Third, when cellular cyclin-cdk kinase activity was inhibited by cyclin-cdk2 inhibitor p21cip1, the phosphorylation of HIRA was blocked. to the products of two genes, and proteins, Hir1p and Hir2p, that are known to play a role in control of cell cycle-regulated transcription of histone genes. Sequence comparisons indicate that HIRA is the best candidate identified to date to be a human ortholog (functional equivalent) of Hir1p and Hir2p. Physique ?Determine11 a shows an alignment of the putative cyclin-cdk2-binding motif of HIRA (amino acids 626 to 633) with the previously characterized cyclin-cdk2-binding motifs of other human cell cycle control proteins. In addition to the RXL motif, the HIRA primary sequence contains 2 putative cyclin-cdk2 phosphorylation sites that conform to the consensus S/TPXK/R (threonine 555 and serine 687), 13 other S/TP motifs that might also serve Rabbit Polyclonal to CDC7 as cyclin-cdk2 phosphorylation sites, and 7 WD repeats (Fig. ?(Fig.1b)1b) (28). Several RXL-containing cyclin-cdk2 substrates stably bind to cyclin-cdk2 complexes in a manner that requires the RXL motif (1, 6, 14, 33, 46, 47, 65). To determine whether HIRA similarly binds to cyclin A, GST fused to residues 421 to 729 of HIRA (GST-HIRA[421C729]) was tested for binding to in vitro-translated 35S-labeled cyclin A. Residues 421 to 729 of HIRA contain the RXL motif and both S/TPXK/R cyclin-cdk2 phosphorylation sites (Fig. ?(Fig.1b).1b). As shown in Fig. ?Fig.2a,2a, GST-HIRA[421C729] efficiently bound to cyclin A whereas GST alone or a HIRA mutant containing a four-alanine substitution in place of the KRKL of the RXL (GST-HIRA[421C729]RXL) did not. Similarly, and as described previously, WT GST-E2F1, but not SB-224289 hydrochloride a mutant lacking the RXL motif (GST-E2F124), bound to cyclin A in this assay (26). All of the WT and mutant proteins were present in the assay mixture at comparable levels (data not shown). SB-224289 hydrochloride Thus, like that of E2F1, HIRA binding to cyclin A was dependent on an intact RXL cyclin-cdk2-binding motif. Open in a separate windows 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[421C729] (lane 2), GST-HIRA[421C729]RXL (lane 3), GST-E2F1 (lane 4), and -GST-E2F124 (lane 5). The bound proteins were fractionated by SDS-PAGE and visualized by autoradiography. Arrowhead, cyclin A. (b) HIRA is usually 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 g of GST-HIRA[421C729] or GST-HIRA[421C729]RXL as substrates, as indicated. The phosphoproteins were SB-224289 hydrochloride fractionated by SDS-PAGE and visualized by autoradiography. Arrowhead, phosphorylated GST-HIRA[421C729]. (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 g of GST-RB[792C928] (lanes 1 to 3 and 7 to 9) and GST-HIRA[421C729] (lanes 4 to 6 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 usually blocked by a peptide made up of 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 g of GST-HIRA[421C729] (lanes 7 to 12) or GST-RB[792C928] (lanes 1 to 6) as the substrate. Kinase assays were performed in the presence of 0.1, 1, or 10 g 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 g 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[792C928]; asterisk, GST-HIRA[421C729]. (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 SB-224289 hydrochloride used in kinase assays with 0.1 or 1 g of the indicated protein fused to GST as the SB-224289 hydrochloride substrate. The phosphoproteins were fractionated by SDS-PAGE and visualized by autoradiography. Arrowheads, relevant GST-pRB fusion proteins. Efficient phosphorylation of p107 and E2F1 in vitro by cyclin-cdk2 kinase requires that each substrate have an intact RXL motif (1). We next asked whether HIRA was phosphorylated in vitro by cyclin-cdk2 kinases and whether this too required an intact RXL cyclin-cdk2-binding motif. Cyclin-cdk2 kinase was immunopurified from asynchronously growing U2OS.