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First published online 15 February 2006
doi: 10.1242/dev.02255
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1 Department of Biochemistry and Howard Hughes Medical Institute, Stanford
University School of Medicine, Stanford, CA 94305-5307 USA.
2 Department of Biological Sciences, Stanford University, Stanford, CA
94305-5020 USA.
¶ Author for correspondence (e-mail: krasnow{at}cmgm.stanford.edu)
Accepted 16 December 2005
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Sprouty (Spry), Corkscrew (Csw), Ptpn11 (SHP-2), Tyrosine phosphatase, Receptor tyrosine kinase (RTK) signaling, Drosophila
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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).
Indeed, many RTK pathways are equipped with positively acting regulators and
negative feedback loops, the balance of which dictates the cellular response
(Perrimon and McMahon, 1999).
Two of the best known but least well understood classes of regulators are the
Corkscrew/SHP-2 family of tyrosine phosphatases that promote RTK signaling and
the Sprouty family of RTK feedback inhibitors, whose genetic and molecular
interactions we explore here.
corkscrew was identified by its requirement for development of the
terminal regions of the embryo. It encodes an SH2 domain-containing protein
tyrosine phosphatase that functions downstream of, and promotes signaling
through, the RTK Torso (Perkins et al.,
1992). A vertebrate homolog, SHP-2 (now known as Ptpn11), was
found to promote PDGFR signaling (Freeman
et al., 1992; Bennett et al.,
1994). Further work demonstrated that Csw and SHP-2 promotes
signaling downstream of many RTKs in a variety of systems
(Perkins et al., 1996)
(reviewed by Feng, 1999), and
that this activity requires their tyrosine phosphate-binding SH2 domains and
tyrosine phosphatase activity (Allard et
al., 1998; Deb et al.,
1998).
How can tyrosine phosphatases promote tyrosine kinase signaling? To account
for their positive effect on signaling, Csw and SHP-2 were postulated to
dephosphorylate either a positive RTK effector that is inactivated by
phosphorylation or a negative regulator that is activated by phosphorylation
(Stein-Gerlach et al., 1998;
Huyer and Alexander, 1999).
This initiated searches for Csw/SHP-2 substrates by genetic screens
(Herbst et al., 1996;
Firth et al., 2000),
biochemical screens using substrate-trapping forms of the phosphatases
(Herbst et al., 1996;
Agazie and Hayman, 2003a), and
tests of candidate proteins. These identified several signal transduction
scaffolding proteins, including DOS, GAB1, GAB2, SHPS1 and IRS1
(Qu, 2002). However,
dephosphorylation of these substrates by Csw/SHP-2 inhibits RTK signaling, so
they cannot account for the positive effects of Csw/SHP-2.
Two other types of substrates may account for some positive effects of
Csw/SHP-2 on RTK signaling. One is the phosphotyrosines on Torso, PDGFR and
EGFR that mediate binding and signal inhibition by RasGAP
(Cleghon et al., 1998;
Ekman et al., 2002;
Agazie and Hayman, 2003b). The
other is the CSK-binding proteins Paxillin and PAG/Cbp, dephosphorylation of
which prevent CSK-mediated inactivation of Src kinase activity and its
positive effect on RTK signaling (Ren et
al., 2004; Zhang et al.,
2004). However, neither of these is likely to be a general target
and other crucial substrates remain to be identified.
Several observations make Sprouty proteins appealing candidates for
Csw/SHP-2 substrates. First, although Sprouty (Spry) was identified by its
role as an Fgf feedback inhibitor in Drosophila tracheal
(respiratory) development (Hacohen et al.,
1998), Spry and its four mammalian homologs, Spry1-Spry4, are now
known to function more broadly as RTK feedback inhibitors that can regulate a
variety of RTK pathways including FGF, EGF, VEGF and PDGF pathways
(Casci et al., 1999;
Kramer et al., 1999;
Reich et al., 1999;
Impagnatiello et al., 2001;
Lee et al., 2001;
Nutt et al., 2001), all of
which are also regulated by Csw/SHP-2
(Perkins et al., 1996;
Feng, 1999). Both Spry and
SHP-2 influence RTK signaling kinetics
(Saxton et al., 1997;
Dikic and Giordano, 2003).
Second, although the mechanism by which Spry proteins modulate signaling is
not understood, like Csw/SHP-2 they localize to the membrane following
signaling and associate with RTK signaling complexes
(Allard et al., 1996;
Herbst et al., 1999;
Qu, 2000;
Hanafusa et al., 2002). Third,
cell culture studies of Spry1 and Spry2 demonstrate that they are
phosphorylated on a conserved tyrosine in response to RTK signaling, and the
modification is required for Spry function
(Hanafusa et al., 2002;
Hall et al., 2003;
Rubin et al., 2003;
Li et al., 2004;
Mason et al., 2004).
