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First published online 8 December 2005
doi: 10.1242/dev.02198
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1 Department of Developmental and Molecular Biology, Albert Einstein College of
Medicine, Bronx, NY 10461, USA.
2 Section of Molecular Cell and Developmental Biology and Institute for Cellular
and Molecular Biology, University of Texas, Austin, TX 78712, USA.
* Author for correspondence (e-mail: rstanley{at}aecom.yu.edu)
Accepted 4 November 2005
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SUMMARY |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Key words: Drosophila embryogenesis, Morphogenesis, Shark tyrosine kinase, Dorsal closure, Dok, Jun kinase
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INTRODUCTION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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).
Embryonic dorsal closure (DC), which represents such an epithelial
morphogenetic event in Drosophila, begins at stage 11 when, following
germband retraction, a dorsal hole appears in the epidermis that is covered by
an extra-embryonic epithelium known as the amnioserosa
(Martinez-Arias, 1993). DC is
the process in which the bilateral dorsal movement of the epidermis over the
amnioserosa, followed by the dorsal suturing of the leading edge (LE) cells,
leads to the complete enclosure of the embryonic viscera. This epidermal sheet
movement is accomplished by the elongation of epidermal cells without their
proliferation or the recruitment of any additional cells
(Martinez-Arias, 1993). DC is
a particularly well-characterized system for the elucidation of signaling
pathways regulating the movement and fusion of epithelial sheets in
development (Martin and Wood,
2002), and may constitute a useful model system for studies of
wound healing (Jacinto et al.,
2001).
Mutants in which the process of DC fails are characterized by the presence
of a dorsal hole in the embryonic cuticle (`dorsal-open' or `dorsal hole'
phenotype) that is due to a failure of closure or suturing of the epidermal
cells (reviewed by Noselli,
1998; Noselli and Agnes,
1999). Mutant identification has enabled multiple signal
transduction pathways to be shown to participate in this process. These
include the Jun amino-terminal kinase (JNK), Dpp and Wingless signaling
pathways. In addition, the cell shape changes that occur during DC are
regulated by the Rho family of small GTPases (reviewed by
Harden, 2002). Finally, DC
requires the involvement of genes encoding membrane proteins such as
canoe and myospheroid, as well as genes encoding
cytoskeletal proteins (Noselli and Agnes,
1999).
Regulation of Dpp expression in LE cells by the JNK cascade is a central
signaling pathway controlling DC. The LE cells, in turn, signal to more
lateral cells in the epidermis through the Dpp receptor, inducing them to
participate in DC (reviewed by Harden,
2002). Mutants in many members of the JNK pathway have been shown
to influence DC and these mutants lead to a loss of DPP expression in the LE
cells (Glise and Noselli,
1997; Stronach and Perrimon,
2002). These mutations can be rescued by expression of a
constitutively activated form of Jun (previously known as c-Jun; Jra -
FlyBase). This group of genes includes misshapen (msn),
which encodes a germinal center kinase, slipper (slpr),
which encodes a mixed lineage kinase
(Stronach and Perrimon, 2002),
hep, which encodes a MAP kinase kinase
(Glise et al., 1995),
bsk, which encodes Drosophila Jun kinase
(Glise et al., 1995;
Riesgo-Escovar et al., 1996),
Djun (Jra - FlyBase)
(Kockel et al., 1997;
Nusslein-Volhard et al., 1984;
Riesgo-Escovar and Hafen,
1997; Riesgo-Escovar et al.,
1996; Sluss et al.,
1996) and kayak, which encodes Dfos
(Jurgens et al., 1984;
Riesgo-Escovar and Hafen,
1997; Zeitlinger et al.,
1997). Several other genes appear to play a role in the activation
of JNK pathway signaling, but are not individually essential, in some cases
because of functional redundancy (reviewed by
Harden, 2002). For example,
although neither Src42A nor Tec29A (Btk29A -
FlyBase) Src kinase mutations individually exhibit a DC defect, Src42A
Tec29A double mutants are DC defective, fail to exhibit LE cell Dpp
expression, and are rescued by the expression of activated Jun
(Tateno et al., 2000).
We have previously identified and characterized Shark (SH2 domain ankyrin
repeat kinase (Ferrante et al.,
1995), and have shown it to be an essential component of the JNK
pathway acting during DC (Fernandez et
al., 2000). Embryonic cuticles produced by
shark1 germ-line clones exhibit a dorsal-open phenotype,
and shark1 flies partially rescued by a hs-shark
transgene produce a split thorax phenotype similar to that produced by
insufficiency for other JNK pathway members
(Glise et al., 1995;
Riesgo-Escovar and Hafen,
1997; Zeitlinger et al.,
1997). shark1 germline clones fail to express
dpp in the LE cells and are rescued by the expression of activated
Jun. These results and ectopic expression studies
(Fernandez et al., 2000)
indicate that Shark functions upstream of JNK in the JNK signaling pathway
regulating DC. To better understand the role of Shark, we performed a yeast
two-hybrid screen for proteins that physically interact with the NH2-terminal
regulatory regions. Here, we describe the characterization of one of the
proteins that interacts with Shark, Drosophila downsteam of kinase
(Ddok), which belongs to the Dok family of adaptor proteins. We show
that Ddok is required for DC, functions upstream of Shark in the JNK
pathway, and is required for the appropriate tyrosine phosphorylation and
localization of Shark to the cell peripheries of LE and lateral epidermal
cells.
