Activation of Erk by binding of SPP to EDG-6. (A) CHO-K1 cells were transiently cotransfected with an HA-tagged Erk2 and EDG-6 or an empty vector. Cells were treated without or with 200 ng/mL PTX for 3 hours, then stimulated with vehicle or 100 nmol/L SPP for 5 minutes. HA-Erk2 was immunoprecipitated from whole cell lysates and assayed for kinase activity using MBP as a substrate as described in “Materials and methods.” Results are typical of 2 independent experiments. (B) ³²P incorporation into MBP was determined by scintillation counting. Data are expressed as picomole per minute per milligram and are the means of 2 separate experiments. White bars indicate the absence of PTX; solid bars, the presence of PTX. (C) CHO-K1 cells stably transfected with EDG-6 were treated without or with PTX for 3 hours, then stimulated with vehicle or 100 nmol/L SPP for 5 minutes, and Erk activation was determined by Western blot analysis with phospho-specific anti-Erk antibody. Blots were then stripped and reprobed with Erk antibody to determine total Erk levels. (D) Binding of SPP to EDG-6 stimulates Elk-1. CHO-K1 cells were cotransfected with the reporter plasmid pFR-Luc and the pcDNA vector alone or with either EDG-6 or EDG-1 inserts. Cells were treated without (white bars) or with 1 μmol/L SPP (solid and hatched bars) in the absence (solid bars) or presence of PTX (hatched bars) for 5 to 6 hours, and luciferase activity was measured as described in “Materials and methods.” Data are means ± SEM.