Mapping of the interacting interfaces on SIRT1 and HIPK2

To characterise the interaction of SIRT1 and HIPK2 more detailed, we performed in vitro GST pulldown assays. To this end, we expressed a GST-SIRT1 fusion protein in E.coli, purified the protein and incubated it with in vitro ³⁵S-labelled HIPK2. ³⁵S-labelled HIPK2 was pulled-down with GST-SIRT1 but not with GST alone (Figure 1c), indicating that SIRT1 and HIPK2 interact in vitro, presumably in a direct fashion.

To map the HIPK2-interacting domain of SIRT1, we generated non-overlapping GST-SIRT1 deletions spanning the regulatory N-terminus (amino acids 1–260), the catalytic SIR domain (aa 261–447) and the regulatory C-terminus (aa 448–747) and performed GST pulldown assays. Our results revealed clear interaction of HIPK2 with the central, catalytic SIR domain (Figure 1d). No interaction of HIPK2 with the C-terminus and only faint interaction with the N-terminal regulatory domain of SIRT1 were detectable. These findings are further supported by co-immunoprecipitation analyses using ectopically expressed Flag-HIPK2 and GFP-SIRT1 truncation mutants (Figure 1e).

To define the SIRT1-interacting interface on HIPK2 we used GST-HIPK2 deletions and performed GST pulldown assays with ³⁵S-labelled SIRT1. The results indicate that both the extreme N-terminus (aa 1–188) and the kinase domain (aa 189–520) of HIPK2 mediate interaction with SIRT1 (Figure 1f). To substantiate these findings we performed coimmunoprecipitation analyses using truncated Flag-HIPK2^1–553 and Flag-HIPK2^551–1191 along with full-length GFP-SIRT1. Weak binding between HIPK2^1–553 and GFP-SIRT1 was observed in the absence of DNA damage, and the interaction was enhanced after DNA damage (Figure 1g). No interaction between GFP-SIRT1 and HIPK2^551–1191 was observed (Figure 1g). In summary, our results indicate that SIRT1 binds via its central SIR domain to the N-terminus and the kinase domain of HIPK2 (Figure 1h).

HIPK2 phosphorylates SIRT1 in vitro

Since HIPK2 is a Ser/Thr-kinase, we next investigated whether SIRT1 is a HIPK2 substrate. To this end, we performed in vitro kinase assays by incubating bacterially expressed, purified GST-SIRT1 and His-HIPK2 proteins. Indeed, SIRT1 was phosphorylated by wild-type His-HIPK2 but not by a kinase-deficient His-HIPK2^K221A point mutant, demonstrating direct phosphorylation of SIRT1 by HIPK2 (Figure 2a).

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Figure 2

HIPK2 phosphorylates SIRT1 in vitro and in cellular context. (a) HIPK2 directly phosphorylates SIRT1. Recombinant 6xHis-HIPK2, kinase-deficient 6xHis-HIPK2^K221A and GST-SIRT1 were purified from E. coli. In vitro kinase assays were performed and SIRT1 phosphorylation as well as HIPK2 autophosphorylation was examined by SDS–PAGE and autoradiography. Protein levels were analysed by either Coomassie Brilliant Blue staining or immunoblotting. (b) HIPK2 phosphorylates SIRT1 at Ser27 and Ser682 in vitro. GST-SIRT1 wild type, SIRT1 S27A, SIRT1 S682A and the double mutant SIRT1 S27A, S682A were incubated with a truncated, catalytically active GST-HIPK2^1–553. In vitro kinase assay was analysed by SDS–PAGE and autoradiography. Protein levels were analysed by Coomassie Brilliant Blue staining. The ratio (amount pSIRT1)/(amount pHIPK2) was quantified by densitometry using the ImageJ software. (c) Recombinant GST-SIRT1 and 6xHis-HIPK2 purified from E.coli were subjected to an in vitro kinase reaction. GST-SIRT1 phosphorylation was analysed by immunoblotting using the indicated antibodies. (d) U2OS cells were retrovirally transduced with a SIRT1-specific shRNA or a control shRNA. Depletion of SIRT1 was examined by immunoblot analysis using the indicated antibodies. The expression of GFP protein was used as a control for the transduction efficiency. (e) HIPK2 phosphorylates SIRT1 in cells. HA-HIPK2 wild type or kinase-deficient point mutant HA-HIPK2^K221A constructs were expressed along with Flag-SIRT1 in 293T cells. Twenty-four hours after transfection cell lysates were analysed by immunoblotting using the indicated antibodies

To map the phosphorylation site(s) we used truncated GST-SIRT1 proteins and GST-SIRT1 proteins where we had exchanged Ser/Thr residues at potential HIPK2 target sites (Ser-Pro/Thr-Pro sites) to Ala. This approach identified two sites on SIRT1, Ser27 and Ser682, to be phosphorylated by HIPK2 (Supplementary Figure S1a and b).

