Departments of Developmental Biology and Genetics, Howard Hughes Medical Institute, Clark Center W252, Stanford University School of Medicine, 318 Campus Drive, Stanford, CA 94305-5439, USA
Author for correspondence (e-mail:
scott{at}cmgm.stanford.edu)
Accepted 10 January 2005
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SUMMARY |
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Key words: Kinesin, Morphogen, Differential gene regulation, Hedgehog, Costal2, Suppressor of Fused, Drosophila
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Introduction |
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Hh signal emanating from the posterior (P) Drosophila wing
imaginal disc induces transcription of decapentaplegic
(dpp), patched (ptc) and anterior
engrailed (en) in stripes of cells within the anterior (A)
compartment of the wing imaginal disc, adjacent to the anteroposterior (AP)
boundary. dpp is induced in a 12- to 15-cell-wide stripe,
ptc in a 10-cell-wide stripe, and en in a 5- to
7-cell-wide stripe (see Fig. S1 in the supplementary material). The regulation
of these target genes is differentially sensitive to Hh dose: modulating the
level of Hh produced by P cells dramatically affects the presence and width of
the stripes of target gene expression
(Strigini and Cohen, 1997
).
Transcriptional activation of all three target genes by Hh is dependent on the
function of the transmembrane protein Smoothened (Smo) in the responding
cells, indicating that Hh acts directly on these target cells and not through
a relay system involving other signaling pathways
(Vincent and Briscoe,
2001
).
Downstream of Smo, the zinc-finger transcription factor Ci controls the
transcriptional responses to Hh (Dominguez
et al., 1996; Methot and
Basler, 2001
). In the absence of Hh, as in anterior disc cells
distant from the Hh source, full-length Ci (CiFL) is
proteolytically processed into a truncated form, CiR, that consists
of the N terminus of Ci and its zinc-finger binding domain. CiR is
a repressor of dpp and hh
(Aza-Blanc et al., 1997
). The
Hh signal opposes the proteolytic processing of Ci, which stabilizes
CiFL, and causes the nuclear accumulation of CiFL, which
is sufficient to induce dpp transcription.
Additional Hh-mediated events that further activate Ci are necessary for
the transcription of ptc
(Ohlmeyer and Kalderon, 1998;
Chen et al., 1999a
;
Methot and Basler, 1999
;
Wang and Holmgren, 1999
;
Wang et al., 2000
). This
activated form, CiACT, has thus far evaded biochemical
characterization, but its existence is suggested by experiments showing that
expression of stabilized CiFL is insufficient to induce
ptc and en transcription to levels comparable with wild-type
levels (Jiang and Struhl,
1998
; Methot and Basler,
1999
; Chen et al.,
1999a
).
Ci exists in the cytoplasm as a component of high molecular weight protein
complexes (Robbins et al.,
1997; Sisson et al.,
1997
). Members of these complexes include Costal2 (Cos2;
cos - FlyBase), which is a kinesin-related protein, Fused (Fu), which
is a Ser/Thr kinase, and Suppressor of Fused [Su(fu)], which is novel
PEST-motif containing protein (Robbins et
al., 1997
; Sisson et al.,
1997
; Monnier et al.,
1998
; Stegman et al.,
2000
; Monnier et al.,
2002
; Stegman et al.,
2004
; Wang and Jiang,
2004
).
Cos2 is an important negative regulator of dpp and ptc in
wing imaginal discs. Loss of Cos2 from anterior cells derepresses ptc
and dpp transcription
(Sanchez-Herrero et al., 1996;
Wang and Holmgren, 2000
;
Wang et al., 2000
) (this
paper), indicating that Cos2 inhibits the positive transcriptional activities
of Ci. It may do this in several ways: Cos2 activity opposes the nuclear
translocation of CiFL (Chen et
al., 1999a
; Stegman et al.,
2000
; Wang et al.,
2000
; Monnier et al.,
2002
), retaining it in the cytoplasm in a microtubule-dependent
manner (Wang and Jiang, 2004
).
Some evidence suggests that Cos2 is also involved in proteolytic processing of
Ci to CiR (Wang and Holmgren,
2000
).
