DNA Damage-Dependent and Independent Phosphorylation of the hRad9 Checkpoint Protein

of analyses indicate that hRad9 is a component of the in and has possible roles in the and Previous studies indicated that hRad9 is modified by phosphorylation, both in the absence of exogenous stress and in response to various genotoxins. In this study, we report the mapping of several sites of constitutive phosphorylation of hRad9 to [S/T]-P-X-[R/P] sequences near the C-terminus of the protein. We also demonstrate that a serine to alanine mutation at residue 272 abrogates an ionizing radiation (IR) induced phosphorylation of hRad9, and further show that phosphorylation at [S/T]-P sites is not a prerequisite for IR induced phosphorylation of serine 272. Finally, we report that hRad9 undergoes cell cycle regulated hyper-phosphorylation in G2/M that is enhanced by IR, but distinct from that on serine 272. Unlike the IR-induced phosphorylation at serine 272, this event is dependent on serine 277 and threonine 292, two C-terminal [S/T]-P sites in hRad9.


Summary
Cell cycle checkpoints are regulatory mechanisms that maintain genomic integrity by preventing cell cycle progression when genetic anomalies are present.
The hRad9 protein is the human homologue of Schizosaccharomyces pombe Rad9, a checkpoint protein required for preventing the onset of mitosis if DNA damage is present or if DNA replication is incomplete. Genetic and biochemical analyses indicate that hRad9 is a component of the checkpoint response in humans, and has possible roles in regulating the cell cycle, apoptosis, and DNA repair. Previous studies have indicated that hRad9 is modified by phosphorylation, both in the absence of exogenous stress and in response to various genotoxins. In this study, we report the mapping of several sites of constitutive phosphorylation of hRad9 to [S/T]-P-X-[R/P] sequences near the C-terminus of the protein. We also demonstrate that a serine to alanine mutation at residue 272 abrogates an ionizing radiation (IR) induced phosphorylation of hRad9, and further show that phosphorylation at [S/T]-P sites is not a prerequisite for IR induced phosphorylation of serine 272. Finally, we report that hRad9 undergoes cell cycle regulated hyper-phosphorylation in G2/M that is 3

Introduction
An organism's genome is under constant stress from a variety of endogenous and exogenous sources. While low frequencies of genetic mutation are tolerated, contributing to genetic diversity, high frequencies are harmful and can lead to cancer (1). At the cellular level, eukaryotes have evolved signal transduction pathways called checkpoints to cope with genetic insults (2)(3)(4). Checkpoints stall progression through the cell cycle, providing time for cellular responses, such as activation and relocalization of DNA repair enzymes to sites of DNA damage or transcriptional activation of specific genes. Checkpoint arrest can also lead to activation of apoptotic pathways perhaps under conditions when cell death is more beneficial to the organism as a whole than repair (reviewed in (5)(6)(7)).
While these data suggest that hRad17 and 9-1-1 are early components of the checkpoint signaling cascade, whether they are responsible for the initial detection of DNA damage still remains unclear. In S.pombe, the Rad3 protein, a PI3 related kinase, phosphorylates Rad26 in response to DNA damage independently of the other checkpoint rads (30), suggesting that it is the initiator of the checkpoint signaling cascade. Two human homologues of Rad3, ATM and ATR, phosphorylate a wide variety of cellular proteins on [S/T]-Q sequences in response to DNA damage (31)(32)(33)(34)(35)(36)(37)(38). Mutations in ATM result in the cancer predisposition syndrome, ataxia telangiectasia (39). Recently, hRad9 has been implicated as an ATM substrate (40).
While the evidence from fission yeast indicate that hRad9 is fulfilling a role in the G2/M transition (11), ATM-dependent phosphorylation of hRad9 occurs regardless of cell cycle position and appears to be important for the G1 DNA damage checkpoint (40). hRad9, through interactions with the anti-apoptotic Bcl-2 and Bcl-xL proteins, can also promote apoptosis, and therefore, appears to have a multi-functional role in responding to genotoxins (41).
Previous studies have indicated that the hRad9 protein is extensively modified by phosphorylation under normal cellular conditions (19) and becomes hyperphosphorylated in response to DNA damage at serine 272 (18,40). Here, we further the current understanding of hRad9 phosphorylation by mapping sites required for its 5 constitutive phosphorylation and by identifying of a novel, cell cycle regulated, ionizing radiation-induced phosphorylation event.