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MATERIALS AND METHODS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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;
Lioubin et al., 1996) as a
BamHI fragment and subcloned into pLexA-Shark at the SacI
site to create pLexA-Shark/Src encoding activated Src kinase driven by the
ADH1 promoter. Proteins from yeast transformed with this vector exhibited a
high degree of tyrosine phosphorylation in anti-phosphotyrosine western blots
(data not shown). A cDNA library from 0- to 21-hour-old Drosophila
embryos (Clontech) cloned into pB42AD (Clontech) was used as the prey. These
bait and prey plasmids were co-transformed into the Saccharomyces
cerevisiae EGY48 (p8op-LacZ) host strain that carries Leu2 and
lacZ under the control of LexA operators. Approximately
5x106 co-transformants were screened on Ura-,
His-, Trp-, Leu- and X-gal plates.
Seventeen independent transformants activated both the lacZ and
Leu2 reporter genes. The pB42AD plasmids were individually isolated
from each of the yeast transformants and sequenced. The cDNA encoding Ddok was amplified by PCR (Expand High Fidelity PCR System, Roche) from the FlyBase clone LD32155, using the following primers: forward, 5'CGGAATTCGCCACCATGGATGTTGAAATACCT-3'; and reverse, 5'-GAGGAGTCTAGATTACTACTTATCGTCGTCATCCTTGTAATCCACTCGCTTCGG-3' (CO2H-terminally tagged Ddok-Flag). The primers used for subcloning Shark were: forward, 5'-GAGCAGAAACTGATCAGCGAGGAGGACCTGAAATGGTAC-3'; and reverse, 5'-CATTTCAGGTCCTCCTCGCTGATCAGTTTCTGCTCGTAC-3' (NH2-terminally tagged Myc-Shark). The PCR products were digested with EcoRI and XbaI (Ddok), or with NotI and XhoI (Shark), and cloned into the pMTA vector (Invitrogen).
Antibody production, immunostaining procedures and cuticle preparation
The region encoding the unique CO2H-terminal (amino acid
residues 241-622) of Ddok was cloned into pGEX-KG
(Guan and Dixon, 1991) at the
EcoRI and NcoI sites. The GST fusion protein was purified
and injected into rabbits (Covance). Rabbit anti-Y927 Shark phosphopeptide
antiserum was raised against a Shark phosphopeptide
ARDPDY(PO4)QNLPELVQTVHIC (pY927 peptide, amino acids 922-940)
(coupled through Cys940 to Keyhole Limpet Hemocyanin). Anti-phosphoY927
antibody (anti-pY927) was purified from antibodies to unphosphorylated
peptides in this antiserum by binding to a pY927 peptide column and eluting
with 0.1 M glycine-HCl (pH 2.0) buffer. Anti-P-Tyr-100 was obtained from cell
signaling. For the immunofluorescence studies, Drosophila Schneider
(S2) cells were grown in Schneider Medium (Invitrogen) containing 10% fetal
bovine serum (Invitrogen) on Polylysine (10 mg/ml)-coated coverslips. They
were transfected (1x107 cells/ml, Ddok-Flag, Myc-Shark) with
Effectine (QIAGEN), induced with CuSO4 for 6 hours and grown for
48-72 hours in six-well plates. The cells were then washed with PBS and fixed
with 4% paraformaldehyde for 20 minutes. Coverslips were blocked with 10% goat
serum in PBS for 20 minutes, the cells incubated with primary (2 hours) and
secondary (1 hour) antibodies with PBS washes before and after secondary
antibody. Anti-Flag (Sigma) and anti-Myc (Invitrogen) antibodies were used at
1:500 dilutions. Immunofluorescence staining of embryos was performed using
the anti-Ddok antiserum at 1:1000, the anti-LacZ antibody and the
anti-Fasciclin III antibody (Developmental Studies Hybridoma Bank) at
dilutions of 1:2000 and 1:40, respectively, and the anti-pY927 antibody at 1
µg/ml. Secondary antibodies for staining S2 cells and embryos were Alexa
488- or Alexa 594 (Molecular Probes)-conjugated mouse or rabbit IgG used at a
dilution of 1:500. Embryos were fixed with 4% paraformaldehyde prior to
immunostaining and devitellinized with methanol (or 80% cold ethanol for
phalloidin staining). Cuticles were prepared as described previously
(Wieschaus and Nusslein-Volhard,
1986), and photographed under bright and dark field using
Ektachrome 160T film.
