TLK1B mediated phosphorylation of Rad9 regulates its nuclear/cytoplasmic localization and cell cycle checkpoint
© Awate and De Benedetti. 2016
Received: 5 October 2015
Accepted: 26 January 2016
Published: 9 February 2016
The Tousled like kinase 1B (TLK1B) is critical for DNA repair and survival of cells. Upon DNA damage, Chk1 phosphorylates TLK1B at S457 leading to its transient inhibition. Once TLK1B regains its kinase activity it phosphorylates Rad9 at S328. In this work we investigated the significance of this mechanism by overexpressing mutant TLK1B in which the inhibitory phosphorylation site was eliminated.
Results and discussion
These cells expressing TLK1B resistant to DNA damage showed constitutive phosphorylation of Rad9 S328 that occurred even in the presence of hydroxyurea (HU), and this resulted in a delayed checkpoint recovery. One possible explanation was that premature phosphorylation of Rad9 caused its dissociation from 9-1-1 at stalled replication forks, resulting in their collapse and prolonged activation of the S-phase checkpoint. We found that phosphorylation of Rad9 at S328 results in its dissociation from chromatin and redistribution to the cytoplasm. This results in double stranded breaks formation with concomitant activation of ATM and phosphorylation of H2AX. Furthermore, a Rad9 (S328D) phosphomimic mutant was exclusively localized to the cytoplasm and not the chromatin. Another Rad9 phosphomimic mutant (T355D), which is also a site phosphorylated by TLK1, localized normally. In cells expressing the mutant TLK1B treated with HU, Rad9 association with Hus1 and WRN was greatly reduced, suggesting again that its phosphorylation causes its premature release from stalled forks.
We propose that normally, the inactivation of TLK1B following replication arrest and genotoxic stress functions to allow the retention of 9-1-1 at the sites of damage or stalled forks. Following reactivation of TLK1B, whose synthesis is concomitantly induced by genotoxins, Rad9 is hyperphosphorylated at S328, resulting in its dissociation and inactivation of the checkpoint that occurs once repair is complete.
The Tousled (Tsl) gene was first identified in the plant Arabidopsis thaliana. Recessive Tsl mutants show defects in leaf and flower development . This was proposed to be linked to a replicative defect during organogenesis, but it may also result from failure to protect the genome from DNA damage [2–4], resulting in developmental aberrations [5, 6]. Animal homologs of Tousled, known as Tousled like kinases (TLKs), are found from Caenorhabditis elegans to mammals. They are generally considered as genes of metazoans and are not found in yeast, although they are present in unicellular trypanosomes . In mammals their activity is cell cycle regulated with maximal activity found in the S-phase. After many years of study, only a few direct “interacting” substrates of TLKs have been identified, namely the histone chaperone Asf1 , histone H3 , Rad9 , and Aurora B kinase . As evident from their substrates, TLKs play a major role in chromatin assembly [10, 11], transcription [4, 12], DNA repair [3, 10, 13], and condensation of chromosomes at mitosis [5, 6]. In humans two structurally similar TLK genes (TLK1 and TLK2) with several splice variants have been identified. A splice variant of TLK1, TLK1B that lacks the first 237 amino acids was identified in our lab. TLK1 and TLK1B interact with similar substrates, are believed to have similar enzymatic functions and are often referred to as TLK1/1B. Our previous studies have shown that translation of TLK1B is induced by DNA damage through the activation of the mTOR-eIF4E pathway. We have shown that elevated expression of TLK1B promotes cell survival after irradiation (IR) or doxorubicin  and UV  by facilitating DNA repair and promoting chromatin assembly after repair. Expression of a dominant-negative mutant of TLK1B renders mammalian cells sensitive to IR . Thus, the human homolog, TLK1B, has invoked interest because of its established role in cell survival after DNA damage [3, 9, 13]. Identification of Rad9 as a substrate for TLK1/1B attributes a direct role of TLK1/1B in DNA repair . Our previous work suggests that TLK1/1B’s chaperone activity, independent of its kinase activity, helps in the recruitment of Rad9 at the break site. We had previously shown some evidence that TLK1/1B kinase activity is important for the dissociation of Rad9-Rad1-Hus1 (9-1-1) complex from a double stranded break (DSB) .