Next, the phosphorylation sites were validated in the SIRT1 full-length context in vitro. Whereas single mutation of Ser27 or Ser682 moderately reduced SIRT1 phosphorylation, simultaneous mutation of both sites led to a profound reduction of SIRT1 phosphorylation by HIPK2 (Figure 2b), identifying these sites as major phosphorylation sites on SIRT1. Database analysis revealed a high degree of conservation of Ser682, in contrast to Ser27, which was found to be less conserved in evolution (Supplementary Figure S1c).

Phosphorylation of SIRT1 at Ser682 by HIPK2

Next, we raised a phosphorylation-specific antibody against the Ser682 phospho-site of SIRT1 and affinity-purified it using the phospho-peptide. Immunoblot analysis showed that the antibody specifically recognized GST-SIRT1 phosphorylated by HIPK2, but not unphosphorylated SIRT1, validating its specificity towards Ser682-phosphorylated SIRT1 (Figure 2c). Furthermore, depletion of SIRT1 in U2OS cells by SIRT1-specific RNA interference abolished the signal of the SIRT1 pSer682 antibody (Figure 2d), indicating its specificity to SIRT1. Together, these results show that the antibody specifically recognizes Ser682-phosphorylated SIRT1.

We next studied Ser27 and Ser682 phosphorylation of ectopically expressed SIRT1. Immunoblot analysis using a commercially available phospho-specific SIRT1 pSer27 SIRT1 antibody and our SIRT1 pSer682 antibody revealed that phosphorylation of SIRT1 at Ser27 and Ser682 is stimulated by wild-type HIPK2 but not by its kinase-deficient form (Figure 2e), indicating that HIPK2 can phosphorylate both sites in the cellular context.

HIPK2 phosphorylates SIRT1 at Ser682 after DNA damage

To determine whether endogenous SIRT1 is phosphorylated at Ser27 and/or Ser682 in after DNA damage, we treated U2OS cells with 0.75μg/ml Adriamycin and analysed the lysates by immunoblotting. Although immunoblotting indicated weak background phosphorylation of SIRT1 at Ser682 in unstressed cells, a robust increase in SIRT1 Ser682 phosphorylation was observed upon Adriamycin treatment (Figure 3a). Furthermore, SIRT1 Ser682 phosphorylation correlated with HIPK2 stabilization, p53 Ser46 phosphorylation and acetylation of p53 at Lys382 (Figure 3a). Unexpectedly, we observed no increase in Ser27 phosphorylation of SIRT1 after DNA damage (Figure 3a). Thus, we focused our analyses on the regulation and function of SIRT1 Ser682 phosphorylation.

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Figure 3

Phosphorylation of SIRT1 at Ser682 by HIPK2 upon DNA damage. (a) SIRT1 Ser682 phosphorylation is linked to DNA damage. U2OS cells were treated with 0.75μg/ml of Adriamycin for the indicated time points. Total cell lysates were analysed by immunoblotting using the indicated antibodies. (b) SIRT1 Ser682 phosphorylation depends on HIPK2. U2OS cells were transfected with HIPK2-targeted siRNA. Twenty-fourhours post-transfection cells were treated with Adriamycin (0.75μg/ml) for 24hours or left untreated. Total cell lysates were analysed by immunoblotting using the indicated antibodies. (c) U2OS cells were treated with sublethal (0.1μg/ml) and lethal (0.75μg/ml) doses of Adriamycin for the indicated time points. Total cell lysates were analysed by immunoblotting using the indicated antibodies. (d) SIRT1 Ser682 phosphorylation is linked to lethal DNA damage. U2OS cells were treated with sublethal (0.1μg/ml) and lethal (0.75μg/ml) doses of Adriamycin for 24hours. Total cell lysates were analysed by immunoblotting using the indicated antibodies

To investigate whether HIPK2 is essential for SIRT1 Ser682 phosphorylation upon DNA damage, we depleted endogenous HIPK2 by RNA interference. Whereas control siRNA-transfected cells showed HIPK2 stabilization and concomittant Ser682 phosphorylation of SIRT1 upon DNA damage (Figure 3b), HIPK2-depleted cells showed a strong reduction in SIRT1 Ser682 phosphorylation (Figure 3b), demonstrating that HIPK2 is essential for SIRT1 Ser682 phosphorylation after DNA damage.

