|
|
|
|
|
|
|
||||
| Home Help Feedback Subscriptions Archive Search Table of Contents | ||||
First published online 18 October 2006
doi: 10.1242/dev.02616
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
1 Department of Pathology, Nagoya University Graduate School of Medicine, 65
Tsurumai-cho, Showa-ku, Nagoya 466-8550, Japan.
2 Department of Genetics and Development, Columbia University, 701 West 168th
Street, New York, NY 10032, USA.
3 Laboratory of Molecular Biochemistry, School of Life Science, Tokyo University
of Pharmacy and Life Science, Tokyo 192-0392, Japan.
4 Division of Molecular Neurobiology, MRC National Institute for Medical
Research, The Ridgeway, Mill Hill, London NW7 1AA, UK.
Author for correspondence (e-mail:
mtakaha{at}med.nagoya-u.ac.jp)
Accepted 6 September 2006
 |
SUMMARY |
|---|
 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Key words: RET tyrosine kinase, GDNF, Enteric nervous system, Protein kinase A, JNK, Mouse
|
INTRODUCTION |
|---|
|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
;
Schuchardt et al., 1994). It
has also been demonstrated that RET signaling is required for the proper
development of parasympathetic neurons and motoneurons as well as
spermatogonia (Airaksinen and Saarma,
2002; Enomoto et al.,
2000; Meng et al.,
2000). In humans, germline mutations of Ret lead to the
development of Hirschsprung's disease, which is characterized by the absence
of enteric ganglia from the distal gastrointestinal tract
(Edery et al., 1994;
Romeo et al., 1994).
RET is activated by the glial-cell-line-derived neurotrophic factor (GDNF)
family of ligands, which include GDNF, neurturin, artemin and persephin
(Airaksinen and Saarma, 2002).
Unlike other receptor tyrosine kinases, glycosyl phosphatidylinositol-linked
cell-surface proteins called GDNF family receptor 
1-4 (GFR 1-4)
are required as ligand-binding components for RET activation
(Airaksinen and Saarma, 2002;
Jing et al., 1996;
Klein et al., 1997;
Treanor et al., 1996).
Gdnf- or Gfr 1-deficient mice also showed
phenotypes similar to Ret-deficient mice, confirming that complex
formation of these three molecules is crucial for the development of the ENS
and the kidney (Cacalano et al.,
1998; Enomoto et al.,
1998; Moore et al.,
1996; Pichel et al.,
1996; Sanchez et al.,
1996; Schuchardt et al.,
1994). In the developing ENS, RET is expressed in migrating
enteric neural crest cells (ENCCs) whereas GDNF is expressed in the mesenchyme
of the gut in a spatially and temporally regulated manner
(Natarajan et al., 2002). In
the developing kidney, RET and GDNF are expressed in the branching ureteric
buds and metanephric mesenchyme, respectively
(Hellmich et al., 1996;
Pachnis et al., 1993). In
addition, it has been reported that RET signaling activated by neurturin,
artemin or persephin plays an important role in survival, proliferation and/or
differentiation of various types of central and/or peripheral neurons
(Airaksinen and Saarma,
2002).
RET can activate a variety of intracellular signaling pathways, including
Ras/mitogen-activated protein kinase (MAPK), PI3K/AKT, RAC1/Jun
NH2-terminal kinase (JNK), p38MAPK and phospholipase C 
pathways (Ichihara et al.,
2004; Kodama et al.,
2005). RET can also activate RHO family GTPases, including RHO,
RAC and CDC42, which are involved in reorganization of the actin cytoskeleton
necessary for cell motility (Barone et al.,
2001; Chiariello et al.,
1998; Maeda et al.,
2004; Murakami et al.,
1999; van Weering and Bos,
1997). Moreover, Encinas et al.
(Encinas et al., 2001)
reported that SRC kinase activity is necessary to elicit optimal GDNF-mediated
signaling, neurite outgrowth and survival.
Recently, we demonstrated that phosphorylation of the serine residue at
codon 696 (S696) of RET via a cAMP-dependent mechanism is required for
RAC1-GEF activation and lamellipodia formation in the human SK-N-MC
neuroectodermal tumor cell line transfected with the human RET gene
(designated MC(RET) cells) (Fukuda et al.,
2002; Fukuda et al.,
2005). When S696 was replaced with alanine (S696A), GDNF-mediated
activation of RAC1-GEF and lamellipodia formation were abolished. We found
that S696 represents a protein kinase A (PKA) phosphorylation site and that
treatment with the PKA inhibitor impaired these GDNF-dependent activities.
Phosphorylation of tyrosine 687 (Y687) and S696 at the juxtamembrane region
appeared to induce opposite effects on lamellipodia formation, which suggested
the possibility that S696 phosphorylation suppresses the signal arising from
phosphorylated Y687 residue (Fukuda et
al., 2002). In addition, we found that the RAC1/JNK signaling
pathway was specifically impaired in S696A cells and that GDNF stimulation
caused G2/M cell-cycle delay in MC(RET) cells but not in S696A cells
(Fukuda et al., 2005).