Rad9 plays a major role in DNA repair, cell cycle checkpoint and apoptosis. Aberrant Rad9 expression has been linked to breast, lung, thyroid, skin and prostate tumorigenesis . Rad9 is a part of 9-1-1 heterotrimeric complex which is required for activation of ATR. Rad9, Rad1 or Hus1 KO mice are embryonic lethal [16, 17]. Loss of Rad9 produces a defect in ATR signaling and increases the sensitivity of the cells towards genotoxic stress . In response to replication stress RPA directs the clamp loader RAD17–replication factor C (RFC) to load the 9-1-1 complex at the 5′ end of the double strand-single strand DNA junctions [19, 20]. Chromatin-bound 9-1-1 complex acts as a scaffold for the recruitment of various DNA repair proteins and polymerases at the DNA damage break site. It ensures filling of gaps and efficient repair of DNA [21, 22]. Recently it has been shown that 9-1-1 complex is required for the recruitment of WRN protein at stalled replication forks and this interaction is important for the fork recovery . WRN belongs to the RecQ family of DNA helicases. Loss of WRN gives rise to a genetic disease known as Werner syndrome (WS). It is characterized by pre-mature ageing and pre-disposition to cancer [24, 25]. Cells derived from the WS patients show a prolonged S-phase, a reduced life-span, and an increase in genomic instability [26, 27]. It has been shown that WRN stabilizes the stalled replication forks and the loss of WRN leads to the fork collapse and increase in DSBs that are repaired through recombination . WRN interacts with the 9-1-1 complex to maintain genomic stability by preventing accumulation of DSBs at the damaged forks .
Activation of the DNA damage induced checkpoint mediates rapid and transient inhibition of TLK activity. This transient inhibition in response to DNA damage requires ATM and Chk1 function. Chk1 directly phosphorylates TLK1 at S695 which is equivalent to S457 of TLK1B . Once TLK1/1B regains its kinase activity it phosphorylates Rad9 at S328  and T355 . Rad9 S328 phosphorylation follows the pattern of TLK1/1B activity wherein it is inhibited immediately after DNA damage and gets phosphorylated when TLK1/1B regains its activity [14, 31]. The reason for this transient inactivation of TLK1/1B still remains a question as there is a lack of direct evidence for the role of this inhibitory phosphorylation with regards to its effect on Rad9 and ATR mediated cell signaling. In order to answer this question we have made an S457A mutant (Mut) of TLK1B that lacks the inhibitory phosphorylation site. In this study we show that Mut TLK1B remains active in the presence of DNA damage, and cells overexpressing it display an altered cell cycle checkpoint and delay in cell cycle progression upon recovery from hydroxyurea (HU). Mut TLK1B overexpressing cells show an increase in phosphorylation of Rad9 at S328. In response to HU mediated replication arrest, Mut TLK1B overexpressing cells show a massive reduction in the formation of Rad9 foci and reduced association of Rad9 with the chromatin. Mut TLK1B expressing cells show reduced association of Rad9 with Hus1 and WRN suggesting an early dissociation of 9-1-1 complex. In response to HU, these cells show an increased accumulation of DSBs which are marked by an increase in association of p-ATM (S1981) and γ-H2AX with the chromatin. Cellular fractionation data of Mut TLK1B overexpressing cells showed an increase in the accumulation of total Rad9 and p-Rad9 (S328) in the cytoplasm. Furthermore, studies with Rad9 S328D phosphomimetic mutant suggest that the phosphorylation of Rad9 at S328 alone is sufficient to prevent Rad9 localization into the nucleus. Our results indicate that in response to replication arrest, transient inhibition of TLK1/1B is crucial to maintain localization of Rad9 into the nucleus at damage sites.