Experimental Procedures
Plasmids -The full length hRad9 cDNA was subcloned into unique XhoI and XbaI restriction sites of the pyDF31 mammalian expression vector. Protein expression from pyDF31 is driven by the strong constitutive SR alpha promoter composed of the SV40 early promoter and a segment of the LTR of Human T-Cell Leukemia Virus (42). All hRad9 point mutants were generated in pyDF31 using the Transformer Site-Directed Mutagenesis Kit (Clontech, Palo Alto, CA) according to the manufacturer's instructions. Three selection primers were used to disrupt unique XhoI, NotI, and EagI restriction sites in pyDF31-hRad9. The sequence of these primers and the hRad9 mutagenic primers are shown in Table 1. Constructs with multiple point mutations were made by sequential mutagenesis reactions or concurrently by using multiple mutagenic primers in the same reaction. The presence of the desired base substitutions were confirmed by DNA sequencing using an automated sequencer (Cortec DNA 6 snnglycerophosphatidylethanol-amine) (Sigma, Oakville ON) and DDAB (dimethyldioctadecyl-ammonium bromide) (Sigma), respectively. The transfection reagent was mixed with 2:g DNA in 3.3ml DMEM (10cm plate) or 0.25:g in 600:l DMEM (6-well plate) and applied to cells for 4 hours at 37°C. The transfection solution was then replaced with DMEM plus 10% FBS, and cells were cultured for an additional 30 to 48 hours prior to lysis.
Cell Synchronization and Flow Cytometry -HeLa cells, cultured as described above, were synchronized in early S-phase by double thymidine block as previously described (43). Cells were cultured to a confluence of approximately 30% and treated with 2mM thymidine for 18 hours. After 18 hours, cells were released from thymidine for 8 hours, treated for an additional 18 hours, and then released for varying lengths of time. hTERT-RPE1 cells were synchronized in early S-phase using a single, 24 hour dose of 5mM thymidine. Mitotic HeLa cells were generated by treatment with 70ng/ml demicolcine (Sigma). Synchronized cell populations were followed by flow cytometry; cells were harvested, resuspended in 1ml of PBS +1% FBS, fixed by addition of 1ml of 100% ethanol, and stored at 4°C for at least 1 hour. After fixing, cells were washed twice in PBS, resuspended in 1ml of PBS + 1% FBS and 0.5mg/ml RNaseA, and incubated for 40 min at 37°C. Cells were then collected by centrifugation and resuspended in PBS + 50µg/ml propidium iodide and 0.1mg/ml RNaseA and analysed using a flow cytometer (Beckman/Coulter EPICS Elite, Mississauga, ON). fluoride HCl (AEBSF), 20:g/ml aprotinin, 4:g/ml leupeptin, 0.7:g/ml pepstatin, 2mM Na 3 VO 4 , 20mM ∃-glycerophosphate and 0.2mM NaF. Lysates were incubated on ice 8 for 1 hour, then centrifuged at 16000g for 20 min at 4°C. Soluble cell lysates were immunoprecipitated with antibodies directed against hRad9 essentially as described above. Agarose was washed 4x with NETN buffer and resuspended in 60:l of 2x electrophoresis buffer and boiled for 5 min prior to SDS-PAGE (10% acrylamide).
The gel was either transferred to nitrocellulose and immunoblotted with antibodies directed against hRad9 or dried using a gel slab dryer (BIORAD, Mississauga, ON).
Protein quantification of the immunoblot was performed using AlphaEase software and a chemiimager (Alpha Innotech Corporation, San Leandro, CA). 32 P quantification was performed using ImageQuant software and a phosphoimager (Molecular Dynamics, Sunnyvale, CA).
Immunoblotting -All samples were boiled for 5 minutes prior to electrophoresis through 8% or 10% acrylamide, as indicated. Proteins were Sequences -The hRad9 protein is constitutively phosphorylated in the absence of DNA damage (19), and becomes additionally phosphorylated when DNA damage is present (18). We have observed that under normal cellular conditions, (i.e. those where cells are not exposed to exogenous stresses) overexpressed hRad9 consists of four species that differ in migration rate on SDS-PAGE as visualized by immunoblot analysis. The slowest migrating of these species, which we have termed hRad9∀, comigrates with the majority of endogenous hRad9 at an apparent molecular weight of approximately 60 kDa ( Figure 1a). In vitro dephosphorylation of exogenous hRad9 causes each of its four migratory forms to collapse into a single band at about 45 kDa, which we have called hRad9 * ( Figure 1b). These data suggest that, relative to endogenous hRad9, a large portion of the overexpressed protein is only partially phosphorylated, and hence has various migratory forms. We have collectively designated all of these partially phosphoryated forms hRad9∃. Previous work in our lab has shown that deleting the C-terminus of exogenous hRad9 can reduce the number of differentially migrating species from four to one (our unpublished results), indicating that the C-terminus of hRad9 is required for constitutive phosphorylation of the protein.