Phosphorylation of SIRT1 at Ser682 is linked to lethal DNA damage

We next studied whether SIRT1 is differentially phosphorylated at Ser682 after sublethal vs lethal DNA damage, similar to the p53 Ser46 phosphorylation mark, a HIPK2 target residue linked to lethal damage.^{38, 39, 49} To this end, we treated U2OS cells with 0.1μg/ml and 0.75μg/ml Adriamycin, respectively. FACS-based analysis in combination with immunoblotting for the cell cycle inhibitor p21 (which is downregulated after lethal damage⁵⁰) and caspase-cleaved PARP confirmed 0.1μg/ml Adriamycin as sublethal and 0.75μg/ml Adriamycin as lethal dose (Figure 3c and Supplementary Figure S2). Interestingly, phosphorylation of SIRT1 at Ser682 was triggered in response to lethal DNA damage, but remained unresponsive to sublethal DNA damage (Figure 3d). In addition, phosphorylation of p53 at Ser46, an established marker for lethal DNA damage,⁴⁹ correlated with SIRT1 Ser682 phosphorylation and was specifically induced after lethal damage (Figure 3d). Phosphorylation of p53 at Ser15 by ATM was detectable both after sublethal and lethal damage (Figure 3d), in accordance with previous studies.^{37, 49} Together, our findings demonstrate that phosphorylation of SIRT1 at Ser682 is linked to lethal DNA damage.

DNA damage-induced phosphorylation of SIRT1 at Ser682 is linked to PML-NBs

Both HIPK2 and SIRT1 can be recruited to PML-NBs upon stress.^{39, 47} In addition, PML-NBs have an important role in regulating p53 posttranslational modifications and pro-apoptotic function.^{46, 51} To address a potential interplay between SIRT1 and HIPK2 at PML-NBs we performed immunofluorescence stainings. Confocal microscopy revealed that ectopic expression of PML IV, a PML isoform known to recruit HIPK2 to PML-NBs,³⁹ stimulated co-localization of SIRT1 and HIPK2 at PML-NBs (Figure 4a).

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Figure 4

Regulation of SIRT1 Ser682 phosphorylation at PML-NBs. (a) PML provokes colocalization of SIRT1 and HIPK2 at PML-NBs. GFP-SIRT1 (green), mCherry-HIPK2 (red) (upper panels) and PML IV (lower panels) were co-expressed in U2OS cells and analysed by immunofluorescence staining and confocal microscopy. DNA is stained by Hoechst33342 (blue). Scale bar, 10μm. (b) Endogenous SIRT1 and HIPK2 co-localize at PML-NBs upon DNA damage. U2OS cells were treated with adriamyin (0.75μg/ml) for 24h. Endogenous PML, HIPK2 and SIRT1 were stained using indirect immunofluorescence. Representative confocal images are shown. Scale bar, 5μm. (c) Phosphorylated SIRT1 localizes to PML-NBs. Flag-PML IV, SIRT1 and HA-HIPK2 were expressed in U2OS cells. pSer682 SIRT1 (anti-pSer682) and PML (anti-Flag) were examined by immunofluorescence staining and confocal microscopy. Scale bar, 10μm. DNA is visualized with Hoechst33342 (blue). Representative cells are shown. (d) Endogenous SIRT1 phosphorylated at Ser682 localizes upon DNA damage to PML-NBs. U2OS cells were treated with adriamyin (0.75μg/ml) for the indicated timepoints and analysed by indirect immunofluorescence staining. Representative confocal images are shown. Scale bar, 5μm. (e) Localization of phosphorylated SIRT1 to PML-NBs is dependent on HIPK2 expression. U2OS cells were transfected with HIPK2-targeted siRNA. Twenty-four hours post-transfection cells were treated with adriamycin (0.75μg/ml) for 6 and 24h or left untreated. Total cell lysates were analysed by immunoblotting using the indicated antibodies. In parallel, cells were analysed by indirect immunofluorescence staining. Representative confocal images are shown. Scale bar, 5μm. (f) Ectopic expression of PML potentiates SIRT1 phosphorylation at Ser682. U2OS cells were transfected with the indicated constructs and cell lysates were analysed by immunoblotting using the indicated antibodies. (g) Depletion of endogenous PML expression by RNA interference results in diminished SIRT1 Ser682 phosphorylation after DNA damage. U2OS cell were treated with the indicated siRNA and treated with Adriamycin (0.75μg/ml) for 24h. Total cell lysates were analysed by immunoblotting using the indicated antibodies

We next aimed to determine the subcellular localization of endogenous SIRT1, HIPK2 and PML proteins in the absence and presence of DNA damage. Confocal microscopy analyses indicated no substantial co-localization of SIRT1 and HIPK2 in unstressed cells (Figure 4b). However, after Adriamycin treatment, endogenous SIRT1 and HIPK2 co-localized at PML-NBs (Figure 4b), suggesting that HIPK2-mediated SIRT1 phosphorylation takes place in association with PML-NBs. Indeed, confocal microscopy indicated that Ser682-phosphorylated SIRT1 localizes to PML-NBs upon overexpression of PML IV (Figure 4c). Furthermore, also a fraction of endogenous SIRT1 phosphorylated at Ser682 localized to PML-NBs after DNA damage (Figure 4d), which is in line with the hypothesis that SIRT1 Ser682 phosphorylation takes place at PML-NBs.