Immunoprecipitation and western blot
S2 cells were cultured in 100-mm diameter tissue culture dishes (Falcon) in
Schneider medium containing 10% fetal calf serum at 22°C. For Src
inhibitor studies, cells were treated with PP2 (10 µM, Calbiochem) for 1
hour. S2 cells transfected (Effectine, QIAGEN) with different plasmids
(
107 cells/dish) were lysed in l ml of lysis buffer (10 mM
Tris-HCl, 50 mM NaCl, 50 mM NaF, 30 mM sodium pyrophosphate, 5 µM
ZnCl2, 1 mM sodium orthovanadate, 1% NP-40, 1 mM Benzamidine, 10
µg/ml leupeptin and 5 µg/ml aprotinin, pH 7.2). The cell lysates were
centrifuged (13,000 g, 30 minutes, 4°C) and Sepharose G
beads (20 µl, packed volume, Pharmacia) and Flag antibody (0.5 µg) were
added to the supernatant, which was rotated at 4°C overnight. The beads
were then washed with lysis buffer (four to five times, 500 µl), the
immunoprecipitated proteins eluted with 10 µl of 3xSDS-PAGE loading
buffer, subjected to SDS-PAGE, transferred to a Nylon 0.2 µm PVDF membrane
and western blotted with primary antibodies at the following concentrations:
RC20H Anti-Ptyr:HRP (Transduction Laboratories), 1:5000; anti-Myc monoclonal
antibody (Invitrogen), 1:5000; rabbit anti-Shark antiserum
(Fernandez et al., 2000),
1:1000; rabbit anti-Ddok,1:1000; anti-Y927 peptide antibody, 1 µg/ml; and
anti-Flag monoclonal antibody (M2 monoclonal, Sigma), 1:5000.
In situ hybridization and RT-PCR
Whole-mount in situ hybridization was performed as described by Tautz and
Pfeifle (Tautz and Pfeifle,
1989), with digoxigenin-labeled RNA probes prepared by in vitro
transcription. The Ddok probe was 1241-1782 bp of cDNA sequence and the Dpp
probe was the entire cDNA. RT-PCR was performed on RNA extracted from
wild-type and DdokPG155/Y males with the following
primers: forward, 5'-GGCTGGATATCTTAATGTGCCGAC-3'; reverse,
5'-GCGGCTCCTGAGTGATCTTCACA-3' (the expected product size was 223
bp).
Genetic protocols
To eliminate both maternal and zygotic Ddok activity, germ-line clone (GLC)
mutants of DdokPG155 were generated as described
previously (Chou et al., 1993).
Virgin females of the genotype y w DdokPG155
FRT101/FM7 were crossed to ovoD1
FRT101/Y; hs-Flp38 males. The larvae were heat shocked for 1
hour at 37°C on days 4, 5 and 6 AEL to induce recombination.
GLC-containing females of the genotype y w DdokPG155
FRT101/ovoD1 FRT101; hs-Flp38 were then
mated to wild-type males. Whenever it was necessary to distinguish GLC mutants
from non-mutant flies a ftz-lacZ balancer was used. For the epistatic
analyses, GLC virgin flies were crossed to males of the following genotypes:
hs-Src42A22.3/hs-Src42A22.3
(Lu and Li, 1999),
hs-shark-10/hs-shark-10
(Fernandez et al., 2000),
hs-SEjunasp/CyO
(Treier et al., 1995) and
pucE69/TM3 (Glise and
Noselli, 1997; Martin-Blanco
et al., 1998). In crosses where heat shock was required,
developmentally synchronized embryos were obtained and heat shocked for 30
minutes at 37°C during stages 9-10. For electron microscopy, a Jeol 6400
scanning electron microscope was used.
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RESULTS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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).
Tyrosine phosphorylated residues mediate interaction with SH2 domains
(Bradshaw and Waksman, 2002)
and there is little tyrosine phosphorylation in Saccharomyces
cerevisiae. To increase the probability of obtaining proteins whose
interactions with Shark rely on the SH2 domains, we modified the yeast
two-hybrid system by co-expressing the activated Src-kinase domain, which has
been found to tyrosine phosphorylate library proteins in yeast
(Keegan and Cooper, 1996;
Lioubin et al., 1996). The
bait plasmid was based on pGilda (Gimeno
et al., 1996) and contained the LexA DNA-binding domain fused to
the shark sequences, together with the DNA sequences encoding an
activated Src-kinase domain (Lioubin et
al., 1996), placed under the control of a constitutive promoter.
The bait plasmid was co-transformed into yeast together with a
Drosophila `prey' library, in which cDNAs from 0-21-hour-old embryos
were fused to the B42 activation domain AD
(Gyuris et al., 1993) (pB42AD;
Clontech). Two out of the 17 clones whose interaction with the bait protein
mediated LexA-dependent lacZ and Leu2 reporter gene
expression were shown to encode overlapping portions of a protein similar to
the Downstream of kinase (Dok) family of proteins
(Carpino et al., 1997;
Di Cristofano et al., 1998;
Ellis et al., 1990;
Lemay et al., 2000;
Yamanashi and Baltimore,
1997). The Drosophila Dok gene (Ddok) has been
annotated in the Drosophila genome (Accession Number CG2079), and is
predicted to encode a protein of 622 amino acids. When the screen was repeated
using the same LexA-Shark-containing plasmid but lacking the Src kinase domain
as bait, the positive clones obtained did not include Ddok sequences. Dok
family proteins share a similar domain structure, comprising a Pleckstrin
homology (PH) domain (Yan et al.,
2002) and a phosphotyrosine-binding (PTB) domain
(Lemmon, 2003), which are
believed to confer association with the plasma membrane and with the tyrosine
phosphorylated motif NPXpY, respectively
(Yan et al., 2002;
Lemmon, 2003). The predicted
Ddok protein has a PH domain (amino acid residues 6-113;
Fig. 1A) and a PTB domain
(amino acid residues 138-234; Fig.
