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-2^C459S 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-2^C459S 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-Spry1^Y53F 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-2^C459S on Spry1 Y53 phosphorylation. HEK293 cells were transfected with plasmids expressing FGFR1c and HA-Spry1, and empty vector or vector expressing dominant-negative SHP-2^C459S 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-2^C459S 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-2^C459S on other Spry1 phosphotyrosines. HEK293 cells were transfected with plasmids expressing FGFR1c and either empty vector or vector expressing dominant-negative SHP-2^C459S as indicated, and plasmids expressing HA-Spry1 (WT, lanes 1,2), HA-Spry1^Y53F (lanes 3, 4), HA-Spry1^Y89F (lanes 5, 6), or HA-Spry1^{Y53F, Y89F} (lanes 7, 8). FGF treatment and subsequent analysis with anti-phosphotyrosine antiserum was as in lanes 4 and 5 (of B). SHP-2^C459S 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.