To determine whether SIRT1 Ser682 phosphorylation at PML-NBs depends on HIPK2, we depleted endogenous HIPK2 by RNA interference and performed confocal microscopy. Robust reduction of the pSer682 SIRT1 signal at PML-NBs was observed upon HIPK2 depletion (Figure 4e), indicating that HIPK2 is essential for SIRT1 Ser682 phosphorylation at PML-NBs. Together, these findings support the conclusion that HIPK2 phosphorylates SIRT1 in association with PML-NBs.

We next analysed the role of PML in HIPK2-mediated SIRT1 phosphorylation. Interestingly, ectopic expression of PML IV substantially potentiated HIPK2-mediated SIRT1 Ser682 phosphorylation (Figure 4f). Accordingly, PML IV expression also enhanced HIPK2-SIRT1 interaction (Supplementary Figure S3).

We also depleted endogenous PML by RNA interference and analysed the effect on SIRT1 Ser682 phosphorylation. PML depletion resulted in a massive reduction of SIRT1 Ser682 phosphorylation after DNA damage (Figure 4g), demonstrating an essential role of PML in HIPK2-mediated SIRT1 phosphorylation. PML depletion also led to reduced p53 Lys382 acetylation (Figure 4g), which is in line with previous reports.^{44, 48} Collectively, our findings unveil an essential role of PML in HIPK2-mediated SIRT1 Ser682 phosphorylation.

Ser682 phosphorylation inhibits SIRT1 activity and facilitates efficient p53 acetylation

To gain insight in the functional consequences of HIPK2-mediated SIRT1 phosphorylation, we analysed p53 acetylation, which is well-established to be negatively regulated by SIRT1.^{7, 13} First, we determined the relevance of endogenous HIPK2 expression in DNA damage-induced p53 Lys373/382 acetylation. To this end, we depleted HIPK2 by RNAi and subsequently treated the cells with a lethal dose of Adriamycin. HIPK2-depletion resulted in a massive reduction in DNA damage-induced p53 Lys382 acetylation (Figure 5a), in line with a previous report.⁵² As expected, also a substantial decrease in the HIPK2 phospho-targets SIRT1 Ser682 and in p53 Ser46^{37, 39, 52} was observed, and caspase-dependent PARP cleavage was absent after HIPK2 depletion (Figure 5a). These results highlight an essential role of HIPK2 in p53 acetylation and commitment to cell death.

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Figure 5

Ser682 phosphorylation regulates SIRT1 deacetylase activity, p53 acetylation and cell death. (a) HIPK2 is important for p53 Lys382 acetylation after DNA damage. HIPK2 was depleted in U2OS cells by RNA interference and cells were treated with Adriamycin (0.75μg/ml) as indicated. Total cell lysates were analysed by immunoblotting using the indicated antibodies. (b) HIPK2 antagonizes SIRT1-mediated deacetylation of p53. Flag-SIRT1, HA-HIPK2, p53 and HA-CBP were expressed in H1299 cells as indicated and total cell lysates were analysed by immunoblotting using the indicated antibodies. GFP expression was used to control the transfection efficiency. Total p53 protein expression levels were adjusted to equal levels to be able to compare p53 acetylation under different conditions. The ratio (amount p53 acK)/(amount p53) was quantified by densitometry using the ImageJ software. (c) SIRT1 Ser682 is required for HIPK2-mediated SIRT1 inhibition. H1299 cells were transfected with the indicated constructs and total cell lysates were analysed by immunoblotting using the indicated antibodies. GFP expression was used to control the transfection efficiency. Total p53 protein expression levels were adjusted to equal levels to be able to compare p53 acetylation under different conditions. The ratio (amount p53 acK)/(amount p53) was quantified by densitometry using the ImageJ software. (d) SIRT1 Ser682 is important for DNA damage-stimulated p53 acetylation. H1299 cells were transfected with the indicated expression constructs. Twenty-four hours after transfection cells were treated with Adriamycin (0.75μg/ml) or left untreated. Total cell lysates were analysed by immunoblotting. GFP expression was used to control the transfection efficiency. Total p53 protein expression levels were adjusted to equal levels to be able to compare p53 acetylation under different conditions. The ratio (amount p53 acK)/(amount p53) was quantified by densitometry using the ImageJ software. (e) SIRT1 Ser682 phosphorylation regulates p53-dependent transcription. U2OS cells were transfected with the indicated expression constructs together with a luciferase PUMA-reporter. Firefly reporter activity was normalized to Renilla reporter activity. Data are shown as means±S.D.; n=3; P<0.05, Student's t-test. (f) U2OS cells were transfected with the indicated expression constructs and expression of p53 target genes: p21, PUMA, BAX, NOXA and p53AIP1 were analysed by qRT-PCR. mRNA expression levels were normalized to the empty vector control. Data are shown as means±S.D.; n=3; P<0.05, Student's t-test. (g) SIRT1 Ser682 phorphorylation potentiates apoptosis. U2OS cells were transfected with the indicated expression constructs. Twenty-four hours after transfection cells were treated with Adriamycin (1μg/ml, 48h) and subsequently analysed by FACS using Annexin V-FITC

