal
specifically in the vulva, while LIN-1 may act as a more general effector
of this signaling pathway.
Table 1. Vulval phenotypes of modified lin-31 transgenes
lin-31 Percent
Transgene Genotype Muva Egl/Vula No.b
- WT 0 0 Many
lin-31(+) WT 0 4±2 >200
lin-1 (+) WT 0 3±3 156
lin-1::lin-31 WT 0 50±7 187
gfp::lin-31 WT 0 3±3 80c
lin-31(PhD) WT 0 42±7 194
lin-31(DBR) WT 5±7 20±5 188
lin-31(VP16) WT 33±6 5±3 162c
- lin-31(n1053) 61 40 Many
lin-31(+) lin-31(n1053) 12±4 10±3 >250
lin-1::lin-31 lin-31(n1053) 23±6 77±6 173
gfp::lin-31 lin-31(n1053) 10±4 9±4 181c
lin31(PhD) lin-31(n1053) 10±4 62±7 202
lin31(VP16) lin-31(n1053) 87±4 ND 195
a Numbers indicate the mean ± 95% confidence interval.
b No. of animals scored
c rol-6+ was used as a transgenic marker. All other strains were generated
using unc-29+.
Table 2. Vulval lineages in animals expressing lin-31(PhD)
Genotype P3.p P4.p P5.p P6.p P7.p P8.p
A. N2 SS or U SS LLTN TTTT NTLL SS
3° 3° 2° 1° 2° 3°
B. lin-31(+); Ex[lin-1::lin-31)
U 3° 3° 1° 3° 3°/ U
U 3° 3° TTN 3° 3°
U /3° 3° 3° 3° 3° 3°
U 3° U 3° 3° 3°
U U 3° 1° 3° 3°
C. lin-31 (+); Ex [lin-31(PhD)]
3° 3° 2° 1° 2° 3°
U 3° 3° LOTT 2° 3°
3° 3° 2° 1° 3° 3°
U 3° U LLNT 3° 3°
U 3° 3° 1° U 3°
D. lin-31 (n1053); Ex [lin-31(PhD)]
3° 3° 3° LTN U 3°
U U NTLL 1° 3° 3°
3° 3° NTLL 1° 3° 3°
U 3° LLTN TLN 3° 3°
U 3° 3° TON 3° 3°
Wild-type vulval lineages are indicated in section (A), with the following
abbreviations : L - longitudinal division, underlining indicates adherence
to the ventral cuticle; O - oblique division, T - transverse division;
N - Pn.px division did not occur, U - Pn.p cell did not divide to give
progeny, S - Pn.px fused with syncitial hypodermis. 1° = TTTT, 2°
= LLTN or NTLL, and 3° = SS or U (for P3.p). Inappropriate or defective
lineages are boxed. Lineages designated 3°/U could not be unambiguously
determined a 3° or U. All animals were lineaged at 20°C.