Signal Transduction Laboratory, Institute of Molecular and Cell Biology, 30 Medical Drive, Singapore 117 609, Republic of Singapore
* Author for correspondence (e-mail: mcbgg{at}imcb.nus.edu.sg)
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Summary |
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Key words: Sprouty, Fibroblast growth factor, Receptor tyrosine kinase, Epidermal growth factor receptor, MAP kinase
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Introduction |
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Bnl has a secondary role in patterning: it induces later programs of
branching in cells near the ends of the primary branches and in this way
contributes to the apical bias in secondary branching. dSpry-/-
mutants cause excessive branching from the stalks of primary branches, close
to the tips where secondary branches normally divide. Furthermore, the
excessive branching appears to result from overactivity of the Bnl pathway.
Molecular evidence presented by Hacohen et al. indicated that spry
encodes a novel inhibitor that limits the range of Bnl signaling and thus
restricts secondary budding to apical positions closest to the FGF signaling
centers (Hacohen et al.,
1998).
The dSpry open reading frame gave a predicted 591-residue (63 kDa)
polypeptide whose most striking feature was a 124-residue C-terminal,
cysteine-rich region (22 cysteine residues), which is flanked by cysteine-free
regions lacking any recognizable protein-protein interaction domain. A
databank search subsequently identified three human homologues that are
separate gene products, designated hSpry1, hSpry2 and hSpry3. hSpry2 is a
315-residue (35 kDa) polypeptide that also contains a cysteine-rich domain,
which shows 51% identity to the similar domain in dSpry
(Hacohen et al., 1998).
Subsequently, four full-length murine Spry proteins (mSpry1-mSpry4) were
cloned and sequenced (Tefft et al.,
1999
; de Maximy et al.,
1999
). At least one short sequence in the N-termini of all Spry
proteins, as well as several isolated sequences or residues distributed
throughout the protein family, is also conserved
(Fig. 1). The conserved
cysteine-rich domain has also been found in another family of proteins, termed
Sprouty-related proteins with an EVH-1
domain (Spreds) (Wakioka et
al., 2001
). The Spred proteins also possess both an N-terminal
enabled/vasodilator-stimulated phosphoprotein (VASP)
homology domain (EVH1) and a c-Kit-binding
domain (KBD) (Fig.
2), which suggests that the two families have different cellular
functions with respect to their interaction with likely binding partners. EVH1
domains bind polyproline peptides with the consensus FPPPP, whereas the KBD
was defined by Wakioka et al. as a common domain on Spred 1 and Spred 2
necessary for binding to the c-Kit RTK
(Wakioka et al., 2001
;
Niebuhr et al., 1997
;
Renfranz and Beckerle,
2002
).
|
|
Hacohen et al. showed that dSpry localizes to the plasma membrane and that
a proportion of the protein can be released into the extracellular environment
and compete with FGF ligand for binding to its receptor
(Hacohen et al., 1998). There
has subsequently been no evidence to support this mode of dSpry or mammalian
Spry action. dSpry is expressed in developing embryos in the tracheal system,
the midline glia and the dorsal vessel, all places where Bnl and Btl are known
to function. It is also expressed in a small subset of VNC neurons, oenocytes
and the eye imaginal disc. Its expression is induced by the Bnl pathway, thus
making the mode of repression a classical feedback mechanism. Tefft et al.
demonstrated that the control of branching by dSpry in the Drosophila
tracheal system has a parallel in mammals
(Tefft et al., 1999
). Using an
antisense oligonucleotide strategy they demonstrated that inhibition of mSpry2
expression in E11.5 murine embryonic cells produces a 72% increase in murine
lung branching. Northern blotting analysis showed that mSpry2 is also
expressed in the adult lung, as well as the heart, brain, skeletal muscle and
kidney.