Here, we investigate the relationship between Csw/SHP-2 and Sprouty in RTK
pathways in vivo and in vitro. We establish a close functional relationship by
manipulating gene activity during RTK signaling in Drosophila
development and in cultured mammalian cells. We use the cell culture system to
demonstrate that SHP-2 controls phosphorylation on the essential tyrosine of
Spry1. Biochemical experiments show that SHP-2 associates in a complex with
Spry1 and can dephosphorylate the essential tyrosine. Substrate-trapping forms
of Csw bind Spry in cultured Drosophila cells and during development.
The results demonstrate that Spry proteins are targets of Csw/SHP-2 tyrosine
phosphatases in Drosophila and vertebrate RTK pathways, and explain
how Csw/SHP-2 can enhance RTK signaling by inactivating a feedback inhibitor.
While this manuscript was in preparation, complementary work showing that
SHP-2 can reduce tyrosine phosphorylation on a Spry protein in PC12 cells was
reported (Hanafusa et al.,
2004).
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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5 null allele was used
(Hacohen et al., 1998).
csw transgenes under sevenless enhancer control were used
for Csw misexpression during eye development: SE-cswG547E,
SE-cswC583S, SE-cswsrc90,
SE-cswG547Esrc90 and SE-cswC583Ssrc90
(Allard et al., 1996;
Allard et al., 1998). We refer
to cswsrc90 as myr-csw. Protein misexpression
with GAL4/UAS system (Brand and Perrimon,
1993) used sev-GAL4
(Brand and Perrimon, 1993),
btl-GAL4 (Shiga et al.,
1996), UAS-GFP (Jarecki et
al., 1999), UAS-cswsrc90
(Johnson Hamlet and Perkins,
2001) and UAS-spry
(Hacohen et al., 1998).
UAS-spryY201F was made by introducing a TAT>TTT
mutation in pUAST-spry (Hacohen
et al., 1998) and establishing transgenic lines by P element
transformation; homozygous viable insertions on chromosome II were used
(insertions 13, 15, 37). Crosses were carried out at 25°C.
Tracheae were visualized by fluorescence microscopy of heat-killed
(65°C for 5 seconds) third instar larvae carrying FRT2A and viable
insertions of btl-GAL4 and UAS-GFP on chromosome II.
Sections (2 µm) through adult eyes were prepared as described
(Tomlinson and Ready,
1987).
Cell culture and transfections
HEK293 cells (ATCC #CRL-1573) were grown in a humidified chamber containing
5% CO2 at 37°C in Dulbecco modified Eagle medium supplemented
with 10% fetal bovine serum (Invitrogen). For transfections,
2x105 log phase cells were cultured in 35 mm plates and
transfected with 0.5 µg of each expression plasmid using Fugene 6 lipid
transfection reagent (Invitrogen). Total DNA per transfection was 1 or 2 µg
depending on number of plasmids used; empty vector was used to keep total DNA
constant.
Drosophila S2 cells were maintained at 22°C in M3 insect
medium (Sigma) or Schneider's medium (Invitrogen) supplemented with 10%
heat-inactivated fetal calf serum. Stably transfected S2 cell lines expressing
Csw, CswC583S and Breathless with C-terminal FLAG epitope
(Herbst et al., 1996;
Toering, 2003) were maintained
in medium containing 200 µg/ml hygromycin.
Expression plasmids
Expression plasmids for HEK293 cells were: pRc/CMV/SHP-2 and
pRc/CMV/SHP-2C459S (Paul Khavari, Stanford) with CMV promoter
driving expression; pEFBOS/mFGFR3 and pEFBOS/mFGFR3K644E
(thanatophoric dysplasia type II mutation)
(Su et al., 1997) with human
EF-1
promoter; pMO/IRES/FGFR1c (David Ornitz, Washington University)
with MoLTR; and pBJ5/ERK2-HA (Gerald Crabtree, Stanford) with SR
promoter. pTA/HA-Spry1, with CMV promoter driving expression of mouse Spry1
with N-terminal HA epitope (YPYDVPDYA), was constructed by PCR amplification
of Spry1 cDNA (Minowada et al.,
1999) using a forward primer encoding initiator methionine, HA
epitope and residues 2-6 of Spry1, and insertion into pTA vector (Invitrogen).