1B). The C-terminal tail of Ddok contains proline-rich stretches,
which, in the mammalian Doks, are known to be docking sites for the Src
homology 3 (SH3) domain-containing Src and Abl kinases
(Master et al., 2003). In
addition, Ddok is predicted to contain five putative sites for phosphorylation
on tyrosine (Fig. 1C), which
may serve as docking sites for SH2 domain-containing proteins. The PH and PTB
domains are the regions exhibiting the greatest sequence similarity among the
mammalian Doks, and between Ddok and the mammalian Doks
(Fig. 1A,B). Ddok and the
mammalian Doks are predicted to have arisen from a single primordial gene
(Fig. 1D).
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74 kDa) in Drosophila embryo lysates
(Fig. 2C), the association of
Ddok with Shark could be detected in untransfected S2 cells
(Fig. 2D). We therefore
investigated the interaction between Ddok and Shark by expressing
epitope-tagged versions of these proteins in S2 cells. Ddok-Flag, when
overexpressed in S2 cells and immunoprecipitated with anti-Flag antibody,
migrated as a single band on SDS-PAGE and was tyrosine phosphorylated
(Fig. 2E, lane 3). When lysates
from Myc-Shark and Ddok-Flag co-transfected cells were used, Shark
(Mr 116 kDa) was co-immunoprecipitated with Ddok,
which exhibited increased tyrosine phosphorylation
(Fig. 2E, lane 4). No tyrosine
phosphorylation of Shark was detected (Fig.
2E, upper panel), suggesting that Shark itself was not activated
by its association with Ddok. However, because Src42A and Src64B were both
recently reported to associate with Ddok in a yeast two-hybrid analysis of
Drosophila proteins (Giot et al.,
2003), we examined the effect of the Src inhibitor PP2, which does
not inhibit Shark tyrosine kinase activity (K. Hong, Y. G. Yeung and E.R.S.,
unpublished). Consistent with the involvement of Src kinases in the
phosphorylation of Ddok, PP2 inhibited Ddok tyrosine phosphorylation in the
presence or absence of transfected Shark
(Fig. 2F).
We also investigated the role of the Ddok tyrosines shown to be required
for the Src-kinase-dependent interaction in the two-hybrid system
(Fig. 2B). In contrast to the
interaction between Ddok and Shark fragments studied in the two-hybrid
analysis, individual Ddok Y427, Y499, Y515 or Y537 mutations in full-length
Ddok exhibited the same degree of association as unmutated Ddok (data not
shown), whereas Ddok Y499, 515F double mutants showed a reduced association
with Shark in S2 cells (Fig.
2E, lane 5). However, the interaction between Shark and Ddok was
not reduced further by the introduction of additional mutations Y427F and
Y537F (Fig. 2E, lanes 6,7).
Thus results from both systems emphasize the importance of Ddok Y499 and Y515.
The lack of effect of the individual Y mutants in the S2 cell system may
reflect the presence of additional interactions due to the use of full-length
constructs and a Drosophila system that is likely to be more
permissive for the participation of third party proteins. As the sequence
surrounding the Ddok Y499 and Y515 comprises an immunoreceptor tyrosine-based
activation motif (ITAM) (Isakov,
1998), these data suggest that the Ddok ITAM plays a dominant, but
not exclusive role in the interaction between Shark and Ddok in S2 cells.
There was no interaction between Ddok and Shark lacking the SH2 domain-ankyrin
repeats-SH2 domain regions (data not shown).
Myc-Shark expressed in S2 cells in the absence of Ddok was present mainly in the cytoplasm with some expression at the cell cortex (Fig. 2G, part b). By contrast, Ddok-Flag expressed in S2 cells localized primarily to the cell cortex with slight cytoplasmic expression (Fig. 2G, part d). When the two proteins were expressed together in S2 cells, there was no change in their distribution and their colocalization was restricted to the cell cortex (Fig. 2G, parts f-h).
Ddok expression pattern
Ddok protein is expressed at all stages of embryonic development, as
demonstrated by staining with an antibody raised against the Ddok-GST fusion
protein. Maternal expression of Ddok protein leads to strong expression in
early stage embryos (see Fig. S1A,B in the supplementary material). Ddok is
highly localized to the cortex of blastoderm embryos at the onset of
cellularization (see Fig. S1B in the supplementary material). During germ band
extension, it is expressed in the cephalic furrow and the proctodeum (see Fig.