Cos2 can also contribute to target gene activation as well. In some cells
near the AP boundary, Cos2 is required for ptc and Hh-dependent
en expression (Wang and Holmgren,
2000; Wang et al.,
2000
). Recent work has shown that Cos2 binds to Smo and links Smo
to the cytoplasmic complex (Jia et al.,
2003
; Lum et al.,
2003
; Ogden et al.,
2003
; Ruel et al.,
2003
). Consistent with this, Cos2 interacts with membranes
differentially in response to Hh signaling
(Stegman et al., 2004
). The
Cos2-Smo interaction is necessary to transduce a response to a high level of
Hh and to turn on en transcription in A cells
(Jia et al., 2003
) and the
ptc-luciferase reporter in cultured cells
(Lum et al., 2003
;
Ogden et al., 2003
;
Ruel et al., 2003
). Thus, Cos2
is both a negative and positive regulator of Hh target genes.
An intriguing feature of Cos2 is its sequence similarity to kinesins,
dimeric molecular motors that bind and move along microtubules carrying
organelles, vesicles, proteins and other cargo to destinations within the
cell. Cos2 binds microtubules in vitro and is released from them in a
Hh-dependent manner (Robbins et al.,
1997; Wang and Jiang,
2004
). Sequence alignments of Cos2 with other kinesins indicate
that it has an N-terminal motor domain, followed by a putative `neck' domain,
a middle region of heptad repeats that form the coiled-coil dimerization
domain, and a unique C-terminal domain that could confer cargo binding
specificity (Sisson et al.,
1997
).
The Cos2 motor domain sequence is quite divergent
(Sisson et al., 1997;
Lawrence et al., 2004
),
indicating that its function may differ from those of classical kinesins.
Despite the sequence differences, within the Cos2 motor domain is a
well-conserved P-loop, a motif that is necessary for binding ATP and
catalyzing the hydrolysis reaction necessary for translocation along
microtubules (Muller et al.,
1999
; Rice et al.,
1999
). The presence of a P-loop in Cos2 suggests that ATPase
activity is important for Cos2 function.
To assess the importance of the putative motor, neck and cargo domains to Cos2 in Hh signaling, we made deletion constructs of Cos2 lacking each domain. In addition, we changed the Ser182 of Cos2 to Asn (S182N) in the P-loop, which in other kinesins gives rise to a dominant-negative form that lacks ATPase activity. Using these mutant forms of Cos2, we investigated the roles of Cos2 and Su(fu) in the regulation of the Hh target genes dpp and ptc. Our data indicate that differential regulation of dpp and ptc occurs by modulation of Cos2 and Su(fu) activities.
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Materials and methods |
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The reporter strain H84 ptc-lacZ (a gift from M. Fietz) is an
nuclear lacZ enhancer trap in the ptc locus.
dpp-lacZ is a transgene constructed from the
dppdisk promoter driving nuclear lacZ
(Sanicola et al., 1995).
UAS-cos2, UAS-cos2GFP, UAS-S182N, UAS-S182N-GFP, UAS-S182T,
UAS-S182T-GFP, UAS-Motor, UAS-
Neck and
UAS-Cos2
C were generated by P-element-mediated
transformation of the constructs described below. To control for position
effects, at least two different insertion lines were used to verify each
result.
Generation of FLP-FRT and FLP-out clones
The heat shock regimen for making cos2- clones was as
described (Sisson et al.,
1997). For making FLP-out overexpression clones, flies were mated
at 20°C, heat shocked 48 hours after egg laying (AEL) for 30 minutes,
allowed to recover at room temperature for 30 minutes, and again heat shocked
for 30 minutes. The heat-shock regimen was conducted on larvae twice a day
(8-12 hours apart) for 3 days.
Plasmid construction
pUAS-cos2 was made by inserting a 3.4 kb fragment encoding the
cos2 open reading frame (ORF) into pUAST
(Brand and Perrimon, 1993).
pUAS-cos2-GFP was made by fusing the GFP ORF from
pBD1010 (gift from Barry Dickson), cut with XhoI (blunted with Klenow
enzyme, NEB) and XbaI, to pUAS-cos2 cut with MamI
and XbaI. This fuses the GFP ORF in frame with the
C-terminal end of Cos2, eight amino acids upstream of the STOP codon. The
fusion amino acid sequence reads as follows: NKIIEGTKM..., where M
is the normal start codon of GFP and Cos2 amino acids are underlined.
