Fig. 1. Reggie-1 is tyrosine phosphorylated in growth-factor-stimulated cells. (A) HeLa and PC12 cells were incubated with pervanadate to inhibit dephosphorylation of tyrosines. Immunoprecipitation of phosphorylated proteins was performed with an anti-Tyr-P antibody (lane PY) or without antibody (lane C), and after stringent washing, endogenous reggie-1 was detected with a specific antibody. (B) HeLa cells were transfected with R1-EGFP or EGFP constructs and immunoprecipitated with anti-GFP. Tyrosine-phosphorylated proteins were detected by western blot with an anti-Tyr-P antibody. R1-EGFP was phosphorylated, whereas the controls (EGFP and SH-R1-EGFP) were not. Membrane association of reggie-1 is necessary for the phosphorylation, because the soluble mutant G2A-R1-EGFP did not carry Tyr-P modifications. Pervanadate was used as indicated. Lane C, nontransfected cells. (C) To demonstrate the physiological tyrosine phosphorylation of reggie-1, HeLa cells were stimulated with EGF in the absence of pervanadate. Stimulation of cells with EGF resulted in a time-dependent tyrosine phosphorylation of reggie-1. (D) EGF-stimulated tyrosine phosphorylation of R1-EGFP could be inhibited by tyrphostin AG1478, which inhibits EGFR phosphorylation (HeLa cells, experiment performed with pervanadate). Antibodies used for the detection by western blot are indicated.