FIG. 2.

All three RSK family members phosphorylate CREB Ser-133 in response to growth factor stimulation. (A) COS cells were transfected with expression constructs for either HA-RSK1, HA-RSK2, or HA-RSK3. Cells were either left untreated or treated with EGF for 10 min. HA-tagged RSKs were immunoprecipitated from lysates of untreated or EGF-treated transfected COS cells with a monoclonal anti-HA antibody (12CA5). Kinase activities were determined by an in vitro kinase assay with CREBtide as a substrate. Fold activation indicates the ratio of kinase activity from EGF-treated cells to that from untreated cells. Data are from three separate experiments. Error bars represent SEM. (B) COS cells were transfected with 1 μg of CMV-Gal4-CREB together with 10 μg of pMT vector (lanes 1 and 2), pMT2-HA-RSK1 (lanes 3 and 4), pMT2-HA-RSK2 (lanes 5 and 6), or pMT-HA-RSK3 (lanes 7 and 8). Transfected COS cells were left untreated (lanes 1, 3, 5, and 7) or treated for 10 min with EGF (30 ng/ml) (lanes 2, 4, 6, and 8). Lysates of transfected COS cells were separated by SDS-PAGE and immunoblotted with anti-PCREB (upper panel) or anti-Gal4 (lower panel) antibodies.