To make pUAS-Neck-GFP, pUAS-cos2-GFP was cut with
NgoMI and re-ligated. This results in a 143 amino acid in-frame
deletion of the neck region of Cos2. The deletion also removes two conserved
microtubule binding sites and 78 amino acids of the motor domain.
To make Motor, two complimentary oligonucleotides were synthesized
such that, when annealed, they form a double stranded oligonucleotide with
NcoI and AatII compatible ends. This
Motor
oligonucleotide encodes a 6xHis tag in frame with the cos2 ORF.
Oligonucleotide sequences are as follows:
5'-CATGCACCACCACCACCACCACGGACGT-3' and
5'-CCGTGGTGGTGGTGGTGGTG-3'. A pBS-KS plasmid containing the
cos2 ORF was cut with NcoI and AatII and the
Motor oligo inserted into this vector. A SpeI fragment from
this
Motor construct was then inserted into
pUAScos2-GFP and pUAS-cos2 to create
pUAS-
Motor-GFP and pUAS-
Motor constructs, respectively. The Cos2
protein encoded by
Motor plasmids is missing the first 313 amino acids
and has a 6xHis tag at the N terminus.
To make pUAS-Cos2C-GFP, two complementary oligonucleotides were
synthesized:
5'-CCGGTGCACCACCACCACCACCACGAGCAGAAGCTTATATCAGAAGAAGATCTGGGTACCTAAGC-3'
and
5'-GGCCGCTTAGGTACCCAGATCTTCTTCTGATATAAGCTTCTGCTCGTGGTGGTGGTGGTGGTGCA-3'.
These oligonucleotides were annealed and cut with KpnI and ligated to
pUAS-cos-GFP cut with SgrAI and KpnI. The resulting
construct encodes Cos2 up to amino acids 1057, after which the C-terminal end
is deleted and replaced by a 6xHis tag followed by fusion in frame with
GFP.
To make S182T and S182N mutant insertions, a PCR-based mutagenesis strategy
was used. The forward primers encode the appropriate point mutation
(underlined): S182T forward primer,
5'-CCAGCGCGGCCAAGGCAAAACCTACACACTCTAC-3'; S182N forward
primer, 5'-CCAGCGCGGCCAAGGCAAAAACTACACACTCTAC-3';
reverse primer, 5'-TGCCATTAACCCCGTACATGAG-3'. PCR products were
cut with BalI and AatII, and ligated to pRV3.9
(Sisson et al., 1997) cut with
BalI and AatII to make pBS-S182T or pBS-S182N, respectively.
To make pUAS-S182T, pUAS-S182N, pUAS-S182T-GFP and pUAS-S182N-GFP, pBS-S182T
or pBS-S182N was cut with BstEII and NheI and inserted the
fragments to pUAS-cos2 and pUAS-cos2-GFP cut with
BstEII and NheI. The resulting plasmids encoded a single
amino acid substitution at amino acids 182 to Thr or Asn, respectively. For
the rescue experiment, pUAS-cos2-GFP were cut with
NheI and XbaI (blunted with Klenow) and ligated this
fragment to pK6.5 (Sisson et al.,
1997
) cut with NheI and MamI. This replaced the
C-terminus of Cos2 with the C-terminal of Cos2-GFP from
pUAS-cos2-GFP described above. Each genomic rescue fragment
was inserted into pCasper4. To make the Cos2
C-GFP rescue construct, the
same strategy was used, starting with pUAS-Cos2
C-GFP instead of
pUAS-cos2-GFP.
Immunohistochemistry and in situ hybridization
Staining was performed using Brower's Fix as described
(http://bender.zoology.wisc.edu/antiweb.html)
using antibodies at the following dilutions: anti-CiFL 2A1 (1:5) (a
gift from Robert Holmgren); anti-ßGal, mouse (Promega) 1:1000;
anti-ßGal, rabbit (Promega) 1:1000; anti-En 4D9, mouse monoclonal (a gift
from Nipam Patel), 1:1000; anti-Myc mouse monoclonal (Sigma) 1:500; anti-Cos2
0.8 rat polyclonal, prepared and used as described previously
(Sisson et al., 1997).
Fluorescent secondary antibodies (Jackson ImmunoResearch Laboratories) were
used at 1:200. In situ hybridization was carried out as described by Johnson
et al. (Johnson et al., 1995
).
dpp and ptc probes: ptc probe was a gift from Alan
Zhu. dpp probe was made with the Genius 4 kit (Promega); the template
was a gift from Michael Hoffman.