Mut TLK1B increases phosphorylation of Rad9 at S328 both in presence and absence of DNA damage
Cells expressing Mut TLK1B show defects in formation of Rad9 foci
Cells expressing Mut TLK1B show reduction in association of Rad9 with the chromatin
Cells expressing Mut TLK1B show an increased accumulation of Rad9 and p-Rad9 S328 in the cytoplasmic fraction
Phosphomimetic Rad9 S328D mutant is sufficient to accumulate Rad9 in the cytoplasm
We next wanted to examine if phosphorylation of Rad9 at S328 is sufficient to alter the nuclear localization of Rad9. In order to do so we generated a phosphomimetic flag-tagged Rad9 S328D mutant (as described in “Methods” section). Fractionation was performed on the cells overexpressing the flag-tagged Rad9 S328D mutant and flag-tagged Wt Rad9. These cells were incubated with or without HU. After fractionating the cytoplasmic, soluble nuclear and chromatin bound proteins the distribution of Rad9 was examined by immunoblotting with either anti-flag antibody to specifically detect overexpressed flag-tagged Wt or Mut Rad9 (Fig. 4b) or anti-Rad9 antibody to detect both the endogenous and overexpressed Rad9 (Fig. 4c). Immunoblotting with anti-flag antibody showed that the overexpressed Rad9 S328D mutant was localized exclusively in the cytoplasm while the overexpressed Wt Rad9 was present in all the three fractions (Fig. 4b). These results confirm that the phosphorylation of Rad9 at S328 is sufficient to accumulate Rad9 in the cytoplasm. As previously mentioned multiple Rad9 bands appear in the chromatin fraction due to phosphorylation of Rad9 at multiple sites. Cellular fractionation results were confirmed by immunolocalization (as described in “Methods” section), which showed that the flag-tagged Rad9 (S328D) localized to the cytoplasm whereas the Wt Rad9 was nuclear (Fig. 4d).
Phosphomimic Rad9-T355D does not affect its nuclear/cytoplasmic localization
Scott Davey`s group has recently found Rad9 T355 as another phosphorylation site of TLK1 . Interestingly T355 lies next to nuclear localization sequence (NLS), which starts at the residue 356 and ends at 364. Bioinformatics analysis with cNLS mapper predicted that Rad9 is localized exclusively in the nucleus, and T355D mutation in Rad9 would cause a partial cytoplasmic accumulation. Our cellular fractionation data of Mut TLK1B overexpressing cells showed an increase in the accumulation of total p-Rad9 (T355) in the cytoplasm but it was unclear if this simply reflected the total Rad9 level, i.e., phosphorylated also at S328 (Additional file 2: Figure S2A). We next wanted to examine if phosphorylation of Rad9 at T355 can alter the nuclear localization of Rad9. In order to do so we generated a phosphomimetic flag-tagged T355D mutant. The Rad9 T355D mutant was also found in the chromatin and nucleoplasmic fractions, although the chromatin to cytoplasmic ratio for the T355D mutant was not identical to the wt Rad9 (Additional file 2: Figure S2B).
Mut TLK1B expressing cells show reduced association of Rad9 with Hus1 and WRN
We next wanted to examine if the overexpressed Rad9 S328D mutant (flag tagged) can interact with WRN and Hus1 since this association should occur largely on chromatin. Immunoprecipitation of the overexpressed Rad9 S328D mutant using flag antibody showed that the interaction of mutant Rad9 with WRN and Hus1 was greatly impaired, while the overexpressed wt Rad9 was able to bind normally (Fig. 5e, f). In this case, the addition of HU did not increase the association of wt Rad9 with WRN and Hus1 probably due to the overexpression of the protein.
