Phosphatidyl inositol 3-kinase-like serine/threonine protein kinases (PIKKs) are required for DNA damage-induced phosphorylation of the 32 kDa subunit of replication protein A at threonine 21

Abstract

Replication protein A (RPA) is a single-stranded DNA (ssDNA) binding protein involved in various processes, including nucleotide excision repair and DNA replication. The 32 kDa subunit of RPA (RPA32) is phosphorylated in response to various DNA-damaging agents, and two protein kinases, ataxia-telangiectasia mutated (ATM) and the DNA-dependent protein kinase (DNA-PK) have been implicated in DNA damage-induced phosphorylation of RPA32. However, the relative roles of ATM and DNA-PK in the site-specific DNA damage-induced phosphorylation of RPA32 have not been reported. Here we generated a phosphospecific antibody that recognizes Thr21-phosphorylated RPA32. We show that both DNA-PK and ATM phosphorylate RPA32 on Thr21 in vitro. Ionizing radiation (IR)-induced phosphorylation of RPA32 on Thr21 was defective in ATM-deficient cells, while camptothecin (CPT)-induced phosphorylation of RPA32 on Thr21 was defective in cells lacking functional DNA-PK. Neither ATM nor DNA-PK was required for etoposide (ETOP)-induced RPA32 Thr21 phosphorylation. However, two inhibitors of the ATM- and Rad3-related (ATR) protein kinase activity prevented ETOP-induced Thr21 phosphorylation. Inhibition of DNA replication prevented both the IR- and CPT-induced phosphorylation of Thr21, whereas ETOP-induced Thr21 phosphorylation did not require active DNA replication. Thus, the regulation of RPA32 Thr21 phosphorylation by multiple DNA damage response protein kinases suggests that Thr21 phosphorylation of RPA32 is a crucial step within the DNA damage response.

Figure 1

Thr²¹ is the major in vitro DNA-PK phosphorylation site in RPA32. One microgram of WT-, T21A- or S33A-RPA14·32 was incubated with purified DNA-PK (60 ng of DNA-PKcs and 20 ng of Ku70/80) in the presence or absence of [γ-³²P]ATP for 10 min. Samples were fractionated on SDS–polyacrylamide gels and RPA32 was visualized by Coomassie staining (RPA32) and autoradiography (³²P-RPA32). RPA32 bands were excised from the gel and ³²P incorporation was quantitated by Cerenkov counting (bottom).

Figure 2

In vitro phosphorylation of RPA32 by DNA-PK. (A) Seventy-two nanograms of WT-, T21A- or S33A-RPA14·32, or 100 ng of tRPA were incubated with purified DNA-PK (as in Fig. 1) in the presence or absence of ATP. Samples were immunoblotted using the Thr²¹ phosphospecific antibody (pThr²¹) either alone (i), in the presence of pThr²¹ blocking peptide (p-peptide) (ii), or in the presence of Thr²¹ mock peptide (peptide) (iii). Alternatively, samples were immunoblotted using a monoclonal RPA32 antibody (RPA32) (iv). (B) tRPA (100 ng) was incubated with purified DNA-PK in the presence or absence of ATP for 5 min, with additional DNA added to the incubation as follows: lanes 1 and 2, none; lane 3, M13 closed circular ssDNA; lane 4, 40 nt ssDNA. Samples were immunoblotted using the Thr²¹ phosphospecific antibody (pThr²¹) or a monoclonal RPA32 antibody (RPA32).

Figure 3

In vitro phosphorylation of RPA32 by ATM. (A) ATM was immunoprecipitated from 1 mg of protein extract from unirradiated ‘C’ cells or cells exposed to 10 Gy IR as described in Materials and Methods. Immunoprecipitates were assayed in the presence of [γ-³²P]ATP and 0.5 µg of PHAS-I, in the presence or absence of 1 µM wortmannin (WM). PHAS-I was visualized by Coomassie staining (PHAS-I) or autoradiography (³²P-PHAS-I). (B) ATM immunoprecipitates from irradiated cells were incubated with 0.75 µg of WT, T21A or S33A RPA14·32, or 1 µg of tRPA, in the presence or absence of 250 µM ATP for 30 min. Samples were immunoblotted using the Thr²¹ phosphospecific antibody (pThr²¹) either in the presence of Thr²¹ mock peptide (peptide) (i), or in the presence of pThr²¹ blocking peptide (p-peptide) (ii). Alternatively, samples were immunoblotted using a monoclonal RPA32 antibody (RPA32) (iii).