Imaginal disc lysates and protein blots
Third instar wing imaginal discs (100-500) were dissected in Clone 8 cell
culture media, made as described
(http://www.stanford.edu/~rnusse/ownpage/protdisccells.html),
supplemented with a cocktail of protease inhibitors: PMSF, benzamidine,
aprotinin, pepstatin, chymostatin, leupeptin (PIs). Discs were rinsed once in
phosphate-buffered saline (PBS) supplemented with PIs and pelleted for 30
seconds in a microfuge at 4°C. Excess PBS was removed, the pellet of discs
was resuspended in 10 µl 6 x SDS lysis buffer (350 mM Tris-Cl, pH
6.8, 10% SDS, 30% glycerol, 0.093 g/ml DTT) and dounce homogenized. PBS (50
µl) was added. SDS-PAGE electrophoresis was carried out using standard
methods with a mini-Protean3 BioRad gel apparatus
(Sambrook and Russell, 2001).
Antibodies used for protein blots were as follows: anti-Su(fu) rat polyclonal
1:1000; anti-BAP111 (gift from Janet Jin, Ophelia Papoulos 1:1000; anti-Dsh
rabbit polyclonal (a gift from K. Willert and R. Nusse) 1:1000;
anti-CiFL 2A1, anti-Ci 1C2, rat monoclonal 1:5 (gifts from Robert
Holmgren); anti-Cos2 0.8 rat polyclonal, prepared and used as described
previously (Sisson et al.,
1997
). Quantitation of protein blots was carried out using
AlphaImager 2000 software (AlphaInnotech)
(Chen et al., 1999a
). For
phosphatase reactions, lysates were treated with AG1 X2 resin (BioRad) to
remove SDS using the method described
(Weber and Kuter, 1971
;
Galko and Tessier-Lavigne,
2000
), spun briefly to pellet resin, and the supernatant was
removed to fresh tubes. Lambda phosphatase (NEB) was added for 2 hours at
30°C, then the proteins were run on a SDS-PAGE gel and immunoblotted.
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Results |
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Expression of S182N in wing discs derepresses dpp expression in A cells
Because the Motor and
Neck deletion constructs had altered
Cos2 activity, we focused our attention on this N-terminal region. In other
kinesin family proteins, the motor and neck domains are necessary for ATP
hydrolysis and the conformational changes necessary for microtubule-based
movement (Woehlke and Schliwa,
2000
). Within the motor domain of Cos2 is a conserved P-loop
motif, which in conventional kinesins is necessary for ATP hydrolysis. In
order to determine whether an intact P-loop is important for normal Cos2
function, we used site-directed mutagenesis to change the conserved Ser at
position 182 to Asn, generating a form of Cos2 designated S182N. Mutation of a
conserved Ser or Thr to Asn at that position in the P-loop has been shown to
give rise to dominant-negative kinesins that dimerizes with their endogenous
kinesin partners, irreversibly bind microtubules, lack ATPase activity and
cannot move (Meluh and Rose,
1990
; Rasooly et al.,
1991
; Blangy et al.,
1998
). These mutant kinesins decorate microtubules in mammalian
cultured cells and inhibit the movements of their endogenous kinesin partners
(Blangy et al., 1998
;
Nakata and Hirokawa,
1995
).
S182T was generated as a control for the S182N mutant protein. This
conservative Ser to Thr change is not expected to alter normal ATPase activity
(Nakata and Hirokawa,
1995).
To determine the effect of S182N production on dpp-lacZ
activation, the FLP-out system (Pignoni
and Zipursky, 1997) was used to generate clones of cells that
express S182N in either the anterior or posterior wing disc
compartments. Strikingly, dpp-lacZ was consistently derepressed in
anterior S182N-expressing clones in a cell-autonomous manner
(Fig. 1K,L). The derepression
of dpp-lacZ occurred regardless of the position of the anterior clone
with respect to the AP boundary (Fig.
1K). The dpp-lacZ expression level in
S182N-expressing clones (Fig.
1K, white arrowhead) is comparable with its Hh-dependent
expression level at the AP boundary (Fig.