S1C in the supplementary material), and in the anterior and posterior midgut
regions. By stage 13, Ddok protein is expressed in the epidermis, malphigian
tubules and hindgut (see Fig. S1F). At later stages of development, it is
expressed in midgut constrictions and the ventral nerve cord (data not shown).
In parallel with the analysis of Ddok protein expression, the mRNA expression
pattern was studied with an anti-sense RNA probe specific for the region of
the Ddok gene encoding its C-terminal tail. The pattern of expression of the
Ddok mRNA was similar to the protein expression pattern (Fig. S1G-L
in the supplementary material). However, the abundance of the mRNA was
observed to decline gradually after stage 13 of embryogenesis. The patterns of
expression of the Ddok mRNA and protein resemble those of the Shark mRNA and
protein (Fernandez et al.,
2000).
A mutation in Ddok affects embryonic DC
The Ddok gene is located in region 7B1 on the X-chromosome. A
screen for lethal mutations on the X-chromosome yielded an insertion of the P
element transgene pGaw in the first intron of the Ddok gene, 387
bp upstream of the second exon (DdokPG155)
(Bourbon et al., 2002)
(Fig. 3A).
DdokPG155 is semilethal in hemizygous males and 
2-3
transposase-mediated P-element-mediated allelic reversion led to the
generation of lines in which males carrying the revertant chromosome were
viable, indicating that the P-element insertion in
DdokPG155 is the basis for lethality
(Bourbon et al., 2002).
DdokPG155/Y males can also be rescued by overexpression of
a Ddok transgene. The rescued flies are normal and fertile. Among the progeny
of the cross DdokPG155/FM7xFM7/Y, 1-2% of
the expected number of DdokPG155/Y males survived to
adulthood, and we considered these flies to be `escapers' of the lethal
phenotype. RT-PCR performed on the DdokPG155 hemizygous
males failed to detect Ddok transcripts
(Fig. 3B). The escapers
exhibited four distinct phenotypes: the majority (50%) exhibited a
phenotype in which the wings are shriveled
(Fig. 3C, part b,c). A similar
wing phenotype of varying severity has also been reported in hep
(hemipterous) mutants (Agnes et
al., 1999). Another commonly observed phenotype is a split thorax
phenotype (30%) (Fig. 3D,
part a,b), accompanied by the loss of bristles in the midline region of the
thorax (Fig. 3D, part b). Both
of these phenotypes are also associated with weak hep alleles
(Glise et al., 1995),
kay mutants that have been partially rescued through transgenic
expression of the kay gene
(Riesgo-Escovar and Hagen,
1997; Zeitlinger et al.,
1997), and with partially rescued Shark1
mutants (Fernandez et al.,
2000). The other phenotypes displayed by
DdokPG155 hemizygous flies include unilateral or bilateral
loss of anterior orbital bristles (<10%) in the head region
(Fig. 3D, part c,d), and
irregularly placed bristles in the eyes
(Fig. 3D, part e,f).
The high maternal expression of Ddok mRNA and protein, and the
ability of embryos hemizygous for DdokPG155 to hatch and
in some cases to survive to adulthood, suggested that maternally expressed
Ddok could support the need for Ddok activity during embryogenesis. To
eliminate the maternal Ddok contribution, germ-line clones (GLC)
homozygous for DdokPG155 were generated
(Chou and Perrimon, 1996). In
cuticle preparations, approximately 50% of the embryos derived from these GLCs
exhibited a severe dorsal open phenotype
(Fig. 4A, part a,b;
Table 1). Those progeny that
did not die as embryos survived to adulthood, and all of them were female.
This indicates that the paternally supplied X-chromosome, carrying the
wild-type copy of Ddok, was capable of rescuing the GLC-associated defect, and
that the embryos that died with a dorsal open phenotype represented the male
progeny of GLC-producing females and were both maternally and zygotically
mutant for Ddok. Immunohistochemical staining with anti-Ddok antiserum
supports the notion that the phenotypes associated with embryos derived from
DdokPG155 GLCs result from drastically reduced levels of
Ddok protein. Although lateral epidermal cells of stage 13 wild-type embryos
showed strong staining with Ddok specific antiserum
(Fig. 4B, part a) at the cell
periphery of the lateral epidermal cells and at the LE, similar staged embryos
that were derived from DdokPG155 GLCs did not express the
Ddok protein, as revealed by immunohistochemical staining
(Fig. 4B, part b). Wild-type
embryos also showed a low level of staining of the aminoserosa.
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). The DC defect in such embryos results from the failure
of epidermal cells to stretch and elongate. Analysis of the epidermal cell
morphology of both wild type and maternal/zygotic
DdokPG155 mutants by staining with anti-Fasciclin III
antibodies revealed that, in contrast to the elongated lateral epidermal cells
observed in wild type embryos (Fig.
