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First published online 15 February 2006
doi: 10.1242/dev.02255

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Sprouty proteins are in vivo targets of Corkscrew/SHP-2 tyrosine phosphatases

Lesley A. Jarvis^1,*,, Stephanie J. Toering^1,*,, Michael A. Simon², Mark A. Krasnow^1,¶ and Rachel K. Smith-Bolton^2,*,

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.

View larger version (66K): [in a new window]   Fig. 1. Spry and Csw/SHP-2 have opposing roles during RTK signaling. (A) Csw, SHP-2 and Spry structures. Open bars, wild-type proteins. SH2 and phosphatase domains of Csw and SHP-2, and the T/SNEY amino acid motif and cysteine-rich domain of Spry proteins are indicated. White gap in Csw phosphatase domain indicates a non-conserved insertion. Mutant forms are indicated below bars: activating mutations (green), inactivating and dominant-negative mutations (red), and neutral mutations (black). myr, N-terminal 90 residues of Src64 including myristylation site. PO3–, phosphotyrosine. (B-E) Effects of spry loss of function and csw gain of function on tracheal development. (B) Fluorescence micrograph of ends of two dorsal branches (DB) from a control spry+ third instar w; btl-GAL4, UAS-GFP larva expressing GFP throughout tracheal system. Dorsal view, anterior upwards. Terminal cells (arrowheads) extend branches anteriorly and laterally. (C) Same view of third instar w; btl-GAL4, UAS-GFP; spry5 larva showing extra terminal cells. (D) Same view of third instar y,w; UAS-myr-csw/btl-GAL4, UAS-GFP larva that expresses myristylated (activated) Csw throughout developing tracheal system. Extra terminal cells are present as in C. (E) Number of DB terminal cells per segment in genotypes shown in B-D. Mean values (±s.e.m.): spry+ (2.2±0.04, n=123 segments), spry5 (3.8±0.1, n=133), btl>myr-csw (3.3±0.06, n=210). (F-H) Effect of spry dose on csw gain- and loss-of-function phenotypes in eye development. (F) Number of R7 cells per ommatidium in SE-myr-csw/+ flies expressing myr-csw in developing eyes (black bars, n=293 ommatidia), and in SE-myr-csw/+; spry5/+ flies (white bars, n=324). Wild type has one R7 cell per ommatidium. myr-csw effect increased when spry dose was reduced. (G) Number of outer photoreceptors per ommatidium in SE-cswG547E/+ flies expressing dominant-negative CswG547E in developing eyes (black bars, n=292) and in SE-cswG547E/+; spry5/+ flies (white bars, n=329). Wild type has six outer photoreceptors per ommatidium. The CswG547E effect was suppressed when spry dose was reduced. (H) Number of outer photoreceptors per ommatidium in SE-cswC583S/+ flies expressing dominant-negative, substrate-trapping CswC583S protein in developing eyes (filled bars, n=582), and in SE-cswC583S/spry5 flies (open bars, n=604). CswC583S effect was not suppressed when spry dose was reduced. (I,J) Effect of SHP-2 and Spry1 on FGF-induced phosphorylation of ERK2 in HEK293 cells. (I) HEK293 cells were transfected with plasmid expressing ERK2 with HA epitope (ERK2-HA) and empty vector or vector expressing Spry1 with FLAG epitope (FL-Spry1), dominant-negative SHP-2C459S or wild-type SHP-2 as indicated. After transfection, bFGF was added for 30 minutes to activate FGF pathway. (Top panels) ERK2-HA immunoprecipitated (IP) from cell lysates with anti-HA antiserum and analyzed on immunoblots with anti-dpERK to show diphosphorylated (active) ERK2-HA or with anti-HA to show total ERK2-HA. (Bottom panels) Immunoblots of whole cell lysates (WCL) probed with anti-FLAG to detect FL-Spry1 or anti-SHP-2 to detect endogenous SHP-2 and SHP-2 from transfected plasmids. Similar results were obtained in three experiments. (J) Effect of Spry1 and dominant negative SHP-2C459S on kinetics of ERK2 activation by FGF. As in I, except ERK2 analysis was carried out at times indicated after FGF addition. Similar results were obtained in two experiments.

