FIG. 2.
The RXL motif of HIRA directs binding to and phosphorylation by cyclin-cdk2 kinases. (a) HIRA binds to cyclin A, and this requires the RXL motif. In vitro-translated 35S-labeled cyclin A was incubated with GST (lane 1), GST-HIRA[421–729] (lane 2), GST-HIRA[421–729]ΔRXL (lane 3), GST-E2F1 (lane 4), and -GST-E2F1Δ24 (lane 5). The bound proteins were fractionated by SDS-PAGE and visualized by autoradiography. Arrowhead, cyclin A. (b) HIRA is phosphorylated by cyclin-cdk2 kinases, and this requires the RXL motif. Extracts of U2OS cells were immunoprecipitated with antibodies to cdk2 (lane 3 to 7) or SV40 large T antigen (control [con.]; lanes 1 and 2) and used in kinase assays with 0.1 or 1 μg of GST-HIRA[421–729] or GST-HIRA[421–729]ΔRXL as substrates, as indicated. The phosphoproteins were fractionated by SDS-PAGE and visualized by autoradiography. Arrowhead, phosphorylated GST-HIRA[421–729]. (c) Phosphorylation of HIRA by purified recombinant cyclin A- and E-cdk2 in vitro. Cyclin A- and E-cdk2 were expressed in and purified from Sf9 cells, and increasing amounts were used to phosphorylate 1 μg of GST-RB[792–928] (lanes 1 to 3 and 7 to 9) and GST-HIRA[421–729] (lanes 4 to 6 and 10 to 12), as indicated. The reactions were stopped by addition of 3× Laemmli sample buffer, and the phosphoproteins were separated by SDS-PAGE. Arrowheads, phosphorylated HIRA and RB; asterisk, autophosphorylated cyclin A. (d) Phosphorylation of HIRA by cyclin-cdk2 is blocked by a peptide containing the RXL motif of E2F1. Extracts of U2OS cells were immunoprecipitated with antibodies to cdk2 (lanes 2 to 6 and 8 to 12) or SV40 large T antigen (control; lanes 1 and 7) and used in kinase assays with 1 μg of GST-HIRA[421–729] (lanes 7 to 12) or GST-RB[792–928] (lanes 1 to 6) as the substrate. Kinase assays were performed in the presence of 0.1, 1, or 10 μg of a 10-residue synthetic peptide that spans the cyclin-cdk2-binding sequence of E2F1 (WT E2F1; PPVKRRLDLE) or in the absence of the peptide, as indicated. As controls, assays were performed in the presence of 10 μg of a peptide of identical amino acid composition but scrambled sequence (Mut E2F1; lanes 6 and 12). The phosphoproteins were fractionated by SDS-PAGE and visualized by autoradiography. Arrowhead, GST-pRB[792–928]; asterisk, GST-HIRA[421–729]. (e) The RXL motif of HIRA potentiates the phosphorylation of another poorly phosphorylated substrate when fused to the C terminus of that substrate. Extracts of U2OS cells were immunoprecipitated with antibodies to cdk2 (lanes 2 to 13) or SV40 large T antigen (control; lane 1) and used in kinase assays with 0.1 or 1 μg of the indicated protein fused to GST as the substrate. The phosphoproteins were fractionated by SDS-PAGE and visualized by autoradiography. Arrowheads, relevant GST-pRB fusion proteins.