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. 2007 Sep;81(18):9653-64.
doi: 10.1128/JVI.00568-07. Epub 2007 Jul 3.

Enhanced phosphorylation of transcription factor sp1 in response to herpes simplex virus type 1 infection is dependent on the ataxia telangiectasia-mutated protein

Satoko Iwahori  1 , Noriko Shirata, Yasushi Kawaguchi, Sandra K Weller, Yoshitaka Sato, Ayumi Kudoh, Sanae Nakayama, Hiroki Isomura, Tatsuya Tsurumi
Affiliations

Enhanced phosphorylation of transcription factor sp1 in response to herpes simplex virus type 1 infection is dependent on the ataxia telangiectasia-mutated protein

Satoko Iwahori et al. J Virol. 2007 Sep.

Abstract

The ataxia telangiectasia-mutated (ATM) protein, a member of the related phosphatidylinositol 3-like kinase family encoded by a gene responsible for the human genetic disorder ataxia telangiectasia, regulates cellular responses to DNA damage and viral infection. It has been previously reported that herpes simplex virus type 1 (HSV-1) infection induces activation of protein kinase activity of ATM and hyperphosphorylation of transcription factor, Sp1. We show that ATM is intimately involved in Sp1 hyperphosphorylation during HSV-1 infection rather than individual HSV-1-encoded protein kinases. In ATM-deficient cells or cells silenced for ATM expression by short hairpin RNA targeting, hyperphosphorylation of Sp1 was prevented even as HSV-1 infection progressed. Mutational analysis of putative ATM phosphorylation sites on Sp1 and immunoblot analysis with phosphopeptide-specific Sp1 antibodies clarified that at least Ser-56