Mut TLK1B expressing cells show increased amount of DNA damage
HU treatment of Mut TLK1B expressing cells show a delay in cell cycle progression and recovery
Mut TLK1B expressing cells display an altered checkpoint control
Tousled Like kinases (TLK) are serine/threonine kinases that play an important role in DNA repair. TLK overexpression is observed in multiple cancers and often corresponds to reduced sensitivity towards radiotherapy or chemotherapy due to the efficient repair in those tumors [32, 55]. Our lab identified TLK1B, which is a splice variant of TLK1 gene from a library of mRNAs that are translationally upregulated by overexpression of translation initiation factor 4E. TLK1B is known to protect cells from genotoxic stress and is translationally upregulated in response to stress and DNA damage via mTOR-eIF4E pathway [13, 56]. At the same time, genotoxic stress leads to Chk1 mediated transient inactivation of TLK . TLK1B promotes repair of damaged DNA in cooperation with Rad9 by facilitating the assembly of repair proteins to the sites of DNA damage. TLKs are the only kinases that phosphorylate Rad9 at S328. Rad9 is aberrantly expressed in prostate, breast, thyroid, skin, lung, and gastric cancers [57, 58]. It plays a major role in cell cycle checkpoint and DNA damage repair. It is essential for genomic stability as frequent chromosomal breakage is observed in cells in which both the Rad9 alleles are inactivated [59, 60]. Rad9, a member of PCNA-like 9-1-1 complex, contains 110 amino acid long C-terminal region that does not share homology with PCNA. This C-terminal region of Rad9 is extensively modified by phosphorylation. Some residues are constitutively phosphorylated while some are transiently phosphorylated in response to DNA damage and cell cycle position [36, 61]. In response to DNA damage, once TLK1B regains its kinase activity it transiently phosphorylates Rad9 at S328. In this study we wanted to elucidate the significance of this phosphorylation.
Our collective work has demonstrated that TLK1B acts as a chaperone for Rad9. In the absence of TLK1 kinase activity, i.e., in presence of DNA damage or when a kinase-dead protein is used, we showed that TLK1B promoted the association of Rad9 with a DSB and presumably also SSB. Once the damage is repaired, the kinase activity recovers and then TLK1B phosphorylates Rad9 at S328, promoting its dissociation from 9-1-1 and export to the cytoplasm, thereby mediating the deactivation of the DDR. In conclusion transient inhibition of the kinase after DNA damage is crucial in retaining 9-1-1 at damage sites until repair is complete, and mutations in the TLK1 gene which can activate the kinase may potentially cause accumulation of DSBs.
Hydroxyurea (Catalog No. H8627), doxorubicin hydrochloride (Catalog No. D1515), thioridazine hydrochloride (Catalog No. T9025), KU-55933 (Catalog No SML1109) and G418 disulfate salt (Catalog No. A1720) were purchased from Sigma. Dulbecco’s modified Eagle’s medium (DMEM) was obtained from Life Technologies (Catalog No. 12100-046), fetal bovine serum (FBS) was obtained from Atlanta Biologicals (Catalog No. 900108). Enhanced chemiluminescence solution was obtained from Thermo-Scientific (Catalog number No. 32106).
Antibodies used in this study were: rabbit α-Actin (Ab1801, Abcam), rabbit α-Rad9 phospho-328 (AP3225a, Abgent), rabbit α-Chk1 phospho-317 (AP3070a, Abgent), rabbit α-Chk1 phospho-345 (sc-17922, Santa Cruz Biotechnology), goat α-Rad9 (sc-10465, Santa Cruz Biotechnology), mouse α-Rad9 (sc-8324, Santa Cruz Biotechnology), donkey α-goat IgG-HRP (sc-2020, Santa Cruz Biotechnology), rabbit α-TLK1 phospho-695 (4121S, Cell Signaling), α-rabbit IgG-HRP (7074S, Cell Signaling), α-mouse IgG-HRP (7076, Cell Signaling), rabbit α-TLK1 (GTX102891, GeneTex), mouse α- H2A.X phospho-139 (05-636, Millipore), mouse α-Flag (F1804, Sigma) and Rabbit α-Flag (F7425, Sigma).
HEK293 cells, obtained from ATCC repository, were maintained in DMEM supplemented with 10 % FBS and 1 % penicillin–streptomycin at 37 °C in a humidified incubator with 5 % CO2. For ATM inhibitor experiment, cells were pretreated with 10 µM KU-55933 for 2 h. 2 mM HU was then added into the media for 16 h. For inhibition of TLK cells were pretreated with 10 µM Thioridazine hydrochloride (THD) for 2 h before addition of 2 mM HU.