Spry1Y53F (TAC>TTC) and Spry1Y89F (TAC>TTC)
mutations were introduced into pTA/HA-Spry1 by site-directed mutagenesis.
pCDNA/mFL-Spry1 was constructed as above except FLAG epitope (DYKDDDDK) and
pCDNA vector (Invitrogen) were used. pCDNA/SHP-2-V5 was constructed in similar
manner by amplifying SHP-2 sequence in pRc/CMV/SHP-2 with reverse primer
encoding V5 epitope (GKPIPNPLLGLDST). Coding sequences of constructs were
verified by DNA sequencing.
Antisera production
Rabbit antisera were raised against an N-terminal peptide of
Drosophila Sprouty (residues 19-37, LPRVHRPRAPEPTLSGVDH) and against
a C-terminal peptide (574-591, RKGDLTPEKRLLDSSPDY) (Biosynthesis, Lewisville,
TX). Sera were affinity purified on columns containing the immobilized peptide
and used at 1:1000 dilution (immunoprecipitation) or 1:2000-1:5000
(immunoblots). Rabbit anti-pY53 mouse Spry1 antiserum was raised against
phosphopeptide GCGSNEpYTEGPSVARRPAPR that includes Spry1 residues 49-66. Eight
week post-immunization bleed was used at 1:2000.
Erk2 assay
Twenty-four hours after transfection of HEK293 cells with pBJ5/ERK2-HA,
human recombinant bFGF (Invitrogen) was added to the medium to 25 ng/ml. At
times indicated after bFGF addition, cells were rinsed with PBS and lysed with
0.4 ml RIPA buffer (50 mM Tris pH 7.4, 1% NP-40, 0.25% Na-deoxycholate, 150 mM
NaCl, 1 mM EDTA) containing protease inhibitors (Complete-Mini, Roche) and 0.2
mM sodium vanadate to inhibit tyrosine phosphatases. HA-ERK2 was
immunoprecipitated by incubating lysate at 4°C with 10 µg anti-HA mAb
(Roche). After 3 hours, 35 µl protein A agarose (Sigma) was added and
incubation continued for 1 hour. Immunoprecipitate was washed three times at
4°C with 1 ml NP40 wash buffer (1% NP40, 1 mM EDTA) and once with 1 ml TNE
(10 mM Tris pH 8, 100 mM NaCl, 1 mM EDTA), boiled in Laemmli loading buffer
and resolved on 10% SDS-PAGE gel. Gel was probed with anti-dpERK mAb (Sigma),
HRP-conjugated secondary antibody and HRP chemistry (Enhanced
Chemiluminescence, Amersham). dpERK levels were quantitated by scanning
densitometry of fluorograms. Blots were reprobed with anti-HA to detect total
ERK2-HA. Expression of other proteins was monitored by SDS-PAGE and
immunoblotting of aliquots of cell lysates.
Spry phosphorylation analysis
HEK293 cells were transfected with pTA/HA-Spry1, pMO/IRES/FGFR1c and the
plasmids noted. Twenty-four hours later, bFGF was added to medium at 200
ng/ml. After 30 minutes, cells were lysed and HA-Spry1 was immunoprecipitated
by incubation with 35 µl anti-HA agarose (Santa Cruz Biotechnology) at
4°C for 4 hours. Immunoprecipitates were washed and separated by SDS-PAGE.
Immunoblots were probed with anti-phosphotyrosine mAb (Upstate Biotechnology)
and HRP immunochemistry. Same conditions were used for analysis of Spry1 Y53
phosphorylation, except cells were treated with bFGF for times noted, HA-Spry1
was immunoprecipitated with anti-HA and immunoblots were probed with
anti-pY53.
To identify Spry1 phosphotyrosines, 2x106 HEK293 cells were transfected with pTA/HA-Spry1 and pMO/IRES/FGFR1c. Forty-eight hours later, bFGF was added to 200 ng/ml. After 30 minutes, cells were lysed, HA-Spry1 was immunoprecipitated with anti-HA, resolved by SDS-PAGE, and stained with Coomassie Blue. HA-Spry1 band was excised, treated with trypsin and proteolytic fragments were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and identified using Mascot MS/MS Ions Search (Mass Spectrometry Lab, Stanford, CA).
To analyze tyrosine phosphorylation of Drosophila Spry, 5x106 exponentially growing S2 cells expressing Breathless-FLAG were treated for 15 minutes with 0.1 mM pervanadate, then washed and lysed. Endogenous Spry was immunoprecipitated with anti-Spry C-terminal antiserum, resolved by SDS-PAGE and probed on immunoblots with anti-phosphotyrosine.