To investigate the in vivo role of RET signaling regulated by PKA, we used targeted mutagenesis in embryonic stem (ES) cells to replace the putative serine phosphorylation site of codon 697 in mouse RET (corresponding to serine 696 in human RET), with alanine (RET S697A mutation). RET S697A mutant mice showed aganglionosis in the distal colon, resulting from migration defects of ENCCs in the developing gut. Consistent with our previous results, GDNF-mediated JNK activation was specifically impaired in neurons derived from the mutant mice. These findings reveal that PKA-dependent RET phosphorylation at S697 regulates the JNK signaling pathway and controls the migration of ENCCs in the developing gut.
|
MATERIALS AND METHODS |
|---|
|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Antibodies
Anti-RET and anti-phospho-RET(pS697) antibodies were developed as described
previously (Fukuda et al.,
2002). Anti-PGP9.5 antibody was purchased from UltraClone Limited.
Anti-ERK, anti-phospho-ERK, Anti-AKT, anti-phospho-AKT, anti-p38MAPK,
anti-phospho-p38MAPK, anti-JNK and anti-phospho-JNK antibodies were purchased
from Cell Signaling Technology. Anti-SRC antibody (GD11) and anti-phospho-SRC
antibody were purchased from Upstate Cell Signaling Solution and Biosource,
respectively. Anti-rabbit Alexa Fluor was purchased from Molecular Probes.
Anti-ß-actin antibody was purchased from Sigma-Aldrich.
Explant cultures
For the chemoattractant assay, E11.5 or E12.5 gut segments prepared from
midgut were cut and transferred onto the surface of collagen gels
(KOKENCELLGEN, Koken) containing 10% fetal bovine serum (FBS). GDNF-soaked
agarose beads (Cibacron, Sigma; 80-110 µm diameter) were placed 500-800
µm from gut segments and cultured for 4 days
(Young et al., 2001). For in
vitro organ culture, embryonic guts were dissected out at E11.5, and cultured
in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% FBS.
Individual guts were cultured in 60 µl hanging drops and fed every day. To
inhibit a specific signaling pathway, 30 µM of MEK1 inhibitor (PD98059), 20
µM of PI3K inhibitor (LY294002), 10 µM of SRC kinase inhibitor (SU6656),
10 µM of JNK inhibitor (SP600125) or 1 µM of PKA inhibitor (KT5720) was
added to the medium.
Primary culture of ENCCs
Primary culture of ENCCs has been described previously
(Barlow et al., 2003). Briefly,
E11.5 guts were dissected and incubated with Collagenase/Dispase (0.5 mg/ml).
Tissues were dissociated into single cells by repeated pipetting and cultured
in F12/DMEM supplemented with 10% FBS. GDNF was added at a concentration of 50
ng/ml. After 3 days, cultured cells were immunostained with anti-PGP9.5
antibody.
Primary culture of dorsal root ganglia
Primary culture of neurons from dorsal root ganglia (DRGs) has been
described previously (Jijiwa et al.,
2004). Briefly, DRGs were obtained from wild-type and mutant mice
at postnatal day 7 and digested with 0.15% collagenase and 0.05% trypsin-EDTA.
Isolated cells were cultured in F12/DMEM supplemented with 10% FBS for 3 days.
After 2 hours of serum starvation, cultured cells were treated with GDNF (100
ng/ml) for 20 minutes, lysed in sodium dodecyl sulfate sample buffer, and
subjected to western blot analysis.
Histology and immunostaining
In explant gut cultures migrated ENCCs, which were differentiated into
neuronal cells, were visualized with anti-PGP9.5 immunostaining as described
previously (Young et al.,
2001). Explants were fixed in 4% paraformaldehyde in
phosphate-buffered saline (PBS). After washing with PBT (PBS+0.1% Triton
X-100) three times, they were incubated for 3 hours with blocking solution
(PBS+2% BSA) and incubated with anti-PGP9.5 antibody (1:1000 dilution) at
4°C overnight. Then explants were washed with PBT several times, incubated
with anti-rabbit Alexa Flour (1:1000 dilution) at 4°C overnight and
analyzed in an Olympus stereomicroscope. To quantify the chemoattractant
response to GDNF, PGP9.5-positive areas were subdivided into quadrants, and
measured using image analysis software (WinROOF).
Whole-mount acetylcholinesterase (ACHE) histochemistry and whole-mount in
situ hybridization were carried out as previously described
(Enomoto et al., 1998;
Natarajan et al., 2002). ENS
cells in the myenteric plexus were stained with cuprolinic blue, and positive
cells were counted under a microscope, and calculated as
number/mm2. Cells were counted in ten randomly selected fields of
each region of the gut from four mice.
|
RESULTS |
|---|
|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
), and excision of the neo gene was confirmed by
Southern blotting (Fig. 1D).
Heterozygous RETS697A mice (S697A mice) were intercrossed to
generate homozygous S697A mice (Fig.
1E). Because a 70 bp insertion containing a single loxP site and a
polylinker sequence was still left in intron 11 in the S697A mice, which may
affect the expression of RET, the protein levels of RET in embryos and newborn
brain stems and stomachs of wild-type and mutant mice were examined by western
blotting. As shown in Fig. 1F,
RET proteins were expressed at comparable levels in wild-type, heterozygous
and homozygous S697A mice. To investigate the phosphorylation status of Serine
697, DRG neurons were isolated and cultured from wild-type and mutant mice,
and analyzed by western blotting with anti-phospho-RET(S697). S697
phosphorylation was clearly observed in wild-type mice but not in mutant mice
(Fig. 1G).
|
|
|
S697A mutation impairs migration of ENCCs in the developing gut
To elucidate why aganglionosis occurred in the distal colon of homozygous
S697A mice, we analyzed the ENS development at embryonic stages E11.5-14.5,
during which ENCCs migrate within the gut towards the distal part of the
hindgut (Young and Newgreen,
2001). At E11.5, RET-positive ENCCs had migrated beyond the cecum
in wild-type mice, while in the homozygous S697A mice they were migrating in
the small intestine but had not yet arrived at the cecum
(Fig. 4A,A',D,D').