With this in mind, we used site directed mutagenesis to identify amino acid residues required for the constitutive phosphorylation of hRad9. Potential phosphorylated residues near the C-terminus of the protein were converted to nonphosphorylatable amino acids. Mutants were expressed in HeLa cells following transient transfection, and screened for migratory shifts as detected by western analysis. The gross overexpression of protein by the strong SR-alpha promoter allowed us to distinguish plasmid derived hRad9 protein from the endogenous protein 10 simply by limiting the exposure time to X-ray film. A trend between those mutants with altered western blot banding patterns soon emerged, as each of these mutants contained disrupted serine or threonine residues followed immediately by a proline (results summarized in Table 2). The [S/T]-P motif is the minimum consensus sequence for the cyclin dependent kinase (CDK) family of kinases (44). We therefore mutated all nine [S/T]-P sequences in hRad9, immunoprecipitated these proteins from transfected HeLa cells, and compared their banding pattern to the wild type protein  (Figure 1d), which is only slightly larger than the predicted molecular weight of 42.5 kDa for hRad9.

hRad9 is Consitutively Phosphorylated on Sites Other Than [S/T]-P
Sequences -The P4A mutant still exhibited a modest mobility shift when treated with alkaline phosphatase (Figure 2a). This suggested the existence of additional sites of constitutive phosphorylation in hRad9. To determine if these additional sites were [S/T]-P sites, whose phosphorylation produced little or no shift on a western blot, we mutated all nine [S/T]-P sites in hRad9 in combination. The migration of the P9A mutant (P4A + T60A, S160A, T292A, S375A, and S380G) was not readily distinguishable from that of the P4A mutant and was still sensitive to phosphatase treatment ( Figure 2a). Therefore, despite being constitutively phosphorylated on at least four [S/T]-P sites, hRad9 is also phosphorylated on sites other than these sequences. We have called the form of hRad9, which completely lacks phosphorylation at [S/T]-P sites but still remains phosphorylated, hRad9,.
To further characterize the constitutive phosphorylation of hRad9, HeLa cells were transfected with a series of hRad9 mutants or empty vector and metabolically labelled with 32 P ortho-phosphoric acid. The hRad9 point mutants, P3A, P4A, P5A (P4A + T292A), P6A (P5A + S375A), and P9A were used in the transfection and following metabolic labelling, the hRad9 protein was immunoprecipitated from these cells. Immunoprecipitated protein was size-fractionated by SDS-PAGE, transferred to nitrocellulose membrane, and immunoblotted with antibodies directed against hRad9 ( Figure 2b; left panel). The amount of hRad9 protein recovered from each immunoprecipitation was defined by densitometric analysis of the immunoblot using a chemiimager, and was used for data normalization as described below. A second, identical gel was also run, dried, and exposed to a phosphor screen and analyzed using a STORM phosphoimager (Figure 2b; right panel). In agreement with Figure 2a, each 12 hRad9 mutant, including P9A, was significantly phosphorylated confirming that the hRad9 protein is consitutively phosphorylated at sites other than [S/T]-P sites.
We went on to quantitate the 32 P signal in Figure 2b using the ImageQuant software program (Figure 2c). Values were normalized to background 32 P in each lane and to protein levels (which were all within 15%) determined above by spot densitometry of the hRad9 immunoblot. Since only the P3A mutant was wild-type at threonine 355, a site we knew to be constitutively phosphorylated (see Figure 1), we expected that P3A should have a stronger signal than each of the other four mutants, and it did. The remaining mutants exhibited smaller differences in relative 32 P activity, which may be a reflection of confounding variables inherent to the experiment, such as protein normalization, rather than legitimate differences in phosphorylation.  (33). Since serine 272 of hRad9 is followed immediately by a glutamine residue, we hypothesized that this amino acid was the site of the previously reported ionizing radiation (IR)-induced phosphorylation of hRad9 (18). In this regard, we observed that a serine to alanine mutation at serine 272 had no effect on constitutive phosphorylation but abrogated the ability of exogenously expressed hRad9 to become phosphorylated in response to ionizing radiation (Figure 3a). While sub-populations of both the ∀ and ∃ forms of wild-type hRad9 (WT) underwent a subtle mobility change when cells were treated 13 with 20 and 40 Gray doses of IR (hRad9∀ to hRad9( and hRad9∃ to hRad9∃(()), the S272A mutant showed no changes in hRad9∀ or hRad9∃ mobility (Figure 3a; upper panel). This shift was confirmed to be the result of phosphorylation, as CIP treatment of these samples yielded co-migrating dephosphorylated proteins (hRad9 * , Figure 3a; lower panel). While the subtle nature of the mobility shift, combined with the complex banding pattern of hRad9 has made this effect difficult to observe by immunoblotting techniques, we have found this result to be reproducible and offer further evidence in support of it in figures 3b and 3c. This observation also confirms a recent report which demonstrated that ionizing radiation induced phosphorylation at this residue, and that this phosphorylation was ATM-dependent (40). This report however, like all previous studies demonstrating IR-induced phosphorylation of hRad9, involved a constitutively phosphorylated protein (18,29). While the purpose of this constitutive phosphorylation remains unknown, it may be potentiating some aspect of hRad9's cellular activity. The observation in Figure 3a that the ∃ forms of wildtype hRad9, like the ∀ form, shift subtly in response to IR, seems to indicate that [S/T]-P phosphorylation of hRad9 is not required for IR-induced phosphorylation at serine 272.
