To identify novel substrates of cyclin A- or E-cdk2 kinases, we performed a search of the SWISSPROT database for proteins that contain a cyclin-cdk2-docking motif (RXL) and consensus cyclin-cdk2 phosphorylation sites (S/TPXK/R). This search was carried out using the PatScan program (www-unix.mcs.anl.gov/compbio/PatScan/HTML/patscan.html
). One protein identified through this search, HIRA, was of particular interest due to its previously described homology to two S. cerevisiae
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. Figure 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 as cyclin-cdk2 phosphorylation sites, and 7 WD repeats (Fig. b) (28
Several RXL-containing cyclin-cdk2 substrates stably bind to cyclin-cdk2 complexes in a manner that requires the RXL motif (1
). To determine whether HIRA similarly binds to cyclin A, GST fused to residues 421 to 729 of HIRA (GST-HIRA[421–729]) was tested for binding to in vitro-translated 35
S-labeled cyclin A. Residues 421 to 729 of HIRA contain the RXL motif and both S/TPXK/R cyclin-cdk2 phosphorylation sites (Fig. b). As shown in Fig. a, GST-HIRA[421–729] 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[421–729]ΔRXL) did not. Similarly, and as described previously, WT GST-E2F1, but not a mutant lacking the RXL motif (GST-E2F1Δ24), 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). Thus, like that of E2F1, HIRA binding to cyclin A was dependent on an intact RXL cyclin-cdk2-binding motif.
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] (more ...)
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 cells and tested for its ability to phosphorylate equal amounts of purified GST-HIRA[421–729] and GST-HIRA[421–729]ΔRXL. Cyclin-cdk2 kinase efficiently phosphorylated the former substrate but not the latter (Fig. b). Thus, cyclin-cdk2 kinase phosphorylates HIRA in vitro and, as for p107 and E2F1, this requires an intact RXL motif. However, cdk2 complexes immunopurified from asynchronously growing cells consist of cyclin A-cdk2 and cyclin E-cdk2. Therefore we directly compared the abilities of purified recombinant cyclin A-cdk2 and cyclin E-cdk2 expressed in Sf9 cells to phosphorylate GST-HIRA[421–729] and, as a control, GST-RB[792–928]. Although under these particular conditions the ability of each kinase to phosphorylate GST-HIRA[421–729] was less then its ability to phosphorylate GST-RB[792–928], both cyclin A- and E-cdk2 efficiently phosphorylated GST-HIRA[421–729] in vitro (Fig. c).
We have previously shown that short synthetic peptides containing functional RXL motifs will compete with the full-length proteins for binding to the cyclin-cdk2 complex (1
). If the RXL motif of HIRA serves to target the protein to the cyclin-cdk2 kinase in a manner similar to that for the RXL motifs of E2F1 and p107, then a peptide containing the RXL motif of E2F1 should block phosphorylation of HIRA in vitro. To test this, kinase assays were performed with cyclin-cdk2 complexes and GST-HIRA[421–729] or GST-RB[792-928] as a substrate in the absence or presence of a short synthetic peptide encompassing the RXL cyclin-cdk2-binding motif of E2F1. The peptide efficiently blocked phosphorylation of both substrates (Fig. d). In contrast, a peptide of identical amino acid composition but of scrambled sequence blocked phosphorylation of neither substrate. Thus the RXL motifs of HIRA and E2F1 compete with one another, suggesting that they are functionally equivalent.
Previously, Harlow and coworkers showed that the cyclin-cdk2-binding domain of E2F1 potentiates phosphorylation of E2F4 (14
). Likewise, we showed that the RXL motifs of E2F1 and p21cip1
potentiated the phosphorylation of a poorly phosphorylated variant of the retinoblastoma tumor suppressor protein (pRB) that is mutated to remove all of its RXL motifs (pRB[792–829]) (2
). Therefore, as a final test of the functionality of the HIRA RXL motif we tested whether it would also promote phosphorylation of pRB[792–829]. As shown previously, when pRB was expressed and purified as a GST fusion protein, its C terminus (GST-RB[792–928]) was efficiently phosphorylated by cyclin-cdk2 in vitro whereas GST-RB[792–829] was not (2
). Phosphorylation was restored when 10 amino acids encompassing the RXL motif of either E2F1 or HIRA were fused to the C terminus of GST-RB[792–829] (GST-RB[798–829]E2F1Cy and GST-RB[792–829]HIRACy, respectively) (Fig. e). In contrast, fusion of either peptide with a mutation in the RXL motif did not restore phosphorylation (GST-RB[792–829]E2F1CyMut and GST-RB[792–829]HIRACyMut). Thus, the RXL of HIRA can promote phosphorylation of a heterologous protein. Taken together these studies clearly show that, at least in vitro, the HIRA RXL is functionally similar to the RXL of a known cyclin-cdk2 substrate, E2F1.