Coimmunoprecipitation assays
HEK293 cells were transfected with plasmids expressing SHP-2-V5, HA-Spry1
and FGFR3 or FGFR3K644E to activate the FGF pathway. After 48
hours, cells were lysed with 0.4 ml NP40 lysis buffer (150 mM NaCl, 1% NP40,
50 mM Tris pH 8) containing protease inhibitors. SHP-2-V5 was
immunoprecipitated with anti-V5 mAb (Invitrogen), washed and resolved by
SDS-PAGE. HA-Spry1 in immunoprecipitate was detected on immunoblots probed
with rabbit anti-HA (Santa Cruz Biotechnology).
To assay Csw-Spry association in stably transfected S2 cells expressing Csw
or CswC583S, 
2x107 exponentially growing
cells in M3 medium were harvested by centrifugation and lysed in 1 ml NP-40
lysis buffer containing protease inhibitors. Csw was immunoprecipitated at
4°C for 2-4 hours with 1 µl anti-Csw-CT
(Allard et al., 1996) and 50%
(v/v) protein G Sepharose beads (Sigma). Beads were washed three times with
lysis buffer, boiled in Laemmli loading buffer and proteins resolved on 8%
SDS-PAGE gels. Endogenous Spry that co-immunoprecipitated was detected on
immunoblots probed with anti-Spry C-terminal. Spry immunoprecipitation was
carried out as above using anti-Spry and protein A beads;
co-immunoprecipitated Csw was detected with anti-Csw-CT.
For Drosophila imaginal discs, 100 pairs of third instar eye-antennal discs of each genotype were dissected into ice-cold PBS (Fig. 5E) or snap frozen and stored at –80°C, homogenized as above and cleared by centrifugation. Csw immunoprecipitation and subsequent analysis were as above.
GST pulldown assay
GST-SHP-2 fusion proteins were expressed in E. coli and purified
as described (O'Reilly et al.,
2000). To prepare lysates containing Spry1, HEK293 cells were
transfected with pTA/HA-Spry1 and pEFBOS/FGFR3K644E to induce
HA-Spry1 phosphorylation. Cells were lysed in NP40 lysis buffer with protease
inhibitors. Lysate was mixed with 100 µl of 50% (v/v) glutathione-Sepharose
beads coated with a GST-fusion protein and incubated at 4°C. After three
hours, beads were washed three times with NP40 wash and once with TNE.
Proteins were separated by SDS-PAGE, and immunoblots were probed with
anti-HA.
SHP-2 phosphatase assay
Phosphatase assay (O'Reilly et al.,
2000) used purified GST-SHP-2 proteins. To prepare phosphorylated
HA-Spry1 substrate, transfected HEK293 cells expressing FGFR1c and HA-Spry1
were treated with bFGF at 200 ng/ml and 0.1 mM pervanadate for 30 minutes.
Cells were lysed in NP40 lysis buffer and HA-Spry1 was immunoprecipitated with
anti-HA. Immunoprecipitates were washed with NP40 wash buffer and TNE, and
resuspended in phosphatase buffer (25 mM HEPES 7.4, 150 mM NaCl, 5 mM EDTA, 10
mM DTT). Phosphatase reactions (25 µl) containing phosphatase buffer, 50 ng
GST-SHP-2 fusion protein, and substrate immunoprecipitated from
2x105 cells, were incubated at 37°C. After 30 minutes,
Laemmli sample buffer was added, and products were resolved by SDS-PAGE.
Immunoblots were probed with anti-phosphotyrosine, anti-pY53 or anti-HA.
ERK2-HA substrate was prepared from pBJ5/ERK2-HA-transfected cells and
analyzed on immunoblots with anti-dpERK.
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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).
csw– mutations also cause tracheal defects among
other embryonic defects (Perkins et al.,
1996; Firth et al.,
2000), and we found that cswG547E, a
dominant-negative allele (Allard et al.,
1996) (Fig. 1A),
enhanced the penetrance of ganglionic branch outgrowth defects in bnl
heterozygous embryos (data not shown), as does partial inactivation of other
Bnl pathway genes (Sutherland et al.,
1996). Furthermore, expression of myr-Csw, an activated
membrane-localized form of Csw containing the c-Src myristylation site
(Allard et al., 1996), in the
developing tracheal system using a btl-GAL4 driver induced
ectopic terminal cells and branches, just like spry null mutations
(Fig. 1B-E). These results
imply that Csw and Spry have opposite effects on the Bnl RTK pathway.