Another hallmark paper showed that in Drosophila, at least, the
role of Spry is not confined to modulation of tissue branching initiated by
FGF. Casci et al. identified dSpry in a genetic screen as an inhibitor of EGF
receptor (EGFR) signaling (Casci et al.,
1999). EGFR triggers cell recruitment in the eye, and the authors
showed that in eyes lacking dSpry there is an excess of photoreceptors, cone
cells and pigment cells. Furthermore, they demonstrated in genetic screens
that signaling from other receptor tyrosine kinases, Torso and Sevenless, is
also inhibited by dSpry. Studies by Kramer et al. reinforced these
observations, demonstrating that overexpression of dSpry in wing veins and
ovarian follicle cells, two systems where EGFR activation is required for
patterning, results in a phenotype that resembles that of EGFR
loss-of-function mutants (Kramer et al.,
1999
). Casci et al. further demonstrated that dSpry localizes to
the inside surface of the plasma membrane and intercepts signaling at a point
between the receptor and activation of Ras. This study implicated Spry as a
likely generic inhibitor of the Ras/MAP-kinase signaling pathway. Subsequent
work from most labs indicates that Spry proteins act intracellularly. Casci et
al. employed an in vitro candidate approach and revealed that dSpry binds to
Drk, the homologue of mammalian Grb2, and Gap1, an
inositol-phospholipid-binding GTPase-activating protein (GAP) that has
activity against Ras (Casci et al.,
1999
). It was therefore postulated that dSpry might sequester
vital components of the Ras/MAP-kinase pathway. The authors further showed
that the binding to Drk and Gap1 is mediated by the non-conserved N-terminal
region of dSpry, whereas the conserved cysteine-rich domain is responsible for
localizing the protein to the membrane. It thus appeared that the Sprys have a
conserved, C-terminal domain responsible for targeting and a divergent
protein-interacting domain through which the different isoforms recruit a
range of proteins to fulfill their physiological functions.
Genetic analysis in Drosophila reinforced these observations
allowing Reich et al. to demonstrate that ectopic expression of dSpry
abolishes the pattern of activated MAP kinase observed in embryos and
specifically in another FGFR system involving heartless (another FGFR1
homologue), as well as EGF-controlled systems, such as wing imaginal discs
(Reich et al., 1999). dSpry
expression is induced by activation of the EGFR pathway in some but not all
cells. The responsive cells include follicle cells of the ovary, the wing
imaginal disc and the eye disc, and these studies deduced that dSpry
intersects the Ras/MAP-kinase pathway at or downstream of the Raf kinase. This
represented the third postulated mode of dSpry action. It was therefore
possible that dSpry might have multiple or different intersection points for
different factors; alternatively some unifying evidence remained to be
discovered.
In another non-mammalian species, Xenopus, Nutt et al.
demonstrated that the expression of xSpry2 is induced by the
FGF/Ras/MAP-kinase pathway (Nutt et al.,
2001). xSpry2 inhibits mesoderm induction and results in
truncation of the anterior-posterior axis. Although blocking the
Ras/MAP-kinase pathway inhibits mesoderm induction, xSpry2 does not seem to
act on this level but, rather, inhibits FGF-dependent calcium signaling.
Collective evidence strongly indicates that Spry isoforms play a role, probably in opposition to FGF signaling pathways, in modeling different varieties of branching tissue during development. Lung, kidney, prostate, blood vessels and breast ducts all seem likely to come under the modulating influence of induced Spry proteins. The question can therefore be asked: how does the branch manager work?
Note that a number of studies have attempted to address the mode of action of Sprys and there has been some diversity in the observations made. Here we emphasize those that appear to have been substantiated by more than one laboratory.
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Unraveling the mechanism of action of mammalian Spry proteins |
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Target of the conserved cysteine-rich domain |
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Inhibition of the mammalian Ras/MAP-kinase pathway |
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Sasaki et al. have observed similar differences in the inhibitory effects
that the mammalian Spry proteins have on the EGF and FGF pathways. They
demonstrated that EGF, FGF and phorbol ester rapidly induce Spry2 and Spry4 in
cultured cells. Overexpression of hSpry2 or hSpry4 inhibited FGF-induced
MAP-kinase activation but did not affect EGF- or PDBu-induced MAP-kinase
activation. They also generated dominant negative point mutants of hSpry2 (Y55
hSpry2) and hSpry4 (Y53 hSpry4). Whereas wild-type hSpry4 inhibits FGF-induced
differentiation in PC12 cells, Y53 hSpry4 enhances differentiation. Previously
the extension of neurites in PC12 cells has been linked to the strength and
duration of a MAP-kinase signal (Marshall,
1995). Two further reports have reinforced the idea that Spry1 or
Spry2 inhibits FGF-induced Ras/MAP-kinase activation (revealing incidentally
that they do not affect either the p38 or JNK MAP-kinase pathways or the Akt
pathway) (Gross et al., 2001
;
Yusoff et al., 2002
). The
former placed the Spry proteins before Ras, while the latter put them at the
level of Raf. In the former study it was demonstrated that expression of
hSpry2 results in formation of less Ras-GTP
(Gross et al., 2001
). This was
not reiterated in the study of Yusoff et al., which showed that when hSpry2
was similarly expressed in cells it inhibited activation of MAP kinase that
was stimulated by the constitutively active V12 Ras mutant
(Yusoff et al., 2002
). In
another study, Leeksma et al. showed that the ubiquitously expressed hSpry4
suppresses insulin- and EGFR-receptor-induced MAP-kinase signaling probably at
the level of or upstream of Ras (Leeksma
et al., 2002
).