To facilitate subsequent studies of the endogenous protein in vivo, antibodies were raised to HIRA as described in Materials and Methods. Four monoclonal (WC15, 19, 117, and 119) and two polyclonal antibodies (D32 and D34) were obtained. As determined by Western blotting, each of these efficiently detected HA-tagged HIRA[421–729] that was ectopically expressed in U2OS cells (Fig. a and data not shown). Furthermore, in extracts of untransfected cells, each antibody detected a polypeptide with a molecular weight corresponding to that predicted for HIRA. Immunoprecipitation followed by Western blot analysis showed that each antibody reacted with the same polypeptide (Fig. b and data not shown), confirming that each antibody recognizes HIRA.
FIG. 3 Characterization of antibodies to HIRA. (a) Monoclonal antibodies to HIRA detect HIRA by Western blotting. U2OS cells were transfected with a plasmid encoding HA-HIRA[421–729] (odd-numbered lanes) or with empty vector (even-numbered (more ...)
Using these antibodies we tested whether endogenous HIRA was phosphorylated in vivo. Extracts derived from U2OS cells were immunoprecipitated with anti-HIRA monoclonal antibody WC15 and the washed immunoprecipitates were treated with or without λ-phosphatase and fractionated by SDS-PAGE. Western blotting with WC119 showed that treatment with λ-phosphatase resulted in increased mobility of HIRA, indicating that the protein was phosphorylated (Fig. a). When HA-HIRA[421–729] was ectopically expressed in U2OS cells, it too was phosphorylated, as determined by a λ-phosphatase-dependent increase in mobility in SDS-PAGE (Fig. b).
FIG. 4 HIRA is phosphorylated in vivo. (a) Endogenous cellular HIRA is a phosphoprotein. Extracts were prepared from asynchronously growing U2OS cells and immunoprecipitated with anti-SV40 large T antigen (419; lane 1) or anti-HIRA (WC15; lanes 2 and 3). The (more ...)
If HIRA is an in vivo substrate of cyclin-cdk2 kinases, then it should be phosphorylated in vivo on residues that are consensus cyclin-cdk2 phosphorylation sites and that are phosphorylated by cyclin-cdk2 kinase in vitro. To test this, we raised a rabbit polyclonal serum (D44) against a phosphopeptide containing a phosphothreonine that corresponds to threonine 555 (one of the two residues that conforms to the cyclin-cdk2 consensus, S/TPXK/R). An enzyme-linked immunosorbent assay showed that the rabbit immune, but not preimmune, serum specifically reacted with the phosphorylated but not the unphosphorylated peptide (data not shown). Subsequently, immunoprecipitation analysis showed that the anti-phosphothreonine 555 serum reacted with GST-HIRA[421–729] only after the protein had been incubated with cyclin A-cdk2 and magnesium-ATP (Fig. a). Thus, the anti-phosphothreonine 555 serum reacted only with GST-HIRA[421–729] phosphorylated by cyclin A-cdk2, presumably on threonine 555. To determine whether threonine 555 was phosphorylated on HIRA in vivo and whether this phosphorylation was dependent on the RXL motif, U2OS cells were transfected with a plasmid encoding HA-HIRA[421–729] or a mutant either lacking the RXL motif (HA-HIRA[421–729]ΔRXL) or containing a substitution of alanine for threonine 555 (HA-HIRA[421–729]T555A). As shown in Fig. b, phosphorylation of HA-HIRA[421–729] on threonine 555 was readily detectable. In contrast, phosphorylation was undetectable on HA-HIRA[421–729]ΔRXL or HIRA[421–729]T555A. Thus ectopically expressed HIRA was phosphorylated on threonine 555 and, like phosphorylation by cyclin A-cdk2 in vitro, this required an intact RXL cyclin-cdk2-binding motif.
FIG. 5 HIRA is phosphorylated on two consensus cyclin-cdk2 phosphorylation sites in vivo. (a) Specificity of phosphothreonine 555-specific antibody D44. GST-HIRA[421–729] (100 ng) was incubated without (lanes 1 and 4) or with (lanes 2, (more ...)