In the developing eye, both csw and spry regulate EGFR
and/or Sevenless RTK pathways that specify the pattern and fates of
photoreceptors and neighboring cells
(Allard et al., 1996;
Allard et al., 1998;
Casci et al., 1999;
Kramer et al., 1999). Indeed,
chromosomal deficiencies encompassing spry were identified in a
screen for suppressors of the eye and wing vein phenotypes caused by a
csw weak loss-of-function allele
(Firth et al., 2000),
indicating that these genes have opposing roles during RTK signaling in the
developing eye and wing. Consistent with this, removing one copy of
spry enhanced the phenotype of extra R7 photoreceptors caused by
eye-specific expression of myr-Csw (Allard
et al., 1996), increasing the number of R7 cells per ommatidium
from 2.1±0.06 (mean ± s.e.m.) to 3.0±0.07
(Fig. 1F). Likewise, removing
one copy of spry suppressed the loss of photoreceptor phenotype
caused by eye-specific expression of dominant negative CswG547E
(Allard et al., 1998),
increasing the number of outer photoreceptors per ommatidium from
3.3±0.05 to 4.1±0.03 (Fig.
1G). Reducing spry dose did not suppress the effect of
another dominant-negative Csw, CswC583S
(Fig. 1H; 5.1±0.03 for
spry+ versus 4.8±0.03 for
spry+/spry5).
CswC583S is a substrate-trapping form of the enzyme that, unlike
CswG547E, can bind its substrates, although it does not hydrolyze
or release them (Herbst et al.,
1996). We consider the implications of this result in the
Discussion.
To compare effects of Spry and Csw/SHP-2 on RTK signal transduction, we assessed their effects on MAPK activation induced by FGF signaling in human embryonic kidney 293 (HEK293) cells, using an antiserum specific for diphosphorylated (activated) MAPK (dpERK). Transient transfection of a Spry1 expression plasmid reduced activation of MAPK in response to basic FGF (bFGF), as did transfection of a plasmid expressing dominant negative SHP-2C459S (Fig. 1I). When both Spry1 and SHP-2C459S were expressed, MAPK activation was almost completely abolished. Spry1 and SHP-2 both predominantly affected the duration of MAPK activation in response to bFGF, rather than the magnitude of the response, consistent with the idea that they regulate the same step in signal transduction (Fig. 1J). Thus, in all three systems investigated, Spry and Csw/SHP-2 proteins regulated the same RTK signaling processes but in opposite directions.
SHP-2 negatively regulates tyrosine phosphorylation on Spry1
To determine the regulatory relationship between Csw/SHP-2 and Spry during
RTK signaling, the effect of SHP-2 on tyrosine phosphorylation of Spry1 was
analyzed in HEK293 cells. FGF signaling induces tyrosine phosphorylation of
Spry proteins in several cell lines including HEK293 cells
(Hanafusa et al., 2002;
Tefft et al., 2002)
(Fig. 2B, lanes 1,4). If Spry1
is a substrate of SHP-2 (Fig.
2A, arrow 2), then inhibiting SHP-2 should increase tyrosine
phosphorylation on Spry1, even though it reduces signaling overall. However,
if SHP-2 functions upstream of Spry1 phosphorylation (arrow 1), then
inhibiting SHP-2 should decrease Spry1 phosphorylation. If SHP-2 acts
downstream or parallel to Spry1 (arrow 3), no change in Spry1 phosphorylation
is expected.
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P (data not shown). Thus, SHP-2 negatively
regulates tyrosine phosphorylation on Spry1.
Overexpression of wild-type SHP-2 reduced Spry1 tyrosine phosphorylation,
consistent with this model (Fig.
2B, lanes 3,6). However, the effect was small and variable,
presumably because SHP-2 levels are not limiting and its phosphatase activity
is controlled by binding via its SH2 domain to scaffolding proteins as in
other contexts (Barford and Neel,
1998).
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) and for Spry2 and Spry4 in HEK293 cells
(Sasaki et al., 2001). Y53 is
also crucial for Spry1 activity in HEK293 cells, because changing it to
phenylalanine (Spry1Y53F) abolished its ability to block bFGF
activation of MAPK (data not shown). To determine whether phosphorylation on
this critical tyrosine residue is regulated by SHP-2, a phospho-specific
antiserum was generated that recognizes the pY53 form of Spry1
(Fig. 2C). Expression of
dominant-negative SHP-2C459S increased phosphorylation on Y53, both
in the absence and presence of added FGF
(Fig. 2D). Thus, SHP-2
regulates phosphorylation of this crucial Spry1 tyrosine. To determine whether SHP-2 regulates phosphorylation of other Spry1 tyrosines, the effects of dominant negative SHP-2 on Spry1Y53F were examined using a general anti-phosphotyrosine antibody. The Y53F mutation did not completely eliminate tyrosine phosphorylation of Spry1, and the residual phosphorylation increased in the presence of dominant negative SHP-2 (Fig. 2E, lanes 1-4). Thus, there is at least one other Spry1 phosphotyrosine regulated by SHP-2.