View larger version (66K): [in a new window]   Fig. 2. SHP-2 regulates Spry phosphorylation. (A) FGF signaling pathway with Spry feedback loop. FGF activates Spry feedback by inducing spry expression (broken line) and stimulating tyrosine phosphorylation of Spry to generate Spry-P. Circled numbers indicate steps by which SHP-2/Csw could promote to increase signal output. (B) Effect of SHP-2 on Spry1 tyrosine phosphorylation. HEK293 cells were transfected with expression constructs for HA-Spry1, FGFR1c and either empty vector or expression constructs for dominant-negative SHP-2C459S or wild-type SHP-2 as indicated. Transfected cells were left untreated (lanes 1-3) or treated with bFGF (lanes 4-6). (Top) Immunoprecipitated HA-Spry1 analyzed on immunoblot with anti-phosphotyrosine antiserum. SHP-2C459S increased HA-Spry1 phosphorylation (lane 5) and SHP-2 reduced it (lane 6). (Middle) Immunoblot reprobed with anti-HA to show total HA-Spry1. (Bottom) Immunoblot of whole cell lysates probed with anti-SHP-2. Similar results were obtained in two experiments. (C) Specificity of phospho-specific Spry1 antiserum. HEK293 cells were transfected with expression plasmids for FGFR1c and HA-Spry1 or HA-Spry1Y53F as indicated. Transfected cells were treated with bFGF for times indicated, and HA-Spry1 was immunoprecipitated and analyzed on immunoblots probed with -pY53 antiserum (top) or anti-HA to show total HA-Spry1 (bottom). (D) Effect of SHP-2C459S on Spry1 Y53 phosphorylation. HEK293 cells were transfected with plasmids expressing FGFR1c and HA-Spry1, and empty vector or vector expressing dominant-negative SHP-2C459S as indicated. Transfected cells were left untreated (lanes 1,2) or treated with bFGF for 60 minutes (lanes 3,4). (Top) Immunoblot of immunoprecipitated HA-Spry1 probed with -pY53. SHP-2C459S increased phosphorylation on pY53. (Middle) Control immunoblot probed with anti-HA. (Bottom) Immunoblot of whole cell lysates probed with anti-SHP-2. Similar results were obtained in two experiments. (E) Effect of SHP-2C459S on other Spry1 phosphotyrosines. HEK293 cells were transfected with plasmids expressing FGFR1c and either empty vector or vector expressing dominant-negative SHP-2C459S as indicated, and plasmids expressing HA-Spry1 (WT, lanes 1,2), HA-Spry1Y53F (lanes 3, 4), HA-Spry1Y89F (lanes 5, 6), or HA-Spry1Y53F, Y89F (lanes 7, 8). FGF treatment and subsequent analysis with anti-phosphotyrosine antiserum was as in lanes 4 and 5 (of B). SHP-2C459S influenced tyrosine phosphorylation on HA-Spry1 when Y53 was altered (lanes 3,4), indicating that SHP-2 also affects other tyrosine(s), notably Y89 (lanes 5-8). Similar results were obtained in three experiments.

View larger version (30K): [in a new window]   Fig. 3. SHP-2 forms a complex with Spry1. (A) Co-immunoprecipitation analysis. HEK293 cells were transfected with plasmids expressing HA-Spry1 (lanes 1-3, 7-9) or HA-Spry1Y53F (lanes 4-6), plasmid expressing SHP-2-V5 (lanes 1-6), and empty vector (lanes 1, 4, 7) or vector expressing FGFR3 (lanes 2, 5, 8) or constitutively-active FGFR3K644E (lanes 3, 6, 9) to increase FGF pathway activity. SHP-2-V5 was immunoprecipitated from cell lysates. (Top) Immunoblot of immunoprecipitates probed with anti-HA to detect co-immunoprecipitated HA-Spry1. (Middle) Blot reprobed with anti-V5 showing SHP-2-V5 in immunoprecipitates. (Bottom) Immunoblot of whole cell lysates probed with anti-HA to show HA-Spry1 expression. Similar results were obtained in three experiments. (B) GST pull-down analysis of SHP-2 interaction with Spry1. HEK293 cells were transfected with plasmids expressing HA-Spry1 and FGFR3K644E to increase HA-Spry1 phosphorylation. Cell lysates were incubated with beads coated with purified GST (lane 1), or GST fused to constitutively active SHP-2E76A (lane 2) or to truncated SHP-2 containing only the SH2 domains (lane 3). (Top) Immunoblot of proteins bound to beads, probed with anti-HA. HA-Spry1 bound to GST-SHP-2E76A (lane 2) and, less well, to GST-SHP-2SH2 (lane 3). Similar results were obtained in three experiments. (Bottom) Coomassie Blue-stained SDS-PAGE gel of purified GST fusion proteins. Positions of full-length proteins are indicated; lower molecular weight forms are presumably breakdown products.