Next we asked whether the endogenous HIRA protein was phosphorylated on threonine 555. Extracts from U2OS cells were immunoprecipitated with an antibody to total HIRA, the anti-phosphothreonine 555-specific serum, or control antibodies. As shown in Fig. c, the phosphoamino acid-specific antibody immunoprecipitated the endogenous protein. However, the phosphoamino acid-specific antibody precipitated a relatively small proportion of total HIRA protein, and this is consistent with the notion that HIRA is phosphorylated on threonine 555 only under certain conditions, e.g., in specific phases of the cell cycle. Taken together, these results show that HIRA is phosphorylated by cyclin A-cdk2 in vitro on a site that is phosphorylated in vivo, threonine 555.
As an alternative approach to identify the residues of HIRA that are phosphorylated in vivo, mass spectrometry was utilized. In particular, we were interested in determining whether other consensus cyclin-cdk2 phosphorylation sites, e.g., serine 687, were phosphorylated. Asynchronously growing U2OS cells were transfected with a plasmid encoding HA-HIRA[421–729], and the ectopically expressed protein was immunopurified with an anti-HA antibody. The protein was purified by SDS-PAGE, extracted from the gel, proteolytically digested with trypsin, and subjected to matrix-assisted laser desorption ionization–time of flight (MALDI-TOF) and liquid chromatography (LC)-MS/MS mass spectrometry. Both approaches showed that the protein was phosphorylated on the residue that corresponds to serine 687 of the full-length protein (Fig. d and data not shown). In this experiment we failed to obtain unambiguous data on the phosphorylation status of threonine 555. This could be due to a number of technical aspects of the mass spectrometry approach and cannot be taken as evidence that this site is not phosphorylated. Taken together, the mass spectrometry- and immunology-based approaches indicate that HIRA is phosphorylated in vivo on both of the consensus cyclin-cdk2 phosphorylation sites, threonine 555 and serine 687.
If HIRA is a substrate of a cyclin-cdk2 kinase in vivo, then its phosphorylation should be dependent on cyclin-cdk2 kinase activity. To test whether this is the case, U2OS cells were transiently transfected with plasmids encoding HA-HIRA[421–729] alone or together with a plasmid encoding cyclin-cdk2 inhibitor p21cip1. When expressed in the absence of p21cip1, HA-HIRA[421–729] migrated as a doublet (Fig. a). The more slowly migrating band results from phosphorylation (Fig. b and data not shown). However, when expressed in the presence of p21cip1, HA-HIRA[421–729] migrated as a single band of higher mobility, indicating that the cyclin-cdk2 inhibitor blocks the phosphorylation of HIRA (Fig. a).
FIG. 6 Phosphorylation of HIRA requires cyclin-cdk2 kinase activity. (a) Phosphorylation of HIRA is inhibited by cyclin-cdk2 inhibitor p21cip1. U2OS cells were transiently transfected with a plasmid encoding HA-HIRA[421–729] (lanes 2 (more ...)
Next we tested whether inhibition of cyclin-cdk2 kinase activity inhibited phosphorylation of the mapped cyclin-cdk2 phosphorylation site, threonine 555, and some of the other potential cdk2 phosphoacceptor sites (specifically threonine residues followed by proline). U2OS cells were transfected with a plasmid encoding residues 520 to 1017 of HIRA (HA-HIRA[520–1017]) in the absence or presence of a plasmid encoding p21cip1. Residues 520 to 1017 were utilized in this experiment because, unlike full-length HIRA and residues 421 to 729, this region of the protein does not perturb the cell cycle (see Fig. ; data not shown). Phosphorylation of threonine 555 was dramatically inhibited by coexpression of p21cip1, as revealed by immunoprecipitation with the phosphothreonine 555-specific antiserum followed by Western blotting with anti-HA (Fig. b). Likewise, p21cip1 inhibited phosphorylation of HA-HIRA[520–1017], as detected by an anti-phosphothreonine-proline-specific antibody (Fig. b). This antibody reacts with most phosphothreonine residues that are followed by proline. Including T555, there are four such threonine residues in HA-HIRA[520–1017]. Taken together, these experiments show that phosphorylation of HIRA in vivo requires cyclin-cdk2 kinase activity, as would be expected if HIRA is a substrate of a cyclin-cdk2 kinase.
FIG. 9 Ectopic expression of HIRA causes arrest in S phase of the cell cycle. (a) U2OS cells were transfected without (−HIRA) or with (+HIRA) a plasmid encoding WT HA-HIRA[1–1017] together with a plasmid encoding cell (more ...)