We next sought to investigate whether HIPK2 regulates p53 acetylation through modulating SIRT1 function. As expected, co-expression of p53 and its acetyltransferase CBP resulted in p53 acetylation (Figure 5b). Ectopic expression of HIPK2 together with p53 and CBP potentiated p53 acetylation and also stimulated p53 Ser46 phosphorylation (Figure 5b), in accordance with a previous report.³⁹ Furthermore, co-expression of wild-type SIRT1 in the presence of p53 and CBP led to an efficient reduction in p53 acetylation (Figure 5b), as reported previously.^{7, 13} Remarkably, ectopic expression of HIPK2 rescued p53 Lys382 acetylation in the presence of ectopically expressed SIRT1, indicating that HIPK2 inhibits p53 deacetylation by SIRT1 (Figure 5b). Of note, the rescuing effect of HIPK2 required the Ser682 residue of SIRT1, as HIPK2 failed to counteract SIRT1-dependent p53 deacetylation of a SIRT1^S682A point mutant (Supplementary Figure S4a). Thus, SIRT1 Ser682 phosphorylation is critical for p53 Lys382 acetylation by antagonizing SIRT1 function.

In line with our results, phosphorylation-mimetic SIRT1^S682D showed reduced p53 Lys382 deacetylation activity, whereas phosphorylation-deficient SIRT1^S682A deacetylated p53 to a comparable extent as wild-type SIRT1 protein (Figure 5c). This effect is specific for the SIRT1 Ser682 phosphorylation-site, as phosphorylation-mimetic SIRT1^S27D did not show this effect but behaved similar to wild-type SIRT1 (Supplementary Figure S4b). Taken together, Ser682 phosphorylation reduces SIRT1 deacetylase function and thus facilitates p53 acetylation.

We next reasoned that efficient p53 Lys382 acetylation upon DNA damage might require inhibition of SIRT1 function through phosphorylation at Ser682. Indeed, in the presence of phosphorylation-deficient SIRT1^S682A the DNA damage-mediated p53 acetylation by Adriamycin was decreased when compared with p53 acetylation levels in the presence of wild-type SIRT1 (Figure 5d). These findings indicate that Ser682 phosphorylation is important for efficient p53 acetylation upon DNA damage.

SIRT1 Ser682 phosphorylation modulates p53-regulated gene expression and apoptosis

To test whether phosphorylation of SIRT1 at Ser682 regulates p53-dependent transcription, we performed Luciferase-reporter assays using the PUMA promoter. Whereas wild-type SIRT1 and phosphorylation-deficient SIRT1^S682A reduced p53-regulated transactivation of the PUMA promoter to a similar extend, phospho-mimetic SIRT1^S682D was less efficient in suppressing p53-dependent transcription (Figure 5e). In addition, RT-qPCR analysis of endogenous p53 target genes including p21, Bax, PUMA, Noxa and p53AIP1 revealed similar results (Figure 5e). Furthermore, phospho-mimetic SIRT1^S682D showed a slight stimulatory effect on the expression of the apoptotic p53 target genes PUMA, NOXA and p53AIP1 (Figure 5f).

Next, we explored the role of Ser682 phosphorylation in DNA damage-induced apoptosis. To this end, we transfected U2OS cells with empty vector, SIRT1 wild-type or SIRT1^S682D expression constructs and analysed DNA damage-induced apoptosis after Adriamycin-treated by FACS analysis. The level of Annexin V-positive cells in Adriamycin-treated cells transfected with empty vector was arbitrarily set to 100%. In contrast to wild-type SIRT1, which suppressed induction of apoptosis upon Adriamycin treatment, phospho-mimetic SIRT1^S682D instead enhanced apoptosis induction in response to Adriamycin treatment (Figure 5g). Taken together, phosphorylation of SIRT1 at Ser682 potentiates apoptotic p53 target gene expression and cell death.