1K, yellow arrowhead). Discs containing large
S182N-expressing clones frequently had dramatic overgrowths of
anterior tissue (Fig. 1K, white
arrow). As with cos2 loss-of-function clones, no ectopic dpp
expression, or disc outgrowth, was observed in posterior compartment clones
(Fig. 1K, blue arrowhead). By
contrast, producing the control S182T protein in clones repressed dpp
expression in a manner comparable to overexpression of wild-type cos2
(Fig. 1M,N).
Anterior wing duplications in cos2 loss-of-function clones arise
because of ectopic dpp expression within those clones
(Capdevila and Guerrero, 1994).
To test whether dpp derepression in S182N-expressing cells gives rise
to adult wing duplications, S182N and S182T mutant proteins were produced
throughout the wing pouch using the MS1096 Gal4 driver. In contrast to
overexpression of wild-type cos2 and cos2GFP, expression of
S182N or S182N-GFP gave rise to anterior wing duplications, with 100%
penetrance and variable expressivity. The range of severity of the phenotypes
generated by S182N expression is shown in
Fig. 2B,C. The anterior wing
duplications observed mimic the phenotypes of hypomorphic alleles of
cos2, which are large duplications, and sometimes triplications, of a
proximal anterior wing structure called the costa (arrow,
Fig. 2A,B), from which
costal2 gets its name (Whittle,
1976
). By contrast, no wing duplications occurred when S182T-GFP
was overexpressed in the wing pouch using the MS109-Gal4 driver
(Fig. 2D). Instead, the
phenotype of S182T expression mimicked overexpression of wild-type
cos2 and cos2GFP (Fig.
2E).
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S182N expression results in the production and stabilization of Ci in its full-length form
CiFL stabilization is sufficient to induce dpp
transcription in the wing (Methot and
Basler, 1999; Chen et al.,
1999b
; Wang et al.,
2000
). To see how S182N expression affects CiFL levels
and stability, discs expressing S182N under the control of the 71B Gal4 driver
were stained with antibodies against CiFL. The pattern of 71B
Gal4-driven expression is shown in Fig.
4D.
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In order to see whether S182N interferes with the proteolytic processing of CiFL into CiR, thus stabilizing CiFL, protein blots of wing disc extracts were stained with an antibody that detects both CiR and CiFL (Fig. 4E,F). Multiple bands representing CiR are observed in the 75 kDa range; these are presumably isoforms of CiR (Fig. 4E). In Hh overexpressing discs (Fig. 4E, lane 2), Ci is stabilized in its full-length form and all CiR isoforms are undetectable. In S182N-expressing discs, the amount of CiFL is increased compared with its level in wild-type, cos2-overexpressing or S182T-expressing discs (Fig. 4E, lane 5, compare with lanes 1, 3, 6). Interestingly, CiR is also present in S182N-expressing discs (Fig. 4E, lane 5 compare with lanes 1, 3, 6). This indicates that proteolytic processing of Ci may persist in the presence of S182N, and that S182N may affect the stability of CiFL independently of Ci processing.
Quantification of the relative amounts of CiR versus total Ci
concentration in S182N-producing discs showed a significant reduction in
relative CiR concentration. Error bars show the standard deviations
of three independent experiments (Fig.
4F). This altered ratio, which favors CiFL, is likely
to account for the ectopic activation of dpp in anterior cells where
S182N is expressed, as stabilization of Ci in its full-length form is
sufficient to activate dpp transcription
(Methot and Basler, 1999).
S182N represses ptc at the AP boundary and does not activate ptc in anterior cells
cos2 is necessary to repress ptc and dpp in A
cells, and overexpression of cos2 represses both ptc and
dpp in cells at the AP border (see Fig. S1 in the supplementary
material), so a dominant inhibitor of Cos2 such as S182N is expected to induce
ptc as well as dpp. To test this hypothesis, discs
expressing S182N were stained for ptc-lacZ expression. Contrary to
expectations for a dominant-negative Cos2 mutant, S182N repressed
ptc-lacZ expression at the AP boundary, instead of inducing extra
ptc-lacZ expression in anterior cells
(Fig. 5B,C). This repressive
activity of S182N may be responsible for the similarities in size and AP
boundary defects in wings producing S182N and wild-type Cos2 or S182T
(Fig. 2C-E). Furthermore,
production of S182N, S182T or Cos2 using strong wing pouch GAL4 drivers or in
FLP-out clones, does not activate ptc in the anterior compartment
away from the AP boundary (Fig.