To identify additional Spry1 phosphotyrosines, HA-Spry1 was purified from FGF-stimulated HEK293 cells and analyzed by tandem mass spectrometry. This confirmed phosphorylation of Y53 and identified Y89 as a second phosphorylation site. Mutation of Y89 to phenylalanine (Spry1Y89F) reduced Spry1 tyrosine phosphorylation in response to FGF (Fig. 2E, lanes 1,5), implying that Y89 is also a major phosphorylation site. However, Spry1Y89F inhibited MAPK activation by FGF signaling in the HEK293 cell assay, demonstrating that phosphorylation of Y89 is not essential for this activity (data not shown). Although dominant-negative SHP-2 increased tyrosine phosphorylation on both Spry1Y53F and Spry1Y89F (Fig. 2E, lanes 3-6), there was little tyrosine phosphorylation and only a small effect of dominant-negative SHP-2 on the Spry1Y53F/Y89F double mutant (Fig. 2E, lanes 7,8). Thus, tyrosines 53 and 89 are the major tyrosine phosphorylation sites on Spry1 and both are regulated by SHP-2.
SHP-2 and Spry1 associate in a complex
If Spry1 is a SHP-2 substrate, the proteins must associate at least
transiently in vivo. To test this, HA-Spry1 and SHP-2 with a V5 epitope
(SHP-2-V5) were co-expressed in HEK293 cells. SHP-2-V5 was immunoprecipitated
from cell extracts, and HA-Spry1 that co-immunoprecipitated was detected on
immunoblots. Little HA-Spry1 was detected in immunoprecipitates from
unstimulated cells (Fig. 3A,
lane 1), comparable with that observed in controls lacking SHP-2-V5 (lanes
7-9). However, when FGF signaling was activated by overexpression of FGFR3, an
association between the two proteins was detected, and more was observed when
constitutively active FGFR3K644E
(Su et al., 1997) was
expressed (lanes 2,3). Similar results were obtained when the antibody
treatments were reversed (data not shown). Thus, FGF signaling induces
formation of a complex containing SHP-2 and Spry1. Complex formation does not
require the major Spry1 tyrosine phosphorylation sites, because it was not
affected by Spry1Y53F or Spry1Y53F/Y89F mutations
(Fig. 3A, lanes 4-6 and data
not shown).
|
). When
purified SHP-2E76A-GST attached to beads was incubated with
extracts of HEK293 cells expressing HA-Spry1, HA-Spry1 bound to the beads,
whereas little bound to control beads coated with GST
(Fig. 3B). HA-Spry1 also bound
to beads coated with a GST-fusion containing only the SH2 domains of SHP-2
(SHP-2SH2), but with reduced efficiency. Thus, SHP-2 and Spry1
associate in a complex, and the interaction is mediated at least in part by
SHP-2 SH2 domains.
SHP-2 dephosphorylates Spry1
To test whether SHP-2 can dephosphorylate Spry1, purified
SHP-2E76A-GST was incubated with HA-Spry1 protein isolated from
FGF-stimulated HEK293 cells. The SHP-2 E76A mutation prevents auto-inhibition
of the phosphatase domain, alleviating the need for factors that might be
necessary in vivo to relieve auto-inhibition
(O'Reilly et al., 2000).
SHP-2E76A-GST eliminated tyrosine phosphorylation on HA-Spry1,
including the crucial phosphotyrosine (Y53), whereas control proteins lacking
the phosphatase domain (SHP-2SH2, SHP-2P) and GST
alone had no effect (Fig.
4A,B). Under identical conditions, SHP-2E76A-GST did
not dephosphorylate ERK2 phosphotyrosine
(Fig. 4A, lower panel). Thus,
Spry1 is a substrate of SHP-2 in vitro.