SIRT1 Ser682 phosphorylation regulates SIRT1–AROS interaction

Having shown that SIRT1 Ser682 phosphorylation inhibits SIRT1 deacetylase activity on p53, we aimed to identify the underlying molecular mechanism. To this end, we first analysed whether Ser682 phosphorylation directly affects the deacetlyase function of SIRT1 by performing in vitro deacetylation assays using bacterially expressed GST-SIRT1, GST-SIRT1^S682A and GST-SIRT1^S682D. No differences in the deacetylase activity of these SIRT1 proteins on acetylated p53 and acetylated Histones were detectable in vitro (Supplementary Figure S5a, b), indicating that Ser682 phosphorylation does not directly affect SIRT1 deacetylase activity. Furthermore, no change in the subcellular localization was detectable (Supplementary Figure S5c), excluding the possibility that differential localization may account for decreased SIRT1 function.

As SIRT1 activity is regulated by interaction with the activator AROS and the inhibitor DBC1,^{19, 20, 21} we analysed whether SIRT1–DBC1 or SIRT1–AROS interaction is modulated through phosphorylation of SIRT1 at Ser682. Strikingly, phospho-mimetic SIRT1^S682D showed a substantially reduced interaction with AROS when compared with wild-type and phospho-deficient SIRT1^S682A (Figure 6a). In contrast, interaction of SIRT1 with its inhibitor DBC1 was neither affected by SIRT1 Ser682 nor by SIRT1 Ser27 phospho-mimetic mutations (Figure 6b). These results suggest that SIRT1 phosphorylation at Ser682 modulates SIRT1–AROS binding.

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Figure 6

Regulation of the SIRT1–AROS complex by SIRT1 Ser682 phosphorylation and DNA damage. (a) SIRT1 Ser682 phoshorylation regulated SIRT1–AROS binding. 293T cells were transfected with the indicated expression constructs. Twenty-four hours after transfection Flag-SIRT1 was precipitated from the lysates and co-immunoprecipitation of AROS was analysed by immunoblotting using the indicated antibodies. Ten percent of the total cell lysate are shown as input control. (b) No effect of SIRT1 Ser682 phosphorylation on SIRT1–DBC1 interaction. 293T cells were transfected with the indicated expression constructs. Flag-SIRT1 was precipitated from the lysates and co-immunoprecipitation of AROS was analysed by immunoblotting using the indicated antibodies. Ten percent of the total cell lysate are shown as input control. (c) Disruption of the endogenous SIRT1–AROS complex after DNA damage. U2OS cells were treated with Adriamycin (0.75μg/ml for 4h) and MG-132 (20μM) or left untreated. Subsequently, endogenous SIRT1 was precipitated from the lysates and co-immunoprecipitation of endogenous AROS was detected by immunoblotting. As control 10% of the total cell lysates were analysed. (d) Reduced interaction of ectopically expressed SIRT1 and AROS after DNA damage. HA-AROS and Flag-SIRT1 were expressed in 293T cells. Twenty-four hours after transfection cells were treated with Adriamycin (0.75μg/ml for 4h) and MG-132 (20μM) or left untreated. Flag-SIRT1 was precipitated from the lysates and co-immunoprecipitation of HA-AROS was examined by immunoblotting. Ten percent of the total cell lysates are shown as input control. (e) Ser682 is required for dissociation of the SIRT1–AROS complex after DNA damage. HA-AROS and the phosphorylation-deficient mutant Flag-SIRT1^S682A were expressed in 293T cells. Cells were treated with Adriamycin (0.75μg/ml for 4h) and MG-132 (20μM) or left untreated and Flag-SIRT1^S682A was precipitated from the lysates and analysed by immunoblotting using the indicated antibodies. Controls correspond to 10% of the total cell lysates