At E12.5, ENCCs arrived at the cecum in the mutant mice
(Fig. 4E,E'). At E14.5,
wild-type ENCCs reached the terminus of the hindgut, while mutant ENCCs were
still migrating in the middle portion of the hindgut
(Fig. 4C,C',F,F'),
where marked reduction of enteric neurons was observed in newborn mutant mice.
Thus, aganglionosis in the distal colon is likely to be due to the impaired
migration of the mutant ENCCs.
It has been reported that GDNF functions as a chemoattractant for the ENCCs
via RET activation, and that GDNF could control the migration of ENCCs in the
developing gut (Iwashita et al.,
2003; Natarajan et al.,
2002; Young et al.,
2001). To test whether the chemoattractant response to GDNF is
affected by the S697A mutation, we cultured gut segments from wild-type and
mutant mice together with GDNF-soaked beads on collagen gels. After 4 days,
ENCCs were visualized by immunostaining with PGP9.5. Wild-type ENCCs migrated
out massively from the gut segments towards GDNF-soaked beads, whereas
migration of mutant ENCCs was extremely weak
(Fig. 5A). To quantify the
chemoattractant response to GDNF, PGP9.5-positive areas containing neurites
and neural cells were measured in the proximal (towards) and distal (away)
quadrants. In wild-type mice, the PGP9.5-positive area on the proximal
quadrant towards the GDNF beads was significantly greater than in the distal
quadrant. In the homozygous S697A mice, the proximal and distal areas were
small and almost equal (Fig.
5B). These findings indicate that the S697A mutation impairs the
GDNF-mediated migration of ENCCs.
|
|
), we
analyzed their formation in the migrating ENCCs towards GDNF-soaked beads.
ENCCs were identified by immunostaining with anti-p75NTR antibody
(data not shown). In ENCCs from wild-type mice, the number of lamellipodia in
the proximal side was significantly greater than in the distal side. In the
homozygous S697A mice, the number of lamellipodia showed no significant
difference in the proximal and distal sides
(Fig. 5C,D). These results
suggest that GDNF-induced lamellipodia decrease in ENCCs from S697A mutant
mice, resulting in their impaired migration.
|
;
Fukuda et al., 2005). To
investigate whether JNK signal is also impaired in RET S697A mutant mice, DRG
neurons, some of which are known to express RET, were collected from wild-type
and mutant mice and stimulated with GDNF. As shown in
Fig. 6A, ERK, AKT, p38MAPK and
SRC were activated by GDNF in DRG neurons from both wild-type and homozygous
S697A mice, whereas JNK activation by GDNF was specifically impaired in DRG
neurons from mutant mice.
As it has been reported that the JNK signaling plays a role in cell
migration or motility (Huang et al.,
2004; Kawauchi et al.,
2003; Meadows et al.,
2004; Pedram et al.,
2001), we hypothesized that the suppressed migration of ENCCs in
the homozygous S697A mice is due to the impaired JNK signaling by the S697
mutant form of RET. To test whether JNK signaling was required for the
chemoattractant response to GDNF, explant experiments were carried out in the
presence of the JNK inhibitor SP600125. As expected, SP600125 markedly
suppressed the chemoattractant response of ENCCs to GDNF
(Fig. 6B,C).
To further analyze the importance of JNK signaling for migration of ENCCs in the developing gut, we cultured guts in vitro in the presence of various kinase inhibitors. Embryonic guts were dissected out at E11.5, and after 3 days of in vitro culture, ENCCs were visualized by PGP9.5 immunostaining. The PI3K inhibitor LY294002 severely impaired the migration of ENCCs in the cultured guts (Fig. 7C), whereas MEK1 inhibitor PD98059 showed little effect on their migration (Fig. 7B). Interestingly, in the guts cultured with the JNK inhibitor SP600125, migration of ENCCs was partly suppressed (Fig. 7D), which mimicked the impaired migration of ENCCs in the distal colon of the homozygous S697A embryos in vivo (Fig. 4F). Although the S697A mutation did not impair the SRC kinase activity (Fig. 6A), the SRC kinase inhibitor, SU6656, partly suppressed their migration (Fig. 7F). To quantify inhibitory effects, the ratios of the distance of ENCC migration to the colon length were measured and calculated. The ratios were 95±2% for control, and 93±2%, 35±3%, 75±3% and 75±2% in the presence of PD98059, LY294002, SP600125 and SU6656, respectively (Fig. 7).
Both the PKA inhibitor and the human RET S696A mutation showed similar
biological effects in the cultured cell lines
(Fukuda et al., 2002). To
investigate the role of PKA in the migration of ENCCs, embryonic guts were
cultured in the presence of the PKA inhibitor KT5720. KT5720 partly impaired
the migration of ENCCs (the ratio of ENCC migration to the colon length was
78±6%), an effect similar to that of the JNK inhibitor
(Fig. 7D,E). From these
results, it seems likely that PKA controls the JNK signaling pathway
downstream of RET via Ser697 phosphorylation, which is required for proper
migration of ENCCs in the developing gut. Although we attempted the organ
culture of the S697A mutant embryonic gut, mutant ENCCs did not migrate well
even without kinase inhibitors under our experimental conditions (data not
shown).