To address this directly, we tested the response to IR of hRad9 P9A, which lacks all phosphorylatable [S/T]-P sites. The P9A hRad9 mutant was expressed in cells that were subsequently irradiated or mock irradiated. P9A was immunoprecipitated from these cells one hour later, treated with phosphatase as indicated, size-fractionated by SDS-PAGE, and immunoblotted with antibodies directed against hRad9 (figure 3b). In response to irradiation, a slower migrating form of hRad9, (hRad9,(()) became readily apparent. Both hRad9, and hRad9,(() increased in mobility and co-migrated when treated with CIP (hRad9 * ) indicating that hRad9,(() 14 was in fact a phosphorylated form of hRad9,. To confirm that the IR-induced phosphorylation of hRad9, was occurring at serine 272, the S272A mutation was introduced into the P9A mutant and these proteins were analyzed for mobility changes following exposure to low and high doses of IR (Figure 3c). Consistent with Figure   3b

hRad9 is Phosphorylated in a Cell Cycle Dependent Manner in HeLa Cells -
Given that hRad9 is phosphorylated on potential CDK consensus sites, we hypothesized that the attachment of these seemingly constitutive phosphate groups may be regulated in a cell cycle dependent manner. Therefore, a double thymidine block was used to generate synchronized cell populations, which were examined for differences in endogenous hRad9 phosphorylation by immunoblotting (Figure 4a The four remaining [S/T]-P sequences in the protein (threonine 60, serine 160, serine 375, and serine 380) contain neither P, R, nor K at position 4 (Table 2). This, in addition to our observations that mutating these residues does not alter the mobility of hRad9 through SDS-PAGE (figures 1c, 2a, 6a, and 6b) or significantly reduce 32 P 19 uptake in metabolically labelled cells (figure 2c) suggests that these four sites are not constitutively phosphorylated.
While we have termed the [S/T]-P phosphorylation sites constitutive, we can not rule out the possibility that phosphorylation at these sites is regulated in a complex manner. Several groups, using different cell lines and antibodies have observed, as we have, that endogenous hRad9 exists primarily as a single species migrating at 60kDa on a western blot (hRad9∀). However, we have occasionally observed bands in the 45-60kDa range when studying endogenous hRad9 that correlate with the partially phosphorylated bands of the overexpressed protein (hRad9∃). Even though we are able to limit the abundance of these bands by increasing the concentration of phosphatase inhibitors in our lysis buffer, or by lysing cells directly in SDS-PAGE sample buffer, we cannot rule out the possibility that constitutively phosphorylated hRad9 intermediates are physiologically significant. Importantly however, no consistent changes were observed in the 45-60kDa bands at different stages of the cell cycle (Figure 4a, Figure 5b and 5c), suggesting that phosphorylation at serine 277, serine 328, serine 336, and threonine 355 remains constant throughout the cell cycle.
Recently, a phosphospecific antibody directed against the serine 272 of hRad9 was used to demonstrate that IR-induced phosphorylation of hRad9 occurs at this site in vivo (40). We have confirmed these findings, using an independent method, by showing that a serine to alanine mutation at residue 272 abrogates hRad9  (Figure 3a). Second, mutational inactivation of the [S/T]-P phosphorylation sites still yield a protein (hRad9,) that is capable of serine 272 phosphorylation in response to IR (Figure 3b and 3c).