4C, part a), the epithelial cells of the
DdokPG155 mutant embryos, like those of JNK pathway mutant
embryos, are rounded (Fig. 4C,
part b). The expression of Dpp in LE cells, regulated through the JNK pathway, is observed as two lateral stripes, one on either side of stage 11 and 13 embryos (Fig. 4D, part a,c). Whole-mount in situ hybridization with a digoxigenin-labeled antisense RNA probe specific for dpp revealed an absence of dpp staining, specifically in the LE cells of stage 11 (Fig. 4D, part b) and stage 13 (Fig. 4D, part d) DdokPG155 GLC mutant embryos, maternally and zygotically mutant for DdokPG155. Loss of Dpp expression in the LE cells of DdokPG155 mutant embryos, together with their dorsal-open phenotype, indicates that Ddok is a component of the JNK signaling contributing to the process of DC.
The effects of the DdokPG155 mutation on DC was further investigated by F-actin staining of epidermis and amnioserosa of DdokPG155/Y and DdokPG155 GLC embryos with phalloidin at 10 hours and 12 hours AEL (Fig. 5). In DdokPG155/Y embryos, the normal intensity and pattern of actin distribution was preserved. However, the actin cable in the LE cells exhibited minor kinking and regional loss of staining in parts (Fig. 5B), compared with wild-type embryos (Fig. 5A). By contrast, despite initially stretching prior to the failure of DC (Fig. 5C, part a), Ddok GLCs showed decreased F-actin staining of all cells and a severe unevenness of the LE, with a loss of staining of the actin cable and of actin protrusions at the LE (Fig. 5C, part a,b).
Ddok is an upstream component of the JNK signaling pathway
The phenotype of DdokPG155 GLC mutant embryos indicates
that Ddok functions in the JNK pathway. To determine whether Ddok acts
upstream or downstream of JNK in the pathway, we tested whether constitutively
active Jun (hs-SEjunasp)
(Treier et al., 1995) could
rescue the DC phenotype of DdokPG155 GLC-derived embryos.
Although 48% of GLC-derived embryos exhibited a dorsal open phenotype, only
31% of embryos from GLC-producing females crossed to
hs-SEjunasp shared this phenotype
(Table 1). The percentage of
rescue observed (19%) is significant, as hs-SEjunasp
fathers are heterozygous for the transgene and only 25% of the unhatched
embryos are expected to contain it. Cuticles of these embryos exhibited a less
severe DC defect (small anterior holes) than the dorsal open phenotype of the
unrescued embryos (Fig. 6B).
These rescued embryos die at late embryonic stage and do not hatch to
larvae.
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; Martin-Blanco et al.,
1998). Males heterozygous for the pucE69
mutation (Glise and Noselli,
1997; Martin-Blanco et al.,
1998) were crossed to DdokPG155 GLC females to
obtain DdokPG155 male embryos heterozygous for
pucE69. In these crosses, we also observed a decrease in
the number of GLC-derived embryos that were DC defective, from 48% to 29% of
progeny (Table 1), and embryos
exhibiting rescue possessed small holes in the cuticle
(Fig. 6C), consistent with
regulation of JNK signaling by Ddok that is upstream of bsk. To
investigate the epistatic relationship between shark and
Ddok, we similarly tested whether the DdokPG155
GLC DC defect could be rescued by overexpression of Shark. Examination of the
cuticles of embryos from the cross of DdokPG155 GLC flies
with males homozygous for hs-shark-10
(Fernandez et al., 2000)
(Fig. 6D), but not
hs-shark-K698R (Fig.
6E), revealed a reduction in the percentage of embryos that were
DC defective, from 48% to 10% (Table
1), and embryos exhibiting rescue possessed small anterior holes
(Fig. 6D). These results
suggest that Shark acts downstream of Ddok in the JNK pathway, and that Shark
kinase activity is required for signaling.
Src family kinases act upstream of Syk family kinases in mammalian cells.
As Shark belongs to the Syk family, we also examined the epistatic
relationship between Src42A and Ddok by crossing DdokPG155
GLC flies to male flies homozygous for hsSrc42A22.3
(Lu and Li, 1999)
(Fig. 6E). The percentage of DC
defective embryos from this cross was not significantly altered (from 48% to
44%) by expression of Src42A, indicating that Src is not capable of rescuing
the Ddok mutant phenotype. Furthermore, an activated form of Src
(UAS-Src42A.CA) (Tateno et al.,
2000) driven by hs-Gal4 failed to rescue
DdokPG155 mutants.