S697A mutation does not impair proliferation, survival and neurite outgrowth of cultured ENCCs
In addition to the chemoattractant response, it is known that GDNF induces
proliferation, survival and differentiation of cultured ENCCs. Thus, we
investigated these biological responses using cultured ENCCs from wild-type
and S697A mutant mice. As shown in Fig.
8A, both wild-type and mutant ENCCs showed remarkable neurite
outgrowth in response to GDNF stimulation. In addition, after 3 days of
culture, the neuron numbers did not show a significant difference between
wild-type and mutant ENCC cultures (Fig.
8B). These findings suggest that proliferation, survival and
differentiation of ENCCs were not significantly impaired by the S697A
mutation.
|
Ret-deficient mice show an aberrant position of superior cervical
sympathetic ganglion (SCG) and abnormal axonal projection
(Enomoto et al., 2001). Thus,
we analyzed the sympathetic system by whole-mount tyrosine hydroxylase
immunostaining in newborn mutant mice. In the homozygous S697A mice, the size
and location of SCG and chain ganglia appeared normal, and axons from ganglia
projected properly (see Fig. S1 in the supplementary material). Moreover, no
abnormalities were observed in the parasympathetic ganglia and motoneurons of
the spinal cord or in the testis (see Fig. S1 in the supplementary
material).
|
DISCUSSION |
|---|
|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
-1 in mice, it has been revealed that RET signals
play important roles in the development of the ENS and the kidney
(Cacalano et al., 1998;
Enomoto et al., 2001;
Moore et al., 1996;
Pichel et al., 1996;
Sanchez et al., 1996;
Schuchardt et al., 1994). The
RET S697A mutant mice that were generated in the present study showed an
obvious defect in ENS development, resulting in an absence of enteric ganglia
in the distal colon, whereas the only kidney defect observed was a very slight
reduction in size. Given that the RET S697A mutant mice lack a serine residue
necessary for PKA-dependent modification of RET signaling, these findings
suggest that this modification is more important in the development of the ENS
than in that of the kidney.
In the nervous system, cAMP has been demonstrated to be a key regulator of
neuronal survival, differentiation and regeneration via cAMP-mediated PKA
activation (Cai et al., 1999).
In addition, PKA appears to be constantly activated in the developing nervous
system (Cai et al., 2001).
Because GDNF has been shown to upregulate cAMP levels
(Cai et al., 1999), it is
reasonable to suggest that GDNF stimulation itself may trigger phosphorylation
of RET S697. In the developing ENS, such a condition may be necessary for the
PKA-dependent activity of RET, which regulates the activation of the JNK
signaling pathway. Moreover, our result is consistent with a recent report
which showed that the inhibition of JNK signaling does not affect ureteric bud
branching in the developing kidney
(Osafune et al., 2006).
Our data suggest that the PI3K pathway is a crucial signal in the migration
of ENCCs. Inhibition of PI3K activity markedly suppressed the migration of
ENCCs in the gut organ culture, whereas inhibition of MEK1 activity showed no
significant effect (Fig. 7B,C).
PI3K signaling induces activation of RHO family GTPases, including RHO, RAC
and CDC42, which are essential for reorganization of the actin cytoskeleton,
and thus for lamellipodia formation and cell motility
(Govek et al., 2005;
Watanabe et al., 2005). In
addition, we recently reported that AKT, a downstream effector of PI3K, plays
an important role in actin reorganization and cell motility via
phosphorylation of Girdin, a new AKT substrate
(Enomoto et al., 2005). Thus,
it is possible that migration of ENCCs is regulated via activation of these
effector proteins downstream of PI3K.
|
|
). Although we did not observe any effect of the MEK1
inhibitor on ENCC migration in our gut culture system, this difference might
be due to the different sensitivities of the different experimental systems,
and/or to the different environmental conditions surrounding ENCCs in the
whole-mount gut culture and in the chemoattractant assay. In accordance with
the present result, we reported that mutant mice with the RET mutation Y1062F,
which showed marked impairment of the PI3K/AKT signaling, developed
aganglionosis in the whole ENS, supporting the view that the PI3K/AKT pathway
is a critical signal for migration of ENCCs in the developing gut
(Jain et al., 2004;
Jijiwa et al., 2004;
Wong et al., 2005). In
addition, SRC kinase activity appears to play a role in the migration of ENCCs
(Fig. 7F), although the S697A
mutation did not impair its activity.
JNK is a serine/threonine protein kinase belonging to the family of MAPKs
(Johnson and Lapadat, 2002).