5 and data not shown). With respect to ptc regulation,
then, S182N activity is similar to wild-type Cos2 and S182T activity
(Fig. 5D,E and see Fig. S1I,J
in the supplementary material).
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To see whether the transcriptional activity of stabilized CiFL in S182N-expressing cells is subject to regulation by Su(fu), we removed one copy of Su(fu) from discs producing S182N and monitored ptc-lacZ expression. The removal of one copy of Su(fu) from discs expressing S182N caused a dramatic induction of ptc-lacZ expression throughout the anterior compartment of the disc (Fig. 5G,H, compare with 5A-C,F,I,J). Su(fu) did not have this effect on discs in which wild-type cos2 or S182T was overexpressed. In S182N discs lacking one copy of Su(fu), the induction of ptc-lacZ consistently appears higher in the dorsal than in the ventral compartment, especially near the AP compartment boundary (Fig. 5H, arrowhead).
The dose of Su(fu) is therefore crucial in determining the transcriptional outcome of S182N expression: if two wild-type copies of Su(fu) are present, S182N represses Hh-dependent ptc-lacZ expression at the AP boundary; if one copy of Su(fu) is inactivated, then S182N activates ptc-lacZ even in A cells far from the Hh source. By contrast, S182N activates dpp-lacZ regardless of Su(fu) copy number (Fig. 1K,L; data not shown), so activation of dpp by S182N is independent of Su(fu). S182T, like Cos2, always represses both target genes, regardless of Su(fu) copy number (Fig. 5D,E,I,J and Fig. 1M,N).
Su(fu) is phosphorylated in response to Hh
In order to determine how Hh signaling might control Su(fu) activity, we
examined Su(fu) protein in wild-type, hh overexpressing and
S182N-expressing discs. Protein blots of wing imaginal disc lysates
revealed that hh-overexpressing discs produce at least two
immunoreactive bands of Su(fu) instead of the single band seen in wild-type,
cos2, S182N and S182T-expressing discs
(Fig. 6A). The additional
slower-migrating Su(fu) band appeared whenever hh was overexpressed,
even in discs co-expressing hh with cos2 or with
S182N (Fig. 6A, lanes
6,7).
|
We conclude that Su(fu) is phosphorylated in response to Hh and that this activity is unperturbed by the presence of excess Cos2 or by the production of S182N. Phosphorylation of Su(fu) may reduce Su(fu) activity, thus allowing Hh target gene induction at the AP border of the wing disc.
S182N inhibits activator as well as repressor functions of Cos2
Cos2 has both activator and repressor functions in Hh signaling
(Wang and Holmgren, 1999;
Wang et al., 2000
;
Lum et al., 2003
;
Ogden et al., 2003
;
Jia et al., 2003
;
Ruel et al., 2003
). Thus far,
we used the dominant-negative mutant S182N to explore the role of Cos2 in
regulating dpp and ptc, target genes that require repression
by Cos2. We now turn to the effect of S182N expression on the target gene
engrailed (en), which requires Cos2 for activation by Hh
(Wang et al., 2000
). While
most en expression is located in the posterior compartment of the
disc, a narrow band of anterior cells, 5-7 cell diameters in width, expresses
en during the late third instar stage
(Blair, 1992
). This en
expression is dependent on high levels of Hh signaling. In cultured cells,
Cos2 has been shown to be required for maximal activation of the Hh pathway
(Lum et al., 2003
;
Ogden et al., 2003
;
Ruel et al., 2003
). To
demonstrate that cos2 is required for the Hh-dependent expression of
en in vivo, loss-of-function cos2 clones were generated
using the FLP-FRT system, and clones falling within the normal anterior
en-expressing zone were examined for en expression. Many
cos2 clones in the wing pouch lacked detectable en
expression, in agreement with a previous study
(Fig. 7)
(Wang et al., 2000
).
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![]() |
Discussion |
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Conventional kinesins require ATPase activity in order to move along
microtubules. Studies have shown that mutation of the conserved Ser or Thr at
a precise position in the P-loop causes the protein to become immobile,
locking itself and its cargo along microtubules prematurely, before the final
intracellular destination for the kinesin has been reached
(Meluh and Rose, 1990;
Rasooly et al., 1991
;
Blangy et al., 1998
).