Decreased expression of tyrosine phosphorylated Shark at the cell periphery in Ddok mutant embryos
As Ddok is expressed at the cell peripheries of LE, lateral epidermal and
S2 cells, and overexpression of Shark rescues DC in Ddok mutant
embryos, one mechanism of action of Ddok could be to localize Shark for
activation. However, there was no discernable difference between wild-type and
Ddok mutant embryos stained with anti-Shark antiserum (data not shown). We
therefore prepared a phosphopeptide antibody (anti-pY927) to a Shark
phosopeptide containing Y927, which is phosphorylated when Shark is activated
in S2 cells (K. Hong, Y. G. Yeung and E.R.S., unpublished). In contrast to the
anti-phosphotyrosine antibody, which recognizes many bands in embryo extract
western blots (Fig. 7A, lane
2), anti-pY927 recognizes only a single band
(Fig. 7A, lane 1) with the
Mr of Shark (116 kDa); this recognition is lost on
prior phosphatase treatment of the blot
(Fig. 7B), indicating that
recognition by anti-pY927 is dependent on phosphorylation. Staining of
wild-type embryos with anti-pY927 revealed that tyrosine phosphorylated Shark
was localized to the cell peripheries of LE and lateral epidermal cells
(Fig. 7C). By contrast, this
intense localization of pY927 Shark was substantially reduced in
DdokPG155 GLC embryos
(Fig. 7D). These data indicate
the involvement of Ddok in the localization and activation of Shark during
DC.
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DISCUSSION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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).
Using a two-hybrid approach we have now identified tyrosine phosphorylated
Ddok to be a Shark-interacting partner, and we have confirmed this interaction
in Drosophila cells in tissue culture. The similar embryonic
expression patterns of Shark and Ddok are consistent with their functional
interaction, which is supported by evidence that Ddok regulates the JNK
signaling pathway. A mutation in Ddok exhibits phenotypes associated with
other JNK pathway members: split thorax and shriveled wings in adults, and DC
defects in embryos. At the cellular level, the failure of JNK signaling in
embryos lacking Ddok results in an absence of Dpp expression in LE cells.
Consequently, epidermal cells in these mutants fail to stretch and elongate as
they should during DC. The rescue of the DC defect of
DdokPG155 GLC mutant embryos by heat shock-mediated
overexpression of an activated form of Jun (hs-cJunasp),
and by a single loss-of-function allele of puc
(pucE69), a phosphatase that negatively regulates JNK,
indicates that Ddok acts upstream of JNK in the pathway. Ddok also appears to
act upstream of Shark, as heat shock-mediated overexpression of Shark
(hs-shark-10) was capable of rescuing the Ddok mutant
phenotype.
The interaction between the Ddok C-terminal and Shark N-terminal region was
shown to be dependent on tyrosine phosphorylation of Ddok by activated Src,
and to be reduced by mutation of each of four Ddok tyrosines to phenylalanine.
Co-expression studies in S2 cells showed that full-length Shark associates
with full-length Ddok, that Ddok is tyrosine phosphorylated and that Ddok
tyrosine phosphorylation, in the presence or absence of Shark, is Src
dependent. Two Ddok tyrosines, Y499 and Y515, are present within an ITAM
consensus sequence. ITAMs consist of two repeats of conserved sequence
(Tyr-X-X-Leu/Ile) spaced by six to eight residues
(Isakov, 1998) and, when the
conserved tyrosines are phosphorylated, serve as binding sites for SH2
domain-containing proteins, including Syk family members. The association of
Shark with tyrosine phosphorylated Ddok was substantially decreased in the
Ddok Y499, 515F double mutant, implying that association of Ddok and Shark is,
in part, mediated by the ITAM involving these Ddok phosphotyrosines.
|
). Studies on mammalian Dok1 plasma membrane recruitment and
tyrosine phosphorylation suggest that activation of PI 3-kinase promotes the
binding of the Dok1 PH domain to phosphoinositides and Dok1 recruitment to the
plasma membrane where it is phosphorylated by Src, Tec and/or Abl family
kinases (Liang et al., 2002;
Nelms et al., 1998;
Songyang et al., 2001;
Suzu et al., 2000;
Woodring et al., 2004;
Zhao et al., 2001). The cell
cortical localization of Ddok (Fig.
2F) and its ability to associate with Src kinases
(Giot et al., 2003) suggest
that it is positioned to respond to Src signaling. By contrast, Shark is
expressed throughout the cytoplasm (Fig.
2F). During DC, increased activation of Src by appropriate
upstream signals could increase Ddok tyrosine phosphorylation, resulting in
increased localization of Shark to the cell cortex, where it becomes tyrosine
phosphorylated and activated. Consistent with this suggestion, pY927 Shark is
localized to the cortex in wild-type embryos, and this localization is
substantially reduced in DdokPG155 GLC embryos. The rescue
of the DdokPG155 DC defect by overexpression of Shark is
easily understood in terms of this cell cortex-localizing function of Ddok, as
overexpression of Shark may obviate the need for its directed
localization.
Relevant to the synergism between Src and Shark in DC postulated above,
Src42A and Shark appear to function together downstream of bullwinkle
in stretch cells to regulate dorsal appendage cell movement in a
JNK-independent fashion (Tran and Berg,
2003). Synergism between Shark and Ddok may take place in stretch
cells, where overexpression of Shark suppressed the bullwinkle-mutant
dorsal appendage phenotype. A similar synergism may take place during
cardiogenesis, which involves Ddok regulation
(Kim et al., 2004).
The other Syk family tyrosine kinases, Syk and the mammalian ZAP-70, both
of which are predominantly localized to the cell cortical region of the T-cell
(Huby et al., 1997), play
important roles in immunoreceptor signaling
(Chu et al., 1998;
Latour and Veillette, 2001).