Antibodies

The following antibodies were used: mouse monoclonal SIRT1 7B7 (Novus Biologicals, LLC, Littleton, CO, USA) for immunofluorescence staining and mouse monoclonal SIRT1 Clone 3H10.2 (Millipore, Merck KGaA, Darmstadt, Germany), rabbit polyclonal SIRT1 (M07-131; Millipore, Merck KGaA) for immunoblotting, RPS19BP1 (AROS, ATLAS Antibodies AB, Stockholm, Sweden), PARP and p53 phospho-Ser46 (BD Pharming, Pharmingen, San Diego, CA, USA), p53 DO-1, GFP FL and PML H-238 (Santa Cruz Biotechnologies Inc., Dallas, TX, USA), Flag M2 (Sigma-Aldrich, St Louis, MO, USA), actin C4 (MP Biomedicals, (LLC, Santa Ana, CA, USA)), HA clones 12CA5 and 3F10 (Roche, F. Hoffmann-La Roche Ltd., Basel, Switzerland), p53 phospho-Ser46, SIRT1 (rabbit Ab), SIRT1 phospho-Ser27 (all Cell Signaling Technologies Inc., Danvers, MA, USA), acetyl-Lys373/382 p53 (Millipore, Merck KGaA). Affinity-purified rabbit HIPK2 antibodies have been described previously.^{33, 39} Phospho-specific SIRT1 pSer682 antibodies were generated by immunizing rabbits with the following KLH-coupled peptide: NH₂-SGTCQ(pS)PSLEC-CONH₂. Rabbit sera were affinity-purified against the phospho-peptide and subsequently non-phosphorylation specific antibodies were removed by a column containing the immobilized non-phospho-peptide. The rabbit SIRT1 phospho-Ser682 antibody will be commercially available soon from Millipore (Merck KGaA).

Expression constructs

Human HIPK2 constructs have been described previously.^{33, 63} SIRT1 expression construct were kindly provided by Robert Weinberg and Renate Voit or generated by standard PCR technology. SIRT1 point mutant constructs were generated by standard PCR. p53 constructs were kindly provided by Wei Gu and Giannino del Sal. DBC1 cDNA was obtained from Invitrogen (Life Technologies GmbH) as Gateway system vector and cloned into destination vectors. AROS cDNA was chemically synthesized (GeneArt Gene Synthesis, Thermo Fisher Scientific Inc., Waltham, MA, USA) and expression constructs were generated by standard DNA cloning. All constructs used in this study were verified by DNA sequencing.

GST pulldown assays

GST fusion protein expression, protein purification and GST-pulldown assays were performed essentially as described.³³ In brief, GST fusion proteins were expressed in the E.coli BL21 strain and purified using glutathione (GSH) sepharose 4B beads (GE Healthcare, GE Healthcare UK Limited, Buckinghamshire, England). 0.2–0.4μg of bead-coupled GST fusion proteins were used for the pulldowns. GST fusion proteins were incubated with proteins generated by in vitro translation using the TNT Coupled Reticulocyte Lysate System (Promega Corporation, Madison, WI, USA). GST-SIRT1, ³⁵S-Flag-HIPK2 pull down was performed in 20mM Tris-HCl, pH7.4, 0.05% NP40, whereas GST-HIPK2, ³⁵S-Flag-SIRT1 pull down in 1 × PBS, 0.05% NP40.

Immunoblotting and immunoprecipitation

Immunoprecipitation and immunoblotting were performed as described previously and proteins were detected by Western Lightning Plus-ECL (PerkinElmer, Waltham, MA, USA) and enhanced chemiluminescence SuperSignal West Dura and Femto (Pierce, Life Technologies GmbH).³³ SIRT1-HIPK2 coimmunoprecipitation was performed in lysis buffer (20mM Tris–HCl pH 7.4, 10% glycerol, 1% NP40, 150mM NaCl, 25mM NaF, 5mM EDTA, 0.1% SDS, 1mM sodium vanadate, 1 × complete protease inhibitor (Roche, F. Hoffmann-La Roche Ltd.), 100μM MG-132). SIRT1–AROS and SIRT1–DBC1 co-immunoprecipitation were performed as described elsewhere.^{20, 21} Immunoprecipitations were performed using the Rabbit IgG and the Mouse IgG TrueBlot System (eBioscience Inc., San Diego, CA, USA) or Anti-Flag M2-Affinity Gel (Sigma-Aldrich) or GFP-trap (ChromoTek GmbH, Planegg-Martinsried, German) according to the manufacturer's instructions. Immunoprecipiatation reactions were incubated for 2h at 4°C on a rotating wheel and washed three times in lysis buffer. Samples were denatured at 95°C for 5min, separated by SDS–PAGE on 8%, 9.5%, 13% SDS gels or on 4–15% Mini-PROTEAN TGX Precast Gels (BioRad, Hercules, CA, USA) and analysed with the indicated antibodies.