Expression of such kinesin mutants specifically inhibits the movement of its
endogenous partner, but not the movements of other kinesins or dyneins along
the microtubule (Blangy et al.,
1998
; Nakata and Hirokawa,
1995
). We used this knowledge about kinesins and the importance of
their P-loops to design the equivalent mutation in Cos2. The mutation of amino
acid 182 of Cos2 to a conserved Thr does not detectably alter the function of
Cos2 in vivo, while mutation of the same residue to Asn clearly interferes
with normal Cos2 activity. This clearly suggests that Cos2 is likely to use
ATPase activity for either locomotion or conformational changes in response to
Hh signaling. The movement of Cos2 along microtubules in vitro has yet to be
demonstrated, but the importance of intracellular localization of various Hh
signaling components has been clearly demonstrated. Among the examples: in
response to Hh, Smo accumulates at the plasma membrane, and associates with
Cos2 and Fu; Ci accumulates in the nucleus in response to Hh signaling; and in
the absence of Hh signal, Smo is located in internal membranes in the
cytoplasm of responding cells, and Ci is continually exported from the
nucleus, phosphorylated by kinases, and processed into CiR by the proteasome.
How do the components arrive at the appropriate places to affect the
appropriate response? As a binding partner for all of these components and as
a kinesin-related protein, Cos2 is in a unique position to orchestrate some of
these events. We propose ideas for how it may accomplish this below.
Differential gene regulation by Hh
Our data suggest that the activities of Cos2 and Su(fu) are independently
regulated by different concentrations of Hh along the gradient that forms from
posterior to anterior (Fig. 8).
In the anterior cells distant from the AP boundary, little or no Hh is
received and target genes are silent. In these cells, Cos2 is required for
proteolytic processing of Ci into its repressor form
(Wang and Holmgren, 1999) and
possibly for the delivery of CiFL for lysosomal degradation. Our
data suggest that Cos2 requires an intact P-loop for its role these events.
Cos2 ATPase activity may be inhibited in cells receiving very low levels of
Hh, preventing Ci proteolysis and stabilizing CiFL. The
stabilization of CiFL results in the activation of dpp.
Nearer the AP border, where higher levels of Hh are received, Su(fu) becomes
phosphorylated, inactivating its negative regulatory hold on Ci, while
inhibition of the ATPase activity of Cos2 continues to allow stabilization of
Ci. In this situation, ptc and dpp are transcribed. Finally,
at the highest levels of Hh signaling adjacent to the AP border, Cos2 is
required for activation of the pathway and the expression of en.
S182N expression, or cos2 over-expression, inhibits the induction of
en by endogenous Hh in these cells. The elements of this model are
addressed below.
|
These results suggest that Cos2 may use its ATPase activity to transport Ci to a location where it becomes phosphorylated in preparation for processing, or to the site of processing itself. Alternatively, the ATPase activity may be important for regulating the conformation of Cos2 and its binding to partners such as Smo, Su(fu), Fu and Ci, which would be a novel role for the P-loop in a kinesin-related protein. The S182N mutation may lock Cos2 in a conformation that changes association with binding partners. For example, S182N may decrease the ability of Cos2 to bind Ci, releasing Ci from the cytoplasm, resulting in an increased level of CiFL in the nucleus and the activation of dpp.
Suppressor of Fused and the regulation of patched transcription
The human ortholog of Suppressor of fused is a tumor suppressor
gene (Taylor et al., 2002).
Su(fu) can associate with Ci, and with the mammalian homologs of Ci, the Gli
proteins, through specific protein-protein interactions
(Monnier et al., 2002
;
Paces-Fessy et al., 2004
).
Through these interactions, Su(fu) controls the nuclear shuttling of Ci and
Gli (Wang and Holmgren, 2000
;
Wang et al., 2000
;
Taylor et al., 2002
), as well
as the protein stability of CiFL and CiR
(Ohlmeyer and Kalderon, 1998
).
Flies homozygous for Su(fu) loss-of-function mutations are normal, so
the importance of Su(fu) becomes evident only when other gene
functions are thrown out of balance, as in a fu mutant background
(Pham et al., 1995
;
Alves et al., 1998
;
Lefers et al., 2001
), with
extra or diminished Hh signaling caused by ptc, slimb and protein
kinase A mutations (Ohlmeyer and
Kalderon, 1998
; Wang et al.,
1999
) or, as we have shown, when altered Cos2 is produced.