Cell cycle analysis
HEK293 cells were seeded in T-25 flasks at a density of 2 × 105 cells/flask. Cells were treated with 2 mM HU for 16 h. They were briefly washed with 1× PBS and fresh media was added to the cells. Cells were allowed to recover for indicated time points. Cells were washed with 1× PBS and trypsinized. Cell suspensions were centrifuged at 1000 rpm for 5 min, and pellets were fixed with ethanol and stained with 50 μg/ml propidium iodide (Sigma, Catalog No. P4170). Percentages of cells within each of the cell cycle compartments (G0/G1, S, or G2/M) were determined using a FACS Calibur flow cytometer (Becton–Dickinson).
To isolate chromatin, ~107cells cells were resuspended in buffer A (10 mM HEPES, [pH 7.9], 10 mM KCl, 1.5 mM MgCl2, 0.34 M sucrose, 10 % glycerol and 1 mM DTT) supplemented with halt EDTA free protease and phosphatase inhibitors (Life Technologies, Catalog No. 78441). Triton X-100 (0.1 %) was added, and the cells were incubated for 5 min on ice. Cytosolic proteins were separated from nuclei by centrifugation (4 min, 1300×g). Nuclei were washed once in solution A, and then lysed in solution B (3 mM EDTA, 0.2 mM EGTA and 1 mM dithiothreitol) supplemented with halt EDTA free protease and phosphatase inhibitors for 30 min. Insoluble chromatin was then separated from soluble nuclear proteins by centrifugation (4 min, 1700×g), washed once in solution B, and collected by centrifugation (1 min, 10,000×g). The final chromatin pellet was resuspended in SDS sample buffer. Samples were sonicated for 15 s. Aliquots of each fraction were separated on sodium dodecyl sulfate–polyacrylamide (SDS-PAGE) gels and blotted onto polyvinylidene difluoride (PVDF) membranes.
Western blot analysis
Cells were lysed in 1X SDS sample buffer. Lysates were sonicated for 15 s and heated at 100 °C for 5 min. Proteins were separated on 6–12 % SDS-PAGE gels and transferred to PVDF membranes (Millipore). Membranes were incubated with PBS containing 0.05 % Tween 20 and 5 % non-fat dry milk to block non-specific binding and were incubated with primary antibodies; membranes were then incubated with appropriate secondary antibodies conjugated to horseradish peroxidase. Immunoreactive bands were visualized using chemiluminescence reagent.
Construction of mammalian expression vectors and generation of stable cell lines
We had cDNA of the human TLK1B cloned into the BK-shuttle vector . To sub-clone TLK1B into pIRES2 vector the TLK1B ORF was amplified by PCR with the primers: 5′-GTACCGGAATTCAAAATTATTCAGACTGATCTC-3′; 5′-TAATTAGGATCCTGGAGGAAAGTCAGTAAGTAATTA-3′, containing an EcoRI and a BamHI tail respectively. The TLK1B PCR product was sub-cloned in the plasmid pIRES2-EGFP, which was cut with the same enzymes. TLK1B S457A mutant was generated from pIRES2-EGFP using the QuikChange Site-Directed Mutagenesis Kit (Stratagene, Catalog No. 200518) with the following primer: 5′-GAGAAGATCAAATGCTTCAGGAAACCTACAC-3′.
The PCR product was transformed in bacteria, and the presence of the nucleotide substitution was confirmed by DNA sequencing. To generate stable cell lines, HEK293 cells were transfected using Lipofectamine 3000 (Life Technologies, L3000001) as per the manufacturer’s protocol, and stably transfected cells were selected with G418 (500 μg/ml). The pIRES2-EGFP permits the translation of both the TLK1B gene cloned into the multiple cloning site and EGFP from the single bicistronic mRNA. After 30 days of selection in G418, cells expressing high levels of GFP were sorted by flow-cytometry.
We had cDNA of the human Flag-tagged Rad9 cloned into an episomal pREP10 vector. Stable expression of this vector requires expression of full length EBNA-1. To generate stable cell lines we transfected HEK293 c-18 cells (ATCC CRL-10852) which stably express full length EBNA-1. Under G418 selection HEK293 c-18 cells stably express EBNA-1 which is required to maintain pREP10 vector episomally. Maintenance of pREP10 vector requires hygromycin selection.