RT-qPCR analysis

Total RNA was collected from cultured cells using TRIzol reagent (Invitrogen, Life Technologies GmbH). After RNA extraction, cDNA was synthesized using High capacity RNA to cDNA kit (Applied Biosystems, Life Technologies GmbH). Primers were used with TaqMan Gene Expression to perform quantitative (real-time) qRT-PCR (Applied Biosystems, Life Technologies GmbH) using a Step-One-Plus real-time PCR system (Applied Biosystems, Life Technologies GmbH). PCR conditions were as follows: step 1: 95°C for 10min; step 2: 40 cycles of 95°C for 15s followed by 60°C for 1min. The following primers were used: CDKN1A (Hs00355782), BBC3 (Hs00248075), BAX (Hs00180269), PMAIP1 (Hs00560402), TP53AIP1 (Hs00223141), HPRT1 (Hs02800695) (Applied Biosystems, Life Technologies GmbH). HPRT1 expression was used as an endogenous control gene to normalize input cDNA. Ratios of the expression level of each gene to that of the reference gene were calculated using the delta-delta-CT-method.

RNA interference

For HIPK2 knockdown either stealth siRNA against human HIPK2 (LifeTechnologies) was used or a conventional HIPK2 siRNA (QIAGEN Sciences, Germantown, MD, USA). For PML depletion the siPML SmartPool (Dharmacon) was used. For control experiments the medium GC-content control stealth siRNA (LifeTechnologies) and a GL2 luciferase siRNA (QIAGEN Sciences) was used. siRNA duplexes (final concentration 100nM) were transfected using Lipofectamin 2000 (LifeTechnologies) as specified by the manufacturer.

Immunofluorescence microscopy

Cells were seeded onto coverslips and treated or transfected with the indicated constructs. Cells were fixed with 4% PFA in PBS for 40min at RT. After washing once with PBS, the cells were blocked in 10% goat serum in PBS for 1h at RT. Cells were incubated with the following antibodies for 1h at 20°C as indicated in the figure legend: rabbit anti phospho-Ser682 SIRT1, rabbit anti-HIPK2, mouse anti-SIRT1, Flag (M2, Sigma-Aldrich). After washing with PBS, cells were incubated with secondary antibodies AlexaFluor488 or AlexaFlour594 donkey anti-rabbit (Invitrogen, Life Technologies GmbH) in PBS with Hoechst 1:1000w/v (Sigma-Aldrich) and mounted on glass slides with Mowiol (Sigma-Aldrich). Images were taken using a confocal laser scanning microscope (FluoView1000, Olympus, Hamburg, Germany) with a × 60 oil objective using the sequential scanning mode. All images were collected and processed using the FluoView Software (Olympus) and ImageJ (National Institutes of Health, Bethesda, MD, USA; http://imagej.nih.gov/ij/).

DNA damage

Cells were incubated with culture medium supplemented with adriamycin (stock dissolved in water) at the specified concentrations for indicated time points. After incubation cells were harvested and processed as indicated.

Apoptosis measurement

Cell were transfected with indicated expression constructs and apoptosis was determined by FACS analysis (Becton, Dickinson and Company, Franklin Lakes, NJ, USA) of 100000 cells using Annexin V-FITC Apoptosis Detection Kit (Sigma-Aldrich) according to the manufacturer's instructions.

In vitro SIRT1 deacetlyase activity assay

In vitro deacetylase assays were carried out by using human recombinant GST-SIRT1 wild-type and mutant proteins that were expressed in E.coli BL21 and purified by standard procedures under non-denaturing conditions as previously described.³³ Subsequently, proteins were eluted by standard protocols. To compare the activity of GST-SIRT1 (0.5–1ng) and mutant GST-SIRT1^S682D (0.5–1ng) the SIRT1 Direct Fluorescent Screening Assay Kit (Cayman Chemical Company, Ann Arbor, MI, USA) was used according to the manufacturer's instruction. 0.5–1ng GST–SIRT1 and 0.5ng Histone preparations⁵³ were used for deacetylase reactions. Reactions were incubated at 30°C for 10–30min. Proteins were separated on 13 or 8% SDS–PAGE, transferred to PVDF membrane and detected by immunoblotting.

In vitro kinase assays

In vitro kinase assays were performed in principle as described previously.⁶⁴ 0.2–0.5μg bacterially expressed and purified 6 × His-HIPK2 proteins or GST-HIPK2 (amino acids 1–551) were incubated in 30μl kinase buffer containing 40μM cold ATP and 5μCi [γ-³²P] ATP and 1-2μg GST-SIRT1 protein. After incubation for 30min at 30°C, the reaction was stopped by adding 5 × SDS loading buffer. After separation by SDS–PAGE, gels were fixed, dried and exposed to X-ray films.

We thank the members of the Hofmann lab for helpful discussions, Fabian Uch, Kathrin Schultheiss and Larissa Brunner for experimental help, and Simon Anderhub for comments on the manuscript. We are grateful to Robert Weinberg, Wei Gu, Tony Kouzarides and Renate Voit for expression constructs. Our work was supported by the Deutsche Forschungsgemeinschaft (SFB 1036) and the Monika Kutzner Stiftung.