QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

Help viewing high resolution images Return to article

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-csw^G547E/+ flies expressing dominant-negative Csw^G547E in developing eyes (black bars, n=292) and in SE-csw^G547E/+; spry⁵/+ flies (white bars, n=329). Wild type has six outer photoreceptors per ommatidium. The Csw^G547E effect was suppressed when spry dose was reduced. (H) Number of outer photoreceptors per ommatidium in SE-csw^C583S/+ flies expressing dominant-negative, substrate-trapping Csw^C583S protein in developing eyes (filled bars, n=582), and in SE-csw^C583S/spry⁵ flies (open bars, n=604). Csw^C583S 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-2^C459S 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-2^C459S 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.

Return to article