We also report the identification a second hyper-phosphorylation event for hRad9 that is cell cycle regulated. We first identified these phospho-forms (hRad9Φ) in HeLa cells that had been synchronized in G2 or mitosis, and found that we could moderately increase their abundance by treating these cells with ionizing radiation. In contrast, we found that hTERT-RPE1 cells, a karyotypically normal human epithelial Perhaps of further interest is the observation that the hRad9∃ forms, whose abundance normally exceed that of the hRad9∀ form when the protein is overexpressed (see figure 1), are all but absent during a demicolcine induced mitotic arrest ( figure 6a). This could be the result of destabilisation or further phosphorylation of hRad9∃ at the G2/M transition. Whether this has any relevance to the endogenous protein however, which exists predominantly in the hRad9∀ form, remains to be seen. 22 There has been much speculation recently that the association of hRad9 with hRad1 and hHus1 results in the formation of a ring-like heterotrimer that encircles the DNA double helix. While the crystal structure of this complex has yet to be solved, structural predictions using the primary amino acid sequence of these three proteins indicate similarity to the PCNA homotrimer, a ring-like complex that acts as a sliding clamp over DNA. In these modelling studies, the entire length of hRad1 and hHus1 are used but only the first 280 amino acids of hRad9 fit the predicted PCNA-like model (24). The SQ and [S/T]-P sites critical for hRad9 phosphorylation sites we have reported here are located at either the very end of this region (serine 272 and serine 277), or C-terminal to it (threonine 292, serine328, serine336, and threonine 355).
Since some data suggest that phosphorylation at these residues may be important for 9-1-1 assembly (18,21), the C-terminus of hRad9 may be acting as a regulatory domain for assembly of this complex. Alternatively, the attachment of these phosphates could influence some other aspect of hRad9 function, such as its proapoptotic role.
While we have now identified two distinct forms of damage dependent hRad9 phosphorylation, mapped at least four sites of constitutive phosphorylation, and characterized the interdependence of these events, many questions remain regarding the nature and function of hRad9 phosphorylation. These include the location of the remaining constitutive sites of phosphorylation and the identification of proteins responsible for the addition and removal of phosphates. In addition, it remains unclear whether the cell cycle regulated phosphorylation of hRad9 occurs in G2 and persists through mitosis or whether these are two separate events. In any event, this is the first evidence linking the hRad9 protein to the G2/M transition, a transition in which the S.pombe Rad9 protein plays an instrumental regulatory role. Furthermore, the 23 discovery that hRad9 undergoes at least two distinct phosphorylation events in response to IR raises other interesting questions. It is known hRad9's hyperphosphorylation in response to IR occurs concurrently with its association with chromatin (29), and though it has yet to be shown directly, there is likely an interdependence between these two events. Which IR-induced phosphorylation of hRad9 coincides with its association with DNA is currently an unresolved issue. 28 fractionated by SDS-PAGE (8%) and immunoblotted with antibodies directed against hRad9. (B) hRad9 protein was immunoprecipitated from P3A, P4A, P5A, P6A, P9A, and empty vector transfected HeLa cells that were metabolically labelled with inorganic 32 P for 18 hours prior to lysis. A portion of each immunoprecipitated, radiolabelled protein was size fractionated by SDS-PAGE, transferred to nitrocellulose, and immunoblotted with antibodies against hRad9 (left panel). A second, identical gel was dried and exposed to a phosphoimager screen for 3 days (right panel). (C) Quantification of the 32 P signal from the right panel of Figure 2b as determined by the Image Quant software program. Values were normalized 32 P background in each lane and to the chemiluminescence signal from the left panel of Figure 2b.  HeLa cells were synchronized in early S-phase using a double thymidine block, released and harvested 0, 2, 6, 7, 8, 9, 10, and 12 hours later. Cells from each time point were either lysed, size-fractionated by SDS-PAGE (10%) and immunoblotted with antibodies against hRad9 (top), or stained with propidium iodide to measure DNA content prior to analysis by flow cytometry (bottom). (B) Sphase and G2/M HeLa cells, harvested 2 and 8 hours after release from a double thymidine block respectively, were lysed and the hRad9 immunoprecipitated. hRad9 protein from each time point was then treated with CIP in the presence or absence of the phosphatase inhibitor ∃-glycerophosphate (∃-GP). Proteins were separated by SDS-PAGE (10%) and immunoblotted as above. (C) Late S-phase HeLa cells, generated from a single thymidine block and release, were left untreated (-), treated with 4 Gray of IR to delay cells in G2 (+ 4 Gy), or 1.7 :g/ml of the microtubule inhibitor demicolcine (+ DC) to arrest cells in mitosis. Cells were harvested 7, 8, 9, and 10 hours after thymidine release (1.5, 2.5, 3.5, and 4.5 hours after IR and demicolcine administration) and used for immunoblotting (top) and flow cytometry (bottom) as in A.