Figure 4

IR-induced RPA phosphorylation is ATM dependent. (i and ii) BT (ATM-proficient) or L3 (ATM-deficient) cells were labeled with [³²P]orthophosphate, exposed to 10 Gy IR or unirradiated ‘C’, with either 100 µM wortmannin (WM) or 10 mM caffeine (CAFF) pre-treatment as described in Materials and Methods. RPA32 was immunoprecipitated from cell extracts and phosphorylated RPA32 was visualized by autoradiography [³²P-RPA32 (i)] or with an RPA32 antibody [RPA32 (ii)]. (iii and iv) Cells were treated as in (i) and (ii) but without [³²P]orthophosphate. Cell extracts were enriched for RPA32 using Affi-gel blue resin as described in Materials and Methods, and RPA32 was immunoblotted using the Thr²¹ phosphospecific antibody [pThr²¹ (iii)] or a RPA32 antibody [RPA32 (iv)].

Figure 5

ATM-dependent and -independent DNA damage-induced phosphorylation of RPA32 Thr²¹. (A) BT (ATM-proficient) or L3 (ATM- deficient) cells were treated with the indicated DNA-damaging agent as described in Materials and Methods. Cell extracts were enriched for RPA32 using Affi-gel blue resin and RPA32 was immunoblotted using the Thr²¹ phosphospecific antibody (pThr²¹) or a RPA32 antibody (RPA32). C, IR, UVC, ETOP, Dox, t-BH and CPT denote control (untreated), ionizing radiation (10 Gy), ultraviolet radiation (254 nm; 20 J/m²), etoposide (68 µM), doxorubicin (10 µM), t-butyl hydroperoxide (500 µM), and camptothecin (100 µM), respectively. (B) BT (ATM-proficient) or L3 (ATM-deficient) cells were treated with a range of CPT or ETOP concentrations and processed as in (A).

Figure 6

Involvement of DNA replication in the DNA damage-induced phosphorylation of RPA32 Thr²¹. (A) BT (ATM-proficient) cells were either exposed to 10 Gy IR or left untreated ‘C’, either with or without 1 h pre-treatment with aphidicolin. Cell extracts were enriched for RPA32 using Affi-gel blue resin and RPA32 was immunoblotted using the Thr²¹ phosphospecific antibody (pThr²¹) or a RPA32 antibody (RPA32). (B) L3 (ATM-deficient) cells were treated with 10 µM CPT, 68 µM ETOP or were left untreated ‘C’, either with or without 1 h pre-treatment with aphidicolin, and processed as in (A).

Figure 7

DNA-PK regulates the CPT-induced RPA32 Thr²¹ phosphorylation. (A) DNA-PKcs-proficient M059K (K) and DNA-PKcs-deficient M059J (J) cells were untreated ‘C’ or treated with 10 µM CPT and analyzed for RPA32 Thr²¹ phosphorylation, as in Figure 5A. (B) M059J/Fus1 (DNA-PKcs-proficient) and M059J/Fus9 (DNA-PKcs-deficient) cells were labeled with [³²P]orthophosphate and were either left without additional treatment ‘C’ or treated with 10 µM CPT, with or without pre-treatment with 100 µM wortmannin. RPA32 was immunoprecipitated from cell extracts and visualized by autoradiography (³²P-RPA32) or with an RPA32 antibody (RPA32).

Figure 8

DNA-PK-independent ETOP-induced RPA32 Thr²¹ phosphorylation. (A) DNA-PKcs-deficient M059J cells were treated with ETOP and analyzed for RPA32 Thr²¹ phosphorylation, as in Figure 5A. (B) L3 (ATM-deficient) cells were untreated ‘U’ or treated with 68 µM ETOP, either with ETOP alone ‘C’ or with pre-treatment with 10 mM CAFF or 100 µM wortmannin, and then analyzed for RPA32 Thr²¹ phosphorylation, as in Figure 5A.