Another prediction, if HIRA is an in vivo substrate of a cyclin-cdk2 kinase, is that the protein should be phosphorylated when these kinases are active. Both cyclin A- and E-cdk2 are active in growing cells but not in quiescent cells (12
). Therefore, we asked whether HIRA was phosphorylated on threonine 555, a known in vitro phosphorylation site of cyclin-cdk2 (Fig. a), in growing but not quiescent cells. Extracts derived from asynchronously growing NIH 3T3 cells and cells that had been deprived of serum for 48 h to induce quiescence were immunoprecipitated with the anti-phosphothreonine 555-specific antiserum (D44) and then Western blotted with the anti-HIRA antibody (WC119). Although the total amounts of HIRA protein in growing and quiescent cells were the same, the protein was phosphorylated on threonine 555 in growing cells only (Fig. a). Next, we asked whether HIRA is phosphorylated on threonine 555 at a time in the cell cycle consistent with it being a cyclin A- or E-cdk2 substrate. Cyclin A- and E-cdk2 are activated in late G1
and S phases after quiescent cells are stimulated to reenter the cell cycle (18
). Extracts derived from quiescent NIH 3T3 cells or cells refed with serum for 4, 8, or 16 h were analyzed for the abundance of HIRA phosphorylated on threonine 555. Although, the total amounts of HIRA in quiescent and stimulated cells did not vary (Fig. c, top), phosphorylated HIRA was only detected 16 h after serum stimulation (Fig. c, bottom). FACS analysis indicated that phosphorylation temporally coincided with entry of the cells into S phase (Fig. b) and, presumably, activation of cyclin A- and E-cdk2.
FIG. 7 HIRA is phosphorylated on threonine 555 in S phase of the cell cycle. (a) Extracts were prepared from asynchronous NIH 3T3 cells growing in 10% fetal bovine serum (FBS) (lanes 1 to 4) or cells grown in 0.1% FBS for 48 h (lanes 5 to 8) (more ...)
The active forms of cyclin A- and E-cdk2 are localized predominantly in the cell nucleus (39
). Therefore, if HIRA is an in vivo substrate of these kinases, then it too should be localized, at least in part, to the cell nucleus. To test whether this is the case, U2OS cells grown on coverslips were fixed and stained with monoclonal antibodies to HIRA or a control antibody. Three separate monoclonal antibodies to endogenous HIRA (WC15, 19, and 119) revealed a specific punctate nuclear staining pattern (Fig. a, b, e, and f and data not shown). To confirm this staining pattern, U2OS cells were transiently transfected with a plasmid encoding HA-HIRA[1–1017] and stained with anti-HA antibody 12CA5. Ectopically expressed HA-HIRA[1–1017] was present predominantly in the nucleus, although in some cells cytoplasmic staining was also observed (Fig. c, d, g, and h). This observation is consistent with previous studies by Lorain and coworkers, who detected endogenous HIRA as a primarily nuclear protein in various other cell lines (32
). Taken together, these observations confirm that HIRA is a predominantly nuclear protein, and this is consistent with it being an in vivo substrate of cyclin A- or E-cdk2.
FIG. 8 HIRA is a nuclear protein. Asynchronously growing U2OS cells grown on coverslips were stained with a control immunglobulin G1 (IgG1) monoclonal antibody (a) or two anti-HIRA monoclonal antibodies together, WC19 and WC119 (both IgG1) (e). DNA was visualized (more ...)
These data clearly show that HIRA is an in vivo substrate of cyclin A- or E-cdk2. As such, it is a likely regulator of progression through the cell cycle. Accordingly, we next tested whether HIRA was able to modulate progression through the cell cycle. Asynchronously growing U2OS cells were transiently transfected with a plasmid encoding HA-tagged HIRA[1–1017], together with a plasmid encoding cell surface marker CD19, to allow identification of the transfected cells. FACS analysis showed that ectopic expression of HA-HIRA[1–1017] in cells caused the percentage of cells in S phase to increase from 45 to 75% (Fig. a). To confirm that this S-phase accumulation was due to an arrest in S phase, cells were again transiently transfected with plasmids encoding HA-HIRA[1–1017] and CD19 and 1 h prior to harvesting they were pulse-labeled with the thymidine analogue 5′-BrdU. FACS analysis was performed to determine whether the transfected cells had incorporated 5′-BrdU. As shown in Fig. b, approximately 41% of the cells transfected with the empty vector and the plasmid encoding CD19 were 5′-BrdU positive. This percentage of cells in S phase is comparable to the value of 45% that was obtained by propidium iodide staining (Fig. a). In contrast, only 3% of the cells ectopically expressing HA-HIRA[1–1017] were 5′-BrdU positive. Thus, although ectopic expression of HA-HIRA[1–1017] caused a dramatic accumulation of cells with DNA content corresponding to that found in S phase (Fig. a), these cells did not incorporate 5′-BrdU (Fig. b). Thus, they were not actively synthesizing DNA and were arrested in S phase.