In this study, we show that Ddok is required for the localization of Shark to
the membrane, where it is tyrosine phosphorylated and probably activated. As
Ddok functions to localize Shark to the plasma membrane, it is important to
determine the mechanism by which Shark is activated. Given the established
role of the Src family kinases Src42A and Tec29 in DC
(Tateno et al., 2000), the Src
dependence of Ddok tyrosine phosphorylation in S2 cells, the failure of
Src42A22.3 and Src42A.CA to rescue the DC defect
of Ddok-deficient embryos, the association of Src42A and Src64B with Ddok
(Giot et al., 2003), and the
fact that Shark is a member of the Syk family of tyrosine kinases, activation
of which is mediated by the Src kinases
(Chan et al., 1994), the
involvement of the Src kinases in the direct activation of Shark is an
intriguing possibility.
|
|
; Carpino et al.,
1997; Yamanashi and Baltimore,
1997), Dok2 (or Dok-R/FRIP)
(Di Cristofano et al., 1998;
Jones and Dumont, 1998;
Nelms et al., 1998), Dok3 (or
Dok-L) (Cong et al., 1999;
Lemay et al., 2000), Dok4,
Dok5 (Grimm et al., 2001) and
Dok6 (Crowder et al., 2004).
Mammalian Doks have been shown to enhance
(Hosooka et al., 2001) and to
inhibit (Di Cristofano et al.,
2001; Liang et al.,
2002; Nelms et al.,
1998; Songyang et al.,
2001; Suzu et al.,
2000) cell proliferation in several cell types. Relevant to our
observation of the involvement of Ddok in DC, a morphogenetic process
involving considerable cell shape changes, Dok1 has also been shown to
activate cell spreading (Hosooka et al.,
2001; Woodring et al.,
2004) and insulin receptor-dependent cell migration
(Noguchi et al., 1999), and
Dok2 is involved in the Tie2-dependent migration of epithelial cells
(Jones et al., 2003). However,
the level of their involvement in the regulation of cell migration and growth
in cultured cells in vivo remains unclear. Dok1-/- mice appear
normal, are fertile and live at least 1 year
(Yamanashi et al., 2000).
Similarly, Dok1-/-; Dok2-/- double mutants are viable,
fertile and born in appropriate Mendelian proportions
(Niki et al., 2004;
Yasuda et al., 2004). These
mice develop myeloproliferative disease at 1 year of age. As the
expression patterns of the mammalian Dok proteins overlap, it is possible that
they act redundantly, and that it will be necessary to eliminate the function
of several Dok genes in order to obtain stronger phenotypic effects.
Considerable insight into the function of Dok proteins may be gained from
studies of Ddok, as it is the unique member of this family in
Drosophila.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/133/2/217/DC1
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ACKNOWLEDGMENTS |
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REFERENCES |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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F mutations (AD-Ddok Y---F) or nothing (AD). Transformed yeast were
plated on replica plates containing X-gal for screening
ß-galactosidase-positive blue colonies (upper panel) and Leu-
plates for screening Leu-positive colonies (lower panel). (B) Y427,
Y499, Y515 and Y537 are involved in the tyrosine phosphorylation-dependent
interaction of Ddok with Shark. The constructs used in A were used to perform
the more sensitive ß-galactosidase assays of yeast cell lysates
(±s.e.m.; n=3; *P<0.05, Student's
t-test, significantly different from LexA-Shark/Src+AD-Ddok).
(C) Ddok antibody detects a single protein in a western blot of
Drosophila embryo extract. (D) Endogenous Shark and Ddok are
associated in S2 cells. (E) Dependence of Shark and Ddok association on
Ddok tyrosine phosphorylation in co-transfected S2 cells. S2 cells were
transfected with pMT vector alone, Myc-Shark alone, Ddok-Flag alone, both
Myc-Shark and Ddok-Flag, or Myc-Shark with each of the following Ddok-Flag
mutants individually: Ddok-M1 (Ddok Y499F,Y515F), Ddok-M2 (Ddok
Y499F,Y515F,Y427F), or Ddok-M3 (Ddok Y499F,Y515F,Y427F,Y537F). Cell lysates
were immunoprecipitated with Flag antibody and subjected to SDS-PAGE and
western blotting with the indicated antibodies. (F) Inhibition of Ddok
tyrosine phosphorylation in S2 cells by the Src inhibitor, PP2. Ddok-Flag or
Ddok-Flag+Shark transfected cells were incubated in medium containing 10 µm
PP2 or solvent (DMSO) for 1 hour prior to the immunoprecipitation of cell
lysates with Flag antibody. (G) Ddok and Shark are colocalized at the
cell cortex. S2 cells were transfected with Myc-Shark alone (a,b), Ddok-Flag
alone (c,d), or co-transfected with Myc-Shark and Ddok-Flag (e-h),
immunofluorescently stained for Myc (red; b,g,h) and Flag (green; d,f,h), and
examined by confocal microscopy. The merged panel (h) shows the colocalization
(yellow).
