FIGURE 3.

GSK-3β phosphorylates the C-terminal domain of p190A in vitro. A, p190A protein sequences from human, mouse, and rat were analyzed using Scansite software (high stringency), and GSK-3 kinase consensus sites were identified corresponding to amino acids Ser-1472, Ser-1476, and Thr-1480. Amino acid Ser-1483 was also identified as a potential p38 MAPK site. The percentile scores correspond to the probability that these putative sites are related to documented consensus sites by chance. B, full-length p190A baculovirus-expressed protein purified from Sf9 insect cells can be phosphorylated by GSK-3β in vitro. The GSK-3β kinase assay was performed in vitro as described under “Experimental Procedures,” and reaction products were resolved by SDS-PAGE and visualized by autoradiography. C, pEBG, pEBG-50AA-WT, pEBG-50AA-1472A, pEBG-50AA-1476A, pEBG-50AA-1480A, and pEBG-50AA-1483A were transfected into COS-7 cells, and the corresponding GST fusion proteins were purified on glutathione beads. In vitro kinase assays, with or without GSK-3β, were performed as indicated. The left panel corresponds to an immunoblot demonstrating levels of the various fusion protein proteins captured from the cell lysates. The right panel corresponds to the kinase reaction products following SDS-PAGE and autoradiography. D, phosphorylation of a domain containing the C-terminal 50 amino acids of p190A in vivo. The GST-50AA fusion proteins derived from p190A were purified either from E. coli (pGEX-KG-encoded) or from COS-7 cells (pEBG-encoded). The proteins on the glutathione beads were mock-treated, treated with λ-phosphatase, or treated with λ-phosphatase in the presence of phosphatase inhibitors (PPI). Then the beads were washed and boiled in sample buffer, and the proteins were resolved by SDS-PAGE and stained with Coomassie Blue. The faster migrating species are presumed to reflect degradation products derived from the GST fusion proteins. E, mass spectrometry analysis identified sites of phosphorylation within the C-terminal 50 amino acids of p190A. pEBG-50 AA-WT was transfected into COS-7 cells and purified as described under “Experimental Procedures.” The proteins were gel-purified, and proteolytically cleaved peptides were analyzed by mass spectrometry. The phosphorylation sites detected in vivo are underscored in three different peptide species that were detected, and these correspond to amino acids 1472, 1476, and 1483 in p190A.