The work discussed above indicates that Spry1, Spry2 and Spry4 inhibit
FGF-induced Ras/MAP-kinase activation in a relatively specific manner and that
in mammals there seems to be some deviation from what is observed in
Drosophila with respect to the effects of Spry proteins on
EGF-induced signaling. Subsequent publications have provided some insight into
the apparent differences between the effects of Spry on EGF- and FGF-activated
signaling, demonstrating that Spry2 interacts with the adaptor c-Cbl
(Egan et al., 2002;
Wong, E. S. et al., 2002
),
which is necessary for the ubiquitylation and endocytosis of EGFR
(Waterman and Yarden, 2001
).
This might provide one mechanism by which inhibition is effected (see
below).
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Binding partners for Spry proteins |
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Binding partners in FGF signaling |
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A handful of upstream MAP-kinase pathway proteins have thus been implicated in binding directly or indirectly to Spry isoforms during FGFR signaling but a unifying mechanism has yet to emerge. In the case of EGFR signaling, however, strong evidence indicates that interaction with Cbl family proteins has a central role in Spry function, as we discuss below.
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The role of Cbl family proteins |
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|
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Mechanism of action of hSpry 2 |
---|
We propose the following hypothesis for the inhibition of EGFR endocytosis (Fig. 4A,B). In this model, hSpry2 binds constitutively to c-Cbl (an N-terminal sequence in hSpry2 binds to the RING finger of c-Cbl). Upon stimulation of RTKs, the conserved tyrosine residue becomes phosphorylated and binds directly to the SH2 domain of c-Cbl, which normally binds at a site within a similar motif on the activated EGFR (Y1045). Following binding to EGFR, c-Cbl assumes its role as an E3 ubiquitin ligase and ubiquitylates the EGFR, targeting it for destruction by the endocytotic machinery of the cell (Fig. 4A). Thus hSpry2, which has a similar sequence around the Y55 residue, binds to the site where receptors are highly concentrated in membrane ruffles and acts as a decoy, `luring' c-Cbl away from the EGFR by direct competition. This would thus inhibit EGFR endocytosis and consequently the downstream pathways, such as the Ras/MAP-kinase pathway, would remain in an activated state (Fig. 4B). The FGFR isoforms do not have a similar targeting sequence for c-Cbl and consequently are immune to this form of binding competition and subsequent attenuation of `normal' downregulatory events (Fig. 4C,D).
|
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Perspectives |
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There is a greater understanding of how Spry2 modulates EGFR endocytosis. How it regulates FGFR-induced MAP-kinase signaling and thus the basis of its developmental function in branching is much less clear. Sequence comparisons suggest that Spry proteins function by a similar mechanism in distantly related species and underscore the importance of the SpryTD and the N-terminal, short tyrosine-containing motif, which is 100% conserved from Drosophila to man. There are several serines that also appear to be conserved, which begs the question of whether these are phosphorylated in a physiological context.
If in mammalian systems Spry activity is confined mainly to FGFR pathways,
a logical target for Spry would be the relatively specific FGFR or FRS2
docking protein. The latter is confined to the FGF and nerve growth factor
(NGF) pathways, and is reported to act directly or indirectly with hSpry2
(Tefft et al., 2002;
Hanafusa et al., 2002
).
However, FRS2 appears to be absent from Drosophila and as such would
not fit into a universal scheme of Spry function. Currently it has been
reasonably well established that phosphorylation of Y55 on hSpry2 is necessary
for its inhibitory effect on the Ras/MAP-kinase pathway. It is likely the
identity of the protein (or proteins) that binds to this site will provide the
key to Spry function. It is plausible that proteins with SH2 or PTB domains
that are key components of the Ras/MAP-kinase pathway are `diverted' onto this
site, which would this take them away from functional complexes. Neither the
SH2 domain of Grb2 nor Shp2 binds directly to the hSpry2 Y55 motif, because
both have strict C-terminal adjunct residues in comparison to the NXYXXXP
conserved sequence found in all Spry proteins. Currently c-Cbl is the only
candidate for binding to hSpry2 Y55 and could fulfill the inhibitory function
by one or more of the roles that have been assigned to this versatile protein
(Clague and Urbe, 2001
;
Rao et al., 2002
;
Shtiegman and Yarden, 2003
)
(C. W. Fong, H. F. Leong, E. S. M. Wong, J. Lim, P. Yusoff and G. R. Guy,
unpublished). How this actually works and what other proteins are involved
will be the topic of further studies.
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