View larger version (30K): [in a new window]   Fig. 4. SHP-2 dephosphorylates Spry1 in vitro. (A) (Top) Phosphorylated HA-Spry1 isolated from transfected HEK293 cells treated with bFGF was incubated alone (lane 2) or with purified GST (lane 3), GST-SHP-2E76A (lane 4), GST-SHP-2SH2 (lane 5) or GST-SHP-2P (lane 6). Lane 1, HA-Spry1 before bFGF addition. Upper blot, immunoblot of products probed with anti-phosphotyrosine to detect phosphorylated HA-Spry1. GST-SHP-2E76A dephosphorylated HA-Spry1 (lane 4). Lower blot, immunoblot probed with anti-HA to detect all HA-Spry1. Similar results were obtained in three experiments. (Bottom) Tyrosine phosphorylated ERK2-HA was prepared and tested as a SHP-2 substrate as above. Upper blot, immunoblot of ERK2-HA incubated alone (lane 8), with GST (lane 9) or with GST-SHP-2E76A (lane 10), probed with anti-dpERK to detect phosphorylated ERK2-HA. Tyrosine phosphorylation site detected by anti-dpERK (Y185) was not affected by GST-SHP-2E76A. Lower blot, same blot reprobed with anti-HA to detect all ERK2-HA. Lane 7, ERK2-HA before bFGF addition. ERK2 and Spry1 have another phosphorylation site near the phosphotyrosine: S50 for Spry1 (data not shown) and T183 for ERK2. Similar results were obtained in two experiments. (B) Experiment as in A (top), except upper blot was probed with anti-pY53.

View larger version (53K): [in a new window]   Fig. 5. Drosophila Spry is phosphorylated and binds a substrate-trapping form of Csw. (A) Tyrosine phosphorylation of Spry. S2 cells expressing Breathless-FLAG were mock-treated (lanes 1, 3) or treated with 0.1 mM pervanadate, a tyrosine phosphatase inhibitor (lanes 2, 4) and endogenous Spry was immunoprecipitated from cell extracts and analyzed on immunoblots. (Lanes 1, 2) Immunoblot probed with anti-phosphotyrosine to detect phosphorylated Spry. (Lanes 3, 4) Immunoblot probed with anti-Spry to detect all Spry (70 kDa isoform, Spry70; doublet of 42 kDa isoforms, Spry42). There is tyrosine phosphorylation of Spry70 and a Spry42 isoform (lane 2). Similar results were obtained in three experiments. (B,C) Requirement of Spry Y201. (B) Section through eye of w; sev-GAL4/UAS-spry fly that expresses Spry under control of sev-GAL4 in developing eye. Ommatidia are disorganized and some (arrowheads) are missing photoreceptors. (C) Similar section of w; sev-GAL4/UAS-spryY201F that expresses SpryY201F under control of sev-GAL4. Ommatidia appear normal. (D) Binding of Spry to substrate-trapping Csw in S2 cells. Whole cell lysates were prepared from transfected S2 cells expressing Csw (lanes 1, 3) or CswC583S, a substrate-trapping form of the enzyme (lanes 2, 4). Aliquots of lysates were directly resolved by SDS-PAGE (lanes 1, 2) or first immunoprecipitated with anti-Csw (lanes 3, 4). Immunoblots were probed with anti-Spry or anti-Csw as indicated. More Spry co-immunoprecipated with CswC583S (lane 4) than with Csw (lane 3). Similar results were obtained in three experiments. (E) Binding of Spry to substrate-trapping Csw in imaginal discs. Eye-antennal discs dissected from third instar transgenic larvae expressing myr-Csw, myr-CswC583S or myr-CswG547E, a dominant-negative Csw that does not function as a substrate trap, were homogenized and directly resolved by SDS-PAGE (lanes 1-3) or immunoprecipitated with anti-Csw and then resolved by SDS-PAGE (lanes 4-6). Immunoblots were probed with anti-Spry or anti-Csw as indicated. Positions of Spry42 and Csw are shown; Spry70 is variably detected in eye disc lysates. More Spry42 co-immunoprecipitated with myristylated CswC583S than with myristylated Csw or CswG547E. A similar result obtained in a repeat experiment, except in this case Spry70 isoform predominated in co-immunoprecipitate.

View larger version (14K): [in a new window]   Fig. 6. Csw/SHP-2-Spry feedback circuit shapes RTK signaling profile. (A) Ligand binding to RTK activates RAS/MAPK cascade, Spry negative-feedback loop (red), and Csw/SHP-2 tyrosine phosphatase (green). Csw/SHP-2 dephosphorylates Spry, inactivating it. This creates a double-negative regulatory circuit in which Csw/SHP-2 increases RTK output by inactivating a feedback inhibitor. (B) Conceptualized plots of RTK kinetics when no feedback (black), Spry feedback (red) or the complete Csw/SHP-2–Spry circuit (green) is operative.