JNK is generally thought to play a role in inflammation, differentiation and
apoptosis. However, recent studies demonstrated that JNK is important for the
migration of several cell types, including keratinocytes, airway epithelial
cells, vascular smooth muscle cells, leukocytes, cortical neurons and Schwann
cells (Huang et al., 2004;
Kawauchi et al., 2003;
Meadows et al., 2004;
Pedram et al., 2001). As well
as the JNK activation with injury stress, JNK activation after stimulation
with transforming growth factorß, interleukin 8, epidermal growth factor
or platelet-derived growth factor can promote cell migration. JNK has been
implicated in the control of the actin cytoskeleton and stress fibers in
Drosophila embryos and mammalian cells
(Kaltschmidt et al., 2002;
Martin-Blanco et al., 2000;
Zhang et al., 2003). In
addition to the JNK-mediated phosphorylation of various well-studied
apoptosis-related proteins and transcriptional factors, JNK directly
phosphorylates a number of cytoskeleton-associated proteins and signaling
molecules, including paxillin, spir, doublecortin, and microtubule-associated
protein 1B and 2, which are suggested to play roles in cell migration
(Chang et al., 2003;
Gdalyahu et al., 2004;
Huang et al., 2003;
Kawauchi et al., 2003;
Otto et al., 2000). In RET
S697A mutant mice, JNK activation was specifically impaired, whereas both
p38MAPK and ERK, which are also members of the MAPK family, were activated by
GDNF. In addition, gut explant culture experiments showed that chemoattractant
response of ENCCs from S697A mice to GDNF and their lamellipodia formation
were impaired and that the JNK inhibitor suppressed ENCCs migration. Although
it is possible that suppressed migration of ENCCs in the homozygous S697A mice
is due to undefined signals impaired by the S697A mutation, the present data
strongly support the view that lack of JNK activation is responsible for the
suppression of ENCC migration in the mutant mice.
We recently demonstrated that GDNF stimulation induced G2/M cell-cycle
delay in MC(RET) cells but not in S696A cells
(Fukuda et al., 2005). As this
result suggested the possibility that RET S697A mutation affects the
proliferation of ENS cells, we analyzed cell numbers in the ENS of the S697A
mutant mice. As shown in Fig.
3, there was no significant difference in the total numbers of ENS
cells in the whole small intestine and the proximal colon between wild-type
and RET S697A mutant mice. Thus, the RET S697A mutation does not appear to
affect cell proliferation in vivo. Moreover, we found that this mutation does
not significantly affect proliferation, survival and neurite outgrowth of
cultured ENCCs. ACHE-stained neural plexus and neural fibers showed normal
number and size in the proximal mutant guts. A previous study reported a
similar phenotype in the miRET51 mice expressing only the long
isoform of RET (de Graaff et al.,
2001). The miRET51 mice showed aganglionosis in the
distal gut due to the delayed migration of ENCCs, although the ENS in the
small intestine of mutant newborns was normal. As patients of Hirschsprung's
disease also have aganglionosis in the distal colon, these mutant mice may
provide useful models to study the molecular mechanisms of development of
Hirschsprung's disease.
The RET tyrosine kinase is evolutionarily close to fibroblast growth factor
receptor, vascular endothelial growth factor receptor, Kit and
platelet-derived growth factor receptor. The 3' halves of the
juxtamembrane regions of RET and these other receptor tyrosine kinases share
significant similarity, whereas the 5' half of the juxtamembrane region,
containing Y688 and S697, is unique in RET. Thus, the juxtamembrane region in
RET may have a unique function. The juxtamembrane region in human RET contains
one tyrosine residue, Y687 (corresponding to Y688 in mouse RET), which was
reported as one of the autophosphorylation sites
(Liu et al., 1996). We
previously demonstrated that S696 is a putative phosphorylation site for PKA,
and that lamellipodia formation impaired by the S696A mutation was rescued
with an additional Y687F mutation, suggesting that S696 and Y687
phosphorylation leads to opposite biological effects
(Fukuda et al., 2002). As Y687
and S696 are located closely, it seems likely that S696 phosphorylation
suppresses Y687 phosphorylation via a conformational change of the
juxtamembrane region. Interestingly, two Hirschsprung's disease-related
Ret mutations (S690P and G691S) have been reported in this
juxtamembrane region (Attie et al.,
1995; Garcia-Barcelo et al.,
2004). The computational prediction (NetPhosK:
http://www.cbs.dtu.dk/services/NetPhosK/)
suggests that both mutations may affect the probability of phosphorylation at
Y687, supporting the idea that a signal from juxtamembrane region via Y687
phosphorylation plays a role in the ENS development.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/133/22/4507/DC1
|
ACKNOWLEDGMENTS |
|---|
|
Footnotes |
|---|
|
REFERENCES |
|---|
|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Airaksinen, M. S. and Saarma, M. (2002). The GDNF family: signalling, biological functions and therapeutic value. Nat. Rev. Neurosci. 3,383 -394.[CrossRef][Medline]
Attie, T., Pelet, A., Edery, P., Eng, C., Mulligan, L. M.,
Amiel, J., Boutrand, L., Beldjord, C., Nihoul-Fekete, C., Munnich, A. et
al. (1995). Diversity of RET proto-oncogene mutations in
familial and sporadic Hirschsprung disease. Hum. Mol.
Genet. 4,1381
-1386.