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|
To determine if Drosophila Spry is tyrosine phosphorylated,
endogenous Spry in Breathless-expressing S2 cells was immunoprecipitated and
probed with anti-phosphotyrosine antiserum
(Fig. 5A). Spry isoforms of 70
kDa and 42 kDa are present in S2 cells
(Toering, 2003). Tyrosine
phosphorylation of the 70 kDa species was detected at low levels in untreated
cells, and phosphorylation of both forms was apparent when cells were treated
with the phosphatase inhibitor pervanadate, implying that Spry phosphorylation
is regulated by an endogenous phosphatase.
The tyrosine crucial for vertebrate Spry function is conserved in
Drosophila Spry (Hanafusa et al.,
2002). To determine if the conserved tyrosine (Y201) is important
for function, transgenes encoding wild-type Spry (UAS-spry) or mutant
Spry with the tyrosine substituted with phenylalanine
(UAS-spryY201F) were expressed using sev-GAL4
driver, and their effects on eye development examined. Spry expression caused
misrotation and disorganization of ommatidia, missing photoreceptors in 5% of
ommatidia, and external roughening of the eye
(Fig. 5B and data not shown).
SpryY201F expression had little or no effect. In two out of three
UAS-spryY201F insertions analyzed, ommatidia had normal
organization and no missing photoreceptors or eye roughening
(Fig. 5C). The same was true of
the third insertion, except it caused a low frequency of photoreceptor loss
(0.4% of ommatidia). Thus, the conserved tyrosine is crucial for biological
activity of Drosophila Spry.
The above results and the genetic interactions between csw and
spry suggest that Csw might regulate Spry directly by
dephosphorylation. In vivo substrates of Csw/SHP-2 can be identified with
substrate-trapping forms of the enzymes, such as CswC583S, which
bind but do not dephosphorylate or release their substrates
(Herbst et al., 1996;
Agazie and Hayman, 2003a). To
determine if CswC583S can trap endogenous Spry in S2 cells,
wild-type Csw and CswC583S were expressed in S2 cells and the
amount of Spry that bound to each was determined by co-immunoprecipitation
(Fig. 5D). A small amount of
both Spry isoforms bound wild-type Csw (lane 3). Substantially more of each
isoform bound CswC583S (lane 4). This implies that Spry is a direct
target of Csw in S2 cells.
To test for a substrate-trapping interaction between CswC583S
and Spry during development, binding was analyzed in a similar manner in
extracts of eye-antennal imaginal discs dissected from transgenic larvae
expressing modified Csw proteins (myr-Csw, myr-CswC583S or
myr-CswG547E). Myristylated forms of Csw were used to facilitate
membrane localization of the enzyme and detection of interaction with
substrates (Allard et al.,
1996). There was low but detectable interaction between myr-Csw
and endogenous Spry (Fig. 5E,
lane 4). Stronger interaction was observed with myr-CswC583S (lane
5). The enhanced interaction was not simply due to stronger interaction
between the SH2 domains of the mutant Csw and a scaffolding protein such as
Dos that cannot be dephosphorylated by it. If so, other catalytically inactive
forms of Csw, such as myr-CswG547E, should show the same enhanced
interaction, which was not observed (lane 6). We conclude that the enhanced
interaction between Spry and myr-CswC583S is a substrate-trapping
effect, providing strong evidence that Spry is a substrate of Csw during eye
development.
|
DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Four lines of evidence support the conclusion that Csw/SHP-2 inactivate Spry proteins by direct binding and dephosphorylation. First, genetic experiments in developing Drosophila eye and trachea and HEK293 cells demonstrated that Csw/SHP-2 and Spry act in the same RTK signaling events but in opposite directions. Indeed, manipulating their activity in opposite directions caused similar Drosophila phenotypes and similar effects on MAPK activation in HEK293 cells, and reducing spry dose suppressed the csw loss-of-function phenotype in the eye and enhanced the gain-of-function phenotype, supporting the idea that they regulate the same step in signaling. Second, molecular epistasis experiments in HEK293 cells demonstrated that SHP-2 functions upstream of, and negatively regulates, phosphorylation of the critical tyrosine residue (Y53) of Spry1. Third, biochemical studies of extracts of HEK293 cells, Drosophila S2 cells, and eye discs demonstrated that Csw/SHP-2 proteins associate in complexes with Spry proteins. Interaction was enhanced in S2 cells and eye discs when a substrate-trapping Csw was used. Interaction involves more than just binding of Csw/SHP-2 to the crucial tyrosine, because complex formation was observed with SHP-2 mutants lacking the phosphatase domain and with a Spry mutant lacking the tyrosine. Finally, purified SHP-2 selectively dephosphorylated Spry1 in vitro. These data support the conclusion that Spry proteins are direct targets of Csw/SHP-2 in all three systems examined.
