1. Volume 25, Issue 5, Pages 725-738 (March 2007)
DYRK2 Is Targeted to the Nucleus and Controls p53 via Ser46 Phosphorylation in the Apoptotic Response to DNA Damage 
Naoe Taira, Keishi Nihira, Tomoko Yamaguchi, Yoshio Miki, Kiyotsugu Yoshida 
Molecular Cell 
Volume 25, Issue 5, Pages (March 2007)
DOI: /j.molcel
Copyright © 2007 Elsevier Inc. Terms and Conditions

2. Figure 1 Identification of Ser46 Kinases
(A) Schematic diagram of the Ser46 kinase screening (see Experimental Procedures section for detail).
(B) In the first screening step, candidates for Ser46 kinases were screened from phage expression libraries derived from cDNAs of human fetal brain (2 × 107 pfu), placenta (2 × 107 pfu), or HeLa cells (4 × 107 pfu) (upper left panel). Phosphorylated p53 was detected by rabbit anti-phospho-p53(Ser46). Positive phages were replated for the second screening (upper right panel). Following the second screening, selected positive clones were infected with a bacterium that either expressed GST-p53(1–92) wild-type (WT) or S46A (lower panels). The arrow indicates a positive plaque.
(C) DYRK2 phosphorylates p53 on Ser46 in vitro. Recombinant His-DYRK2 was incubated with purified GST-p53(1–92)WT or GST-p53(1–92)S46A and ATP. Reaction products were analyzed by immunoblotting (IB) with rabbit anti-phospho-p53(Ser46) (top panel), mouse anti-phospho-p53(Ser46) (second panel), anti-GST (third panel), or anti-His (bottom panel).
(D) Lysates from COS-7 cells transfected with Flag vector, Flag-DYRK2, or the Flag-DYRK2(K178R) mutant were immunoprecipitated with anti-Flag agarose. The immunoprecipitates were incubated with purified GST-tagged p53 and ATP. Reaction products were analyzed by immunoblotting with rabbit anti-phospho-p53(Ser46) (upper panel), anti-GST (middle panel), or anti-Flag (lower panel).
Molecular Cell  , DOI: ( /j.molcel )
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3. Figure 2
DYRK2 Phosphorylates p53 at Ser46 in Response to Genotoxic Stress
(A) Lysates from 293T cells cotransfected with Flag-p53 and GFP vector, GFP-DYRK2, or the GFP-DYRK2(K178R) mutant were immunoprecipitated with anti-Flag agarose. Immunoprecipitates were then subjected to immunoblot analysis with rabbit anti-phospho-p53(Ser46) (top panel), mouse anti-phospho-p53(Ser46) (second panel), anti-Flag (third panel), or anti-GFP (bottom panel).
(B) U2OS cells were transfected with GFP vector or the GFP-DYRK2(K178R) mutant and then treated with 2 μg/ml adriamycin (ADR) for 24 hr. Lysates were analyzed by immunoblotting with mouse anti-phospho-p53(Ser46) (top panel), anti-p53 (second panel), anti-PCNA (third panel), or anti-GFP (bottom panel).
(C) U2OS cells were transfected with the scramble siRNA or DYRK2 siRNA to reduce endogenous DYRK2 expression. At 48 hr posttransfection, cells were left untreated or treated with ADR for 24 hr. Cell lysates were subjected to immunoblot analysis with mouse anti-phospho-p53(Ser46) (top panel), anti-p53 (second panel), anti-DYRK2 (third panel), or anti-PCNA (fourth panel). Total RNAs were analyzed by RT-PCR using DYRK2-specific (fifth panel), GAPDH-specific (sixth panel), or p53AIP1-specific (bottom panel) primers.
Molecular Cell  , DOI: ( /j.molcel )
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4. Figure 3 DYRK2 Translocates into the Nucleus in Response to DNA Damage
(A) Localization of p53 and DYRK2 in U2OS cells. U2OS cells were transfected with GFP vector, GFP-DYRK2, or GFP-DYRK2(K178R) and then treated with 20 μM etoposide for 24 hr. Cells were stained with anti-GFP (FITC) and anti-p53 (rhodamine) antibodies. The nuclei were stained with DAPI.
(B) U2OS cells were transiently transfected as in (A). The localization of DYRK2 was scored according to whether it was higher in the nucleus (closed bars), evenly distributed between the nucleus and the cytoplasm (dotted bars), or higher in the cytoplasm (open bars). Results are the mean ± SD of values obtained from five fields of 30–100 cells in each of three independent experiments. Statistical analysis was performed with the Student's t test, and p < 0.05 (asterisk) was defined as significant.