Rad9 S328D and T355D mutant was generated using the QuikChange Site-Directed Mutagenesis Kit (Stratagene, Catalog No. 200518) with the following primers: 5′-CTGCCCTCCATTTCCCTTGACCCTGGCCCCCAG-3′ (Rad9 S328D)5′- CAGTACAGTGCCTGGGGATCCCCCACCCAAGAAGTTC-3′ (Rad9 T355D).
The PCR product was transformed in bacteria, and the presence of the nucleotide substitutions were confirmed by DNA sequencing. To generate stable cell lines, HEK293 c-18 cells were transfected using Lipofectamine 3000 as per the manufacturer’s protocol, and cells were selected under G418 (250 μg/ml) and hygromycin (50 μg/ml) challenge for 30 days.
Cells were grown on culture slides. To remove soluble proteins and fix chromatin-bound proteins, cells were pre-extracted in buffer1 (1 % Triton X-100, 10 mM HEPES pH 7.4, 10 mM NaCl and 3 mM MgCl2) supplemented with halt EDTA free protease and phosphatase inhibitors for 5 min at 4 °C. Cells were then fixed in 4 % paraformaldehyde for 10 min at 4 °C and then treated in buffer 2 (0.5 % Triton X-100, 20 mM HEPES pH 7.4, 50 mM NaCl, 3 mM MgCl2 and 300 mM sucrose) supplemented with halt EDTA free protease and phosphatase inhibitors for 5 min at 4 °C. Cells were then blocked for 1 h in SuperBlock solution.
To look at Rad9 cellular localization cells were fixed with methanol and then rehydrated with PBS and blocked for 1 h in SuperBlock solution. Staining with primary antibody was performed overnight at 4 °C in blocking solution, whereas species specific fluorescein/Texas red conjugated secondary antibody (Vector Labs) was applied for 1 h at RT, followed by counterstaining with DAPI. All the primary antibodies were used at a 1:250 dilution, whereas the secondary antibodies were employed at a 1:500 dilution. Fluorescence images were captured using a Zeiss Axioskop 2 microscope.
For co-immunoprecipitation (CoIP) experiment, cells were lysed in the lysis buffer (1 % Triton X-100, 0.5 % Na-Doxycholate, 150 mM NaCl, 2.5 mM MgCl2, 1 mM EGTA, 1 mM EDTA, 20 mM Tris/HCl pH 8.0) supplemented with halt EDTA free protease and phosphatase inhibitors. Cell lysates were sonicated for 20 cycles using Diagenode Bioruptor and then incubated with 250 U of benzonase. In total 1.5 mg of cell lysate was precleared with protein A/G Sepharose beads and then incubated overnight at 4 °C with rabbit polyclonal anti-Rad9 (4 μg; Santa Cruz Biotech) or anti-Rabbit IgG, and tumbled with 70 μl of protein A/G Sepharose beads for 4 h at 4 °C. After extensive washing in CoIP buffer, proteins were eluted by boiling treatment in 2X electrophoresis sample buffer prior to Western blotting analysis.
Neutral comet assay was performed using comet assay kit from TREVIGEN (Catalog# 4250-050-K) following manufacturer’s instructions. DNA was stained with SYBR Gold and fluorescence images were captured using a Zeiss Axioskop 2 microscope. Tail moments were quantified for each cell using ImageJ OpenComet plugin. 50 comet images were measured for each treatment.
Statistical analysis of data was done using one way analysis of variance (ANOVA) with Sigma Stat statistical software. A p value of 0.05 or less was considered significant.
SA and ADB designed the experiments. SA performed the experiments. SA and ADB analyzed the data and wrote the manuscript. Both authors read and approved the final manuscript.
This work was supported by a Grant from the Feist Weiller Cancer Center from LSUHSC, Shreveport. We want to thank Dr. Scott Davey for the kind gift of p-Rad9(T355) antibody. We wish to thank Dr. Abhijit Rath for technical help.
Availability of data and materials
Materials used in this work are available in limited quantities upon request. Raw data images for the microscopy work will be available upon request.
The authors declare that they have no conflict of interests.
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