Barlow, A., de Graaff, E. and Pachnis, V. (2003). Enteric nervous system progenitors are coordinately controlled by the G protein-coupled receptor EDNRB and the receptor tyrosine kinase RET. Neuron 40,905 -916.[CrossRef][Medline]
Barone, M. V., Sepe, L., Melillo, R. M., Mineo, A., Santelli, G., Monaco, C., Castellone, M. D., Tramontano, D., Fusco, A. and Santoro, M. (2001). RET/PTC1 oncogene signaling in PC Cl 3 thyroid cells requires the small GTP-binding protein Rho. Oncogene 20,6973 -6982.[CrossRef][Medline]
Cacalano, G., Farinas, I., Wang, L. C., Hagler, K., Forgie, A., Moore, M., Armanini, M., Phillips, H., Ryan, A. M., Reichardt, L. F. et al. (1998). GFRalpha1 is an essential receptor component for GDNF in the developing nervous system and kidney. Neuron 21,53 -62.[CrossRef][Medline]
Cai, D., Shen, Y., De Bellard, M., Tang, S. and Filbin, M. T. (1999). Prior exposure to neurotrophins blocks inhibition of axonal regeneration by MAG and myelin via a cAMP-dependent mechanism. Neuron 22,89 -101.[CrossRef][Medline]
Cai, D., Qiu, J., Cao, Z., McAtee, M., Bregman, B. S. and
Filbin, M. T. (2001). Neuronal cyclic AMP controls the
developmental loss in ability of axons to regenerate. J.
Neurosci. 21,4731
-4739.
Chang, L., Jones, Y., Ellisman, M. H., Goldstein, L. S. and Karin, M. (2003). JNK1 is required for maintenance of neuronal microtubules and controls phosphorylation of microtubule-associated proteins. Dev. Cell 4,521 -533.[CrossRef][Medline]
Chiariello, M., Visconti, R., Carlomagno, F., Melillo, R. M., Bucci, C., de Franciscis, V., Fox, G. M., Jing, S., Coso, O. A., Gutkind, J. S. et al. (1998). Signalling of the Ret receptor tyrosine kinase through the c-Jun NH2-terminal protein kinases (JNKS): evidence for a divergence of the ERKs and JNKs pathways induced by Ret. Oncogene 16,2435 -2445.[CrossRef][Medline]
de Graaff, E., Srinivas, S., Kilkenny, C., D'Agati, V., Mankoo,
B. S., Costantini, F. and Pachnis, V. (2001). Differential
activities of the RET tyrosine kinase receptor isoforms during mammalian
embryogenesis. Genes Dev.
15,2433
-2444.
Edery, P., Lyonnet, S., Mulligan, L. M., Pelet, A., Dow, E., Abel, L., Holder, S., Nihoul-Fekete, C., Ponder, B. A. and Munnich, A. (1994). Mutations of the RET proto-oncogene in Hirschsprung's disease. Nature 367,378 -380.[CrossRef][Medline]
Encinas, M., Tansey, M. G., Tsui-Pierchala, B. A., Comella, J.
X., Milbrandt, J. and Johnson, E. M., Jr (2001). c-Src is
required for glial cell line-derived neurotrophic factor (GDNF) family
ligand-mediated neuronal survival via a phosphatidylinositol-3 kinase
(PI-3K)-dependent pathway. J. Neurosci.
21,1464
-1472.
Enomoto, A., Murakami, H., Asai, N., Morone, N., Watanabe, T., Kawai, K., Murakumo, Y., Usukura, J., Kaibuchi, K. and Takahashi, M. (2005). Akt/PKB regulates actin organization and cell motility via Girdin/APE. Dev. Cell 9, 389-402.[CrossRef][Medline]
Enomoto, H., Araki, T., Jackman, A., Heuckeroth, R. O., Snider, W. D., Johnson, E. M., Jr and Milbrandt, J. (1998). GFR alpha1-deficient mice have deficits in the enteric nervous system and kidneys. Neuron 21,317 -324.[CrossRef][Medline]
Enomoto, H., Heuckeroth, R. O., Golden, J. P., Johnson, E. M. and Milbrandt, J. (2000). Development of cranial parasympathetic ganglia requires sequential actions of GDNF and neurturin. Development 127,4877 -4889.[Abstract]
Enomoto, H., Crawford, P. A., Gorodinsky, A., Heuckeroth, R. O., Johnson, E. M., Jr and Milbrandt, J. (2001). RET signaling is essential for migration, axonal growth and axon guidance of developing sympathetic neurons. Development 128,3963 -3974.[Medline]
Fukuda, T., Kiuchi, K. and Takahashi, M.
(2002). Novel mechanism of regulation of Rac activity and
lamellipodia formation by RET tyrosine kinase. J. Biol.
Chem. 277,19114
-19121.
Fukuda, T., Asai, N., Enomoto, A. and Takahashi, M.
(2005). Activation of c-Jun amino-terminal kinase by GDNF induces
G2/M cell cycle delay linked with actin reorganization. Genes
Cells 10,655
-663.
Garcia-Barcelo, M., Sham, M. H., Lee, W. S., Lui, V. C., Chen,
B. L., Wong, K. K., Wong, J. S. and Tam, P. K. (2004). Highly
recurrent RET mutations and novel mutations in genes of the receptor tyrosine
kinase and endothelin receptor B pathways in Chinese patients with sporadic
Hirschsprung disease. Clin. Chem.
50, 93-100.
Gdalyahu, A., Ghosh, I., Levy, T., Sapir, T., Sapoznik, S., Fishler, Y., Azoulai, D. and Reiner, O. (2004). DCX, a new mediator of the JNK pathway. EMBO J. 23,823 -832.[CrossRef][Medline]
Govek, E. E., Newey, S. E. and Van Aelst, L.
(2005). The role of the Rho GTPases in neuronal development.
Genes Dev. 19,1
-49.