(C) U2OS cells were transfected with the scramble siRNA or p53 siRNA followed by treatment with ADR. Nuclear and cytoplasmic lysates were subjected to immunoblot analysis with indicated antibodies.
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5. Figure 4 ATM Regulates DYRK2-Mediated Phosphorylation of p53 at Ser46
(A) HCT116 cells were transfected with the scramble siRNA or ATM siRNA1 and then left untreated or treated with ADR for 24 hr. Cell lysates were analyzed by immunoblot analysis with mouse anti-phospho-p53(Ser46) (top panel), anti-p53 (second panel), anti-ATM (third panel), or anti-PCNA (bottom panel). The signals were scanned to compare the amount of phosphorylation induced by ADR treatment in ATM siRNA1-transfected cells with that of scramble-transfected cells. The amount of phosphorylation was normalized by dividing its value by the value for the amount of total p53. We found that 0.38 was the ratio of the amount for phosphorylation with ATM siRNA1:phosphorylation with scramble siRNA.
(B) 293T cells were cotransfected with Flag-DYRK2 or the Flag-DYRK2(K178R) mutant and the scramble siRNA or ATM siRNA1; the transfected cells were treated with ADR for 24 hr. Lysates were immunoprecipitated with anti-Flag agarose, then immunoprecipitates were incubated with purified GST-tagged p53 and ATP. Reaction products were analyzed by immunoblotting with rabbit anti-phospho-p53(Ser46) (top panel), anti-GST (second panel), anti-ATM (third panel), anti-Flag (fourth panel), or anti-PCNA (bottom panel).
(C) HCT116 cells cotransfected with Flag vector or Flag-DYRK2 and scramble siRNA or ATM siRNA1 were left untreated or treated with ADR for 24 hr. Cell lysates were subjected to immunoblot analysis with mouse anti-phospho-p53(Ser46) (top panel), anti-p53 (second panel), anti-ATM (third panel), anti-Flag (fourth panel), or anti-PCNA (bottom panel).
Molecular Cell  , DOI: ( /j.molcel )
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6. Figure 5
DYRK2 Induces p53-Dependent Apoptosis in Response to DNA Damage
(A and B) U2OS cells were transfected with the scramble siRNA, DYRK2 siRNA, or p53 siRNA followed by treatment with ADR. Apoptotic cells were quantified by TUNEL assays (A). The results are represented as the percentage of TUNEL-positive cells. The data indicate the mean ± SD from three independent experiments each performed in triplicate (B). Control cells, open bars; ADR-treated cells, closed bars. Total RNAs were subjected to RT-PCR analysis using DYRK2-specific (upper panel), p53-specific (middle panel), or GAPDH-specific (lower panel) primers.
(C and D) SaOS-2 cells, which are p53 null lines, were cotransfected as indicated and treated with ADR for 24 hr. Apoptotic cells were analyzed by TUNEL assays as described above. Control cells, open bars; ADR-treated cells, closed bars. Cell lysates and total RNA were analyzed by immunoblotting and RT-PCR, respectively.
Molecular Cell  , DOI: ( /j.molcel )
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7. Figure 6
Involvement of DYRK2 on p53 Phosphorylation at Ser46 and Induction of Apoptosis in Response to Various Genotoxic Stress
(A) U2OS cells were left untreated or treated with UV for the indicated dose for 24 hr. Apoptotic cells were quantitated by TUNEL assays. The results are represented as the percentage of TUNEL-positive cells. The data indicate the mean ± SD from two independent experiments each performed in triplicate. Cell lysates were analyzed by immunoblotting with mouse anti-phospho-p53(Ser46) (upper panel), anti-p53 (middle panel), or anti-tubulin (lower panel).
(B) U2OS cells were transfected with the scramble siRNA, DYRK2 siRNA, or HIPK2 siRNA followed by treatment with ADR or 2.5 J/m2 UV for 24 hr. Apoptotic cells were quantitated by TUNEL assays as described above. Control cells, open bars; ADR-treated cells, dotted bars; UV-treated cells, closed bars. Cell lysates and total RNA were analyzed by immunoblotting and RT-PCR, respectively. C, control cells; A, ADR-treated cells; U, UV-treated cells.
(C) A proposed model for DYRK2-mediated apoptosis in the cellular response to DNA damage. Upon exposure to genotoxic stress, p53 is stabilized and activated by phosphorylation at Ser15 and Ser20. Cytoplasmic DYRK2 is activated and targeted to the nucleus and then phosphorylates p53 at Ser46. Ser46 phosphorylation of p53 triggers induction of apoptosis by upregulation of apoptosis-related gene expression, such as p53AIP1.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2007 Elsevier Inc. Terms and Conditions