One genetic result did not readily fit with the model that Csw functions by
inactivating Spry by dephosphorylation. Whereas reduction of spry
dose suppressed the eye phenotype of a hypomorphic csw allele
(Firth et al., 2000) and
dominant-negative CswG547E, consistent with the model, it
did not suppress the milder phenotype of dominant-negative
CswC583S (Fig.
1G,H). This catalytically inactive, substrate trapping form of Csw
has unusual properties: it behaves in a dominant-negative fashion, interfering
with wild-type Csw function, but also retains some wild-type Csw function
because it partially rescues other dominant-negative and hypomorphic
csw alleles (Allard et al.,
1998). This residual activity of CswC583S is proposed
to result from its ability to partially mimic the effect of dephosphorylating
a substrate by binding to it tightly
(Allard et al., 1998). Spry
binds CswC583S and could be such a substrate
(Fig. 5D,E). If so, this could
explain the lack of suppression of CswC583S phenotype by reduction
in spry dose: decreasing spry levels would not reduce
spry function under conditions in which it is already trapped in an
inactive or partially inactive form by CswC583S.
|
Why does a signaling pathway induce both a feedback inhibitor and a protein
that inactivates it? One possibility is that this double-negative circuit
provides a mechanism for rapidly resetting the signaling system: the inhibitor
terminates signaling and the deactivator restores the inhibitor to its
original (inactive) state, readying the cell for another round of signaling.
This may be important when cells experience successive waves of signaling,
such as the waves of EGFR and Sevenless signaling in eye development
(Freeman, 1996).
Another possibility is that the double-negative circuit allows precise
control of the signal output profile (Fig.
6B). In the absence of feedback, the response to a signal is
simple and sustained, increasing monotonically until reaching saturation
(black curve). If a basic negative-feedback system is operative, the magnitude
and duration of the response are limited, generating a parabolic response
profile (red curve). However, if the pathway contains both a feedback
inhibitor (Spry) and an inducible component (Csw/SHP-2) that deactivates it,
this creates more complex output profiles, such as the irregularly shaped
curve observed for MAPK activation following FGFR activation in HEK293 cells
(Fig. 1J; Fig. 6B, green curve). By
altering activity of individual feedback components, other complex profiles
can be generated (Fig. 1J). If
cells can distinguish different profiles, as some cells distinguish different
calcium oscillations (Lewis,
2003), this could lead to different outcomes. The shape of the RTK
response profile could be as important to outcome as the magnitude and
duration of the response. In a similar way, differential induction of
individual components of a double-negative feedback circuit can transform
simple signaling gradients into complex spatial patterns of signal output.
The Csw/SHP-2–Sprouty circuit does not operate in all RTK signaling processes
Although our results imply that the Csw/SHP-2-Spry circuit operates in a
variety of RTK signaling processes, it is unlikely to operate in all such
events. Csw and SHP-2 are widely expressed and required in many and perhaps
all RTK signaling processes, whereas Spry genes are expressed in more limited
domains during development and appear to function in only a subset of such
processes. For example, expression of spry genes in
Drosophila and mouse embryos is largely confined to FGF signaling
centers (Hacohen et al., 1998;
Minowada et al., 1999).
In RTK signaling processes where Spry proteins are not expressed, Csw/SHP-2
must stimulate signaling by dephosphorylating other substrates, such as the
autophosphorylation site on EGFR and Torso/PDGFRs that recruits RasGAP
(Cleghon et al., 1998;
Ekman et al., 2002;
Agazie and Hayman, 2003b) or
sites on Src kinase regulators (Ren et
al., 2004; Zhang et al.,
2004). In some pathways, such as the Torso pathway, more than one
Csw/SHP-2 substrate is likely to be present and regulated by the enzyme
(Cleghon et al., 1998;
Casci et al., 1999). The
specific substrates present in each cell should alter signaling kinetics in
different ways, creating a rich diversity of output profiles. It will be
interesting to determine the extent and importance of this diversity, and
whether alterations in signal output profile and Spry feedback inhibition
contribute to pathogenesis of human diseases associated with misregulation of
SHP-2 activity (Tartaglia et al.,
2001; Musante et al.,
2003).
|
ACKNOWLEDGMENTS |
|---|
|
Footnotes |
|---|
Present address: Department of Radiation Oncology, Stanford University
School of Medicine, Stanford, CA 94305-5307, USA
Present address: Biology Department, Wartburg College, Waverly, IA 50677,
USA
Present address: Department of Molecular and Cell Biology, University of
California, Berkeley, CA 94720, USA
|
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