We found that to activate ptc transcription in the wing disc, two
conditions have to be met simultaneously: CiFL must be stabilized,
and the activity of Su(fu) must be reduced. Removal of
Su(fu) changes S182N from a ptc repressor into a
ptc activator. Removal of Su(fu) may result in the
modification, activation or relocalization of CiFL, or in further
sensitizing the system to stabilized CiFL. In Su(fu)
homozygous animals, the quantity of CiFL and CiR
proteins is greatly diminished, and Su(fu) mutant cells are more
sensitized to the Hh signal (Ohlmeyer and
Kalderon, 1998). The lower levels of both CiFL and
CiR in mutant Su(fu) cells may contribute to the
sensitivity of these cells to Hh, as a small Hh-driven change in the absolute
concentration of either form of Ci would result in a significant change in the
ratio between the two proteins. Both CiFL and CiR bind
the same enhancer sites (Muller and
Basler, 2000
), so their relative ratio is likely to be important
in determining target gene expression. S182N expression tips the ratio of
CiFL to CiR toward CiFL, and reducing the
absolute quantities of both Ci isoforms by removing Su(fu) will
enhance this effect. Furthermore, Su(fu) binds Ci and sequesters it in the
cytoplasm in a stoichiometric manner
(Methot and Basler, 2000
;
Wang et al., 2000
;
Wang and Jiang, 2004
).
Reducing the amount of Su(fu) should release more CiFL to
the nucleus to activate ptc.
Phosphorylation of Su(fu) in response to Hh
The activity of Su(fu) must be regulated or overcome so that target genes
can be activated at the right times and places in response to Hh. We have
shown that the regulation of Su(fu) activity may occur by Hh-dependent
phosphorylation. A phosphoisoform of Su(fu), Su(fu)-P, was detected in discs
where GAL4 was used to drive extra Hh expression
(Fig. 6). At high
concentrations of Hh, the phosphorylation of Su(fu) is not antagonized by
overexpression of cos2 or either of the cos2 mutants,
suggesting that phosphorylation of Su(fu) occurs independently of Cos2
function. During the preparation of this manuscript, it was reported that one
kinase involved in the phosphorylation of Su(fu) is the Ser/Thr kinase Fused,
a well-established component of Hh signal transduction
(Lum et al., 2003). It is not
known whether the phosphorylation of Su(fu) by Fu is direct or indirect.
The phosphorylation state of Su(fu) may be an important factor in determining Hh target gene activity. Phosphorylation of an increasing number of Su(fu) molecules with increasing Hh signal may gradually release Ci from all of the known modes of Su(fu)-dependent inhibition, such as nuclear export and recruitment of repressors to nuclear Ci, leading to higher levels of CiFL in the nucleus and the activation of Hh target genes such as ptc.
en activation
We used anterior en expression as an in vivo reporter of high
levels of Hh signaling. In agreement with a previous report, we find that
cos2 mutant cells at the AP boundary fail to activate en,
suggesting that Cos2 plays a positive regulatory role in en
regulation. S182N, S182T and Cos2 overexpression mimics the cos2
loss-of-function condition with respect to en: en remains
off in these cells. One interpretation of these data is that all the Cos2
proteins are able to associate with another pathway component, such as Smo,
and overproduction of any of them inactivates some of the Smo in
non-productive complexes not capable of activating en.
A Cos2 protein lacking the C-terminal region provides repressor activity
In contrast to the activity of all the other mutations we generated,
deletion of the C terminal domain created a protein (Cos2C) that
repressed normal dpp, ptc and en expression in the wing disc
(Fig. 1; data not shown). In
this in vivo assay, Cos2
C acted just like wild-type Cos2. A similar
deletion has been shown to retain function in cell culture assays
(Lum et al., 2003
). We further
showed that this mutant, expressed under the control of its endogenous
promoter, could rescue the lethality and wing duplication phenotypes of a
cos2 loss-of-function allele over a cos2 deficiency. The
results of the rescue experiment bring up a new possibility: that the
C-terminal domain of Cos2, and the Cos2-Smo interaction via the C terminus of
Cos2, is not necessary for repressor activities of Cos2. Alternatively,
Cos2
C could complement or boost the activity of the hypomorphic allele
cos211, which was used for the rescue experiment.
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Supplementary material |
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ACKNOWLEDGMENTS |
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Footnotes |
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