ATM-dependent phosphorylation of heterogeneous nuclear ribonucleoprotein K promotes p53 transcriptional activation in response to DNA damage

Abstract

Previous work has established that heterogeneous nuclear ribonucleoprotein K (hnRNP K) is stabilized in an ATM-dependent manner in response to DNA damage and acts as a cofactor for p53-mediated transcription. Here, we show that in response to DNA damage caused by ionizing radiation, hnRNP K is phosphorylated in an ATM-dependent manner. Furthermore, our data indicate that ATM-dependent hnRNP K phosphorylation is required for its stabilization and its function as a p53 transcriptional cofactor in response to DNA damage. These findings thereby establish hnRNP K as an ATM target and help define how ATM orchestrates p53-dependent transcriptional responses in response to genotoxic stress.

Keywords: ATM; DNA damage; p53; phosphorylation; transcription.

Figure 1. ATM-dependent phosphorylation of hnRNP K. (A) ATM-mediated phosphorylation of hnRNP K was assessed in U2OS cells with a phospho-ATM/ATR substrate antibody (see “Materials and Methods”). U2OS cells were transfected with an empty vector (Vector), a vector expressing wild-type HA-hnRNP K (WT), a vector expressing the Ser/Thr→Ala mutant HA-hnRNP K (4A) or vector expressing wild-type HA-hnRNP K in the presence of ATM inhibitor (WT +ATMi). Cells were untreated or treated with IR (15 Gy) for 1 h, and immunoprecipitation was performed with HA antibody followed by western blotting with phospho-ATM/ATR substrate antibody. Western blotting with the HA antibody was performed to assess transfection efficiency. (B) ATM-mediated phosphorylation of hnRNP K was assessed as in (A) in U2OS cells transfected with the WT HA-hnRNP K and treated with either GFP siRNA (WT+Control si) or ATM siRNA (WT+ATMsi). A parental plasmid vector (Vector) was used as a control. Chk2 phosphorylation was assessed with a Chk2 phospho Thr-68 specific antibody to monitor ATM function, while an ATM antibody was used to check efficiency of siRNA depletion. Anti-tubulin antibody (Tubulin) was used as a loading control. (C) Schematic of the domain organization of hnRNP K with the locations of the four SQ/TQ motifs described in the text (see Fig. S1 for these in the context of the hnRNP K sequence). (D) As in (A), except that additional hnRNP K constructs were also employed in which Thr-390, Thr-174, Thr-440 or Ser-121 were individually regenerated in the 4A mutant background.

Figure 2. ATM-dependent phosphorylation of hnRNP K controls its stabilization, its HDM2-mediated ubiquitylation and its interaction with HDM2. (A) Stabilization of hnRNP K was assessed by western blotting (with monoclonal anti-HA antibody) in U2OS cells transfected with an empty plasmid vector (Vector), a vector expressing wild type HA-hnRNP K (WT), a vector expressing the quadruple Ser/Thr→Ala mutant HA-hnRNP K (4A) or the vector expressing the wild-type HA-hnRNP K in the presence of 10 μM ATM inhibitor KU-55933 (WT +ATMi). In all cases, cells were untreated (-) or treated (+) with IR (15 Gy) for 1 h. An anti-tubulin antibody was used as loading control. (B) hnRNP K-HDM2 interaction was analyzed by IP in U2OS cells transfected as above and left untreated or treated with IR (15 Gy) for 1 h. Immunoprecipitation was with anti-HA antibody followed by western blotting with HDM2 antibody or HA antibody (hnRNP K). Inputs for both proteins are shown and represent 5% of the material used. (C) Ubiquitylation of hnRNP K was analyzed in U2OS cells transfected as above, treated or not with IR (15 Gy). Where indicated, cells were treated with the proteasome inhibitor MG132 (30 mM) for 3 h prior to IR treatment. Where indicated, U2OS cells transfected with vector expressing WT HA-hnRNP K were treated with 10 μM of ATM inhibitor (ATMi) or wortmannin (10 μM) or DMSO. Cell extracts were analyzed by immunoprecipitation with anti-HA antibody followed by western blotting with the same antibody. Bands representing ubiquitylated forms of hnRNP K (Ub-hnRNP K) and the immunoglobulin light-chain (Ig) are indicated.

Figure 3. ATM-dependent phosphorylation of hnRNP K promotes p53 transcriptional activity following IR. (A) Expression from hnRNP K constructs was assessed by western blotting with the indicated antibodies in cells treated or not with control siRNA (Contr) or hnRNP K siRNA (K). As indicated, these cells also contained control parental vector (Vector), or constructs expressing wild-type hnRNP (WT), siRNA resistant wild-type hnRNP K (WT/siR), 4A mutant hnRN K (4A) or siRNA resistant 4A hnRNP K (4A/siR). Anti-tubulin antibody was used as loading control. (B) p53-dependent induction of p21/WAF1 protein was assessed 6 h following IR by western blotting in cells treated with the indicated siRNAs and containing the indicated constructs. (C) U2OS cells stably transfected with a luciferase reporter gene under the control of multiple p53 binding sites (PG13 U2OS) were transfected with the indicated constructs and then IR (15 Gy) treated or mock IR treated, as indicated. Luciferase activity was measured 18 h afterwards. (D) U2OS cells were transfected with the indicated constructs, treated with control of hnRNP K siRNAs and 48 h later, they were mock treated or exposed to IR (15 Gy) and incubated for 2 h. Samples were then subjected to ChIP with anti-p53 antibody, and precipitated DNA was subjected to PCR with primers covering the p53-response elements of the p21/WAF1 promoter.