Hellmich, H. L., Kos, L., Cho, E. S., Mahon, K. A. and Zimmer, A. (1996). Embryonic expression of glial cell-line derived neurotrophic factor (GDNF) suggests multiple developmental roles in neural differentiation and epithelialmesenchymal interactions. Mech. Dev. 54,95 -105.[CrossRef][Medline]
Huang, C., Rajfur, Z., Borchers, C., Schaller, M. D. and Jacobson, K. (2003). JNK phosphorylates paxillin and regulates cell migration. Nature 424,219 -223.[CrossRef][Medline]
Huang, C., Jacobson, K. and Schaller, M. D.
(2004). MAP kinases and cell migration. J. Cell
Sci. 117,4619
-4628.
Ichihara, M., Murakumo, Y. and Takahashi, M. (2004). RET and neuroendocrine tumors. Cancer Lett. 204,197 -211.[CrossRef][Medline]
Iwashita, T., Kruger, G. M., Pardal, R., Kiel, M. J. and
Morrison, S. J. (2003). Hirschsprung disease is linked to
defects in neural crest stem cell function. Science
301,972
-976.
Jain, S., Naughton, C. K., Yang, M., Strickland, A., Vij, K.,
Encinas, M., Golden, J., Gupta, A., Heuckeroth, R., Johnson, E. M., Jr et
al. (2004). Mice expressing a dominant-negative Ret mutation
phenocopy human Hirschsprung disease and delineate a direct role of Ret in
spermatogenesis. Development
131,5503
-5513.
Jijiwa, M., Fukuda, T., Kawai, K., Nakamura, A., Kurokawa, K.,
Murakumo, Y., Ichihara, M. and Takahashi, M. (2004). A
targeting mutation of tyrosine 1062 in Ret causes a marked decrease of enteric
neurons and renal hypoplasia. Mol. Cell. Biol.
24,8026
-8036.
Jing, S., Wen, D., Yu, Y., Holst, P. L., Luo, Y., Fang, M., Tamir, R., Antonio, L., Hu, Z., Cupples, R. et al. (1996). GDNF-induced activation of the ret protein tyrosine kinase is mediated by GDNFR-alpha, a novel receptor for GDNF. Cell 85,1113 -1124.[CrossRef][Medline]
Johnson, G. L. and Lapadat, R. (2002).
Mitogen-activated protein kinase pathways mediated by ERK, JNK, and p38
protein kinases. Science
298,1911
-1912.
Kaltschmidt, J. A., Lawrence, N., Morel, V., Balayo, T., Fernandez, B. G., Pelissier, A., Jacinto, A. and Martinez Arias, A. (2002). Planar polarity and actin dynamics in the epidermis of Drosophila. Nat. Cell Biol. 4, 937-944.[CrossRef][Medline]
Kawauchi, T., Chihama, K., Nabeshima, Y. and Hoshino, M. (2003). The in vivo roles of STEF/Tiam1, Rac1 and JNK in cortical neuronal migration. EMBO J. 22,4190 -4201.[CrossRef][Medline]
Klein, R. D., Sherman, D., Ho, W. H., Stone, D., Bennett, G. L., Moffat, B., Vandlen, R., Simmons, L., Gu, Q., Hongo, J. A. et al. (1997). A GPI-linked protein that interacts with Ret to form a candidate neurturin receptor. Nature 387,717 -721.[CrossRef][Medline]
Kodama, Y., Asai, N., Kawai, K., Jijiwa, M., Murakumo, Y., Ichihara, M. and Takahashi, M. (2005). The RET proto-oncogene: a molecular therapeutic target in thyroid cancer. Cancer Sci. 96,143 -148.[CrossRef][Medline]
Lewandoski, M. and Martin, G. R. (1997). Cre-mediated chromosome loss in mice. Nat. Genet. 17,223 -225.[CrossRef][Medline]
Liu, X., Vega, Q. C., Decker, R. A., Pandey, A., Worby, C. A.
and Dixon, J. E. (1996). Oncogenic RET receptors display
different autophosphorylation sites and substrate binding specificities.
J. Biol. Chem. 271,5309
-5312.
Maeda, K., Murakami, H., Yoshida, R., Ichihara, M., Abe, A., Hirai, M., Murohara, T. and Takahashi, M. (2004). Biochemical and biological responses induced by coupling of Gab1 to phosphatidylinositol 3-kinase in RET-expressing cells. Biochem. Biophys. Res. Commun. 323,345 -354.[CrossRef][Medline]
Martin-Blanco, E., Pastor-Pareja, J. C. and Garcia-Bellido,
A. (2000). JNK and decapentaplegic signaling control
adhesiveness and cytoskeleton dynamics during thorax closure in Drosophila.
Proc. Natl. Acad. Sci. USA
97,7888
-7893.
Meadows, K. N., Bryant, P., Vincent, P. A. and Pumiglia, K. M. (2004). Activated Ras induces a proangiogenic phenotype in primary endothelial cells. Oncogene 23,192 -200.[CrossRef][Medline]
Meng, X., Lindahl, M., Hyvonen, M. E., Parvinen, M., de Rooij,
D. G., Hess, M. W., Raatikainen-Ahokas, A., Sainio, K., Rauvala, H., Lakso, M.
et al. (2000). Regulation of cell fate decision of
undifferentiated spermatogonia by GDNF. Science
287,1489
-1493.
Moore, M. W., Klein, R. D., Farinas, I., Sauer, H., Armanini, M., Phillips, H., Reichardt, L. F., Ryan, A. M., Carver-Moore, K. and Rosenthal, A. (1996). Renal and neuronal abnormalities in mice lacking GDNF. Nature 382, 76-79.[CrossRef][Medline]
Murakami, H., Iwashita, T., Asai, N., Iwata, Y., Narumiya, S. and Takahashi, M. (1999). Rho-dependent and -independent tyrosine phosphorylation of focal adhesion kinase, paxillin and p130Cas mediated by Ret kinase. Oncogene 18,1975 -1982.[CrossRef][Medline]
Natarajan, D., Marcos-Gutierrez, C., Pachnis, V. and de Graaff, E. (2002). Requirement of signalling by receptor tyrosine kinase RET for the directed migration of enteric nervous system progenitor cells during mammalian embryogenesis. Development 129,5151 -5160.[Medline]
Osafune, K., Takasato, M., Kispert, A., Asashima, M. and
Nishinakamura, R. (2006). Identification of multipotent
progenitors in the embryonic mouse kidney by a novel colony-forming assay.
Development 133,151
-161.
Otto, I. M., Raabe, T., Rennefahrt, U. E., Bork, P., Rapp, U. R. and Kerkhoff, E. (2000). The p150-Spir protein provides a link between c-Jun N-terminal kinase function and actin reorganization. Curr. Biol. 10,345 -348.[CrossRef][Medline]
Pachnis, V., Mankoo, B. and Costantini, F. (1993). Expression of the c-ret protooncogene during mouse embryogenesis. Development 119,1005 -1017.[Abstract]
Pedram, A., Razandi, M. and Levin, E. R.
(2001). Natriuretic peptides suppress vascular endothelial cell
growth factor signaling to angiogenesis. Endocrinology
142,1578
-1586.
Pichel, J. G., Shen, L., Sheng, H. Z., Granholm, A. C., Drago, J., Grinberg, A., Lee, E. J., Huang, S. P., Saarma, M., Hoffer, B. J. et al. (1996). Defects in enteric innervation and kidney development in mice lacking GDNF. Nature 382, 73-76.[CrossRef][Medline]
Romeo, G., Ronchetto, P., Luo, Y., Barone, V., Seri, M., Ceccherini, I., Pasini, B., Bocciardi, R., Lerone, M., Kaariainen, H. et al. (1994). Point mutations affecting the tyrosine kinase domain of the RET proto-oncogene in Hirschsprung's disease. Nature 367,377 -378.[CrossRef][Medline]
Sanchez, M. P., Silos-Santiago, I., Frisen, J., He, B., Lira, S. A. and Barbacid, M. (1996). Renal agenesis and the absence of enteric neurons in mice lacking GDNF. Nature 382, 70-73.[CrossRef][Medline]
Schuchardt, A., D'Agati, V., Larsson-Blomberg, L., Costantini, F. and Pachnis, V. (1994). Defects in the kidney and enteric nervous system of mice lacking the tyrosine kinase receptor Ret. Nature 367,380 -383.[CrossRef][Medline]
Treanor, J. J., Goodman, L., de Sauvage, F., Stone, D. M., Poulsen, K. T., Beck, C. D., Gray, C., Armanini, M. P., Pollock, R. A., Hefti, F. et al. (1996). Characterization of a multicomponent receptor for GDNF. Nature 382, 80-83.[CrossRef][Medline]
van Weering, D. H. and Bos, J. L. (1997). Glial
cell line-derived neurotrophic factor induces Ret-mediated lamellipodia
formation. J. Biol. Chem.
272,249
-254.
Watanabe, T., Noritake, J. and Kaibuchi, K. (2005). Regulation of microtubules in cell migration. Trends Cell Biol. 15,76 -83.[CrossRef][Medline]
Wong, A., Bogni, S., Kotka, P., de Graaff, E., D'Agati, V.,
Costantini, F. and Pachnis, V. (2005). Phosphotyrosine 1062
is critical for the in vivo activity of the Ret9 receptor tyrosine kinase
isoform. Mol. Cell. Biol.
25,9661
-9673.
Young, H. M. and Newgreen, D. (2001). Enteric neural crest-derived cells: origin, identification, migration, and differentiation. Anat. Rec. 262, 1-15.[CrossRef][Medline]
Young, H. M., Hearn, C. J., Farlie, P. G., Canty, A. J., Thomas, P. Q. and Newgreen, D. F. (2001). GDNF is a chemoattractant for enteric neural cells. Dev. Biol. 229,503 -516.[CrossRef][Medline]
Zhang, L., Wang, W., Hayashi, Y., Jester, J. V., Birk, D. E., Gao, M., Liu, C. Y., Kao, W. W., Karin, M. and Xia, Y. (2003). A role for MEK kinase 1 in TGF-beta/activin-induced epithelium movement and embryonic eyelid closure. EMBO J. 22,4443 -4454.[CrossRef][Medline]
This article has been cited by other articles:
|
|
 |
|
  A. Boulay, M. Breuleux, C. Stephan, C. Fux, C. Brisken, M. Fiche, M. Wartmann, M. Stumm, H. A. Lane, and N. E. Hynes The Ret Receptor Tyrosine Kinase Pathway Functionally Interacts with the ER{alpha} Pathway in Breast Cancer Cancer Res., May 15, 2008; 68(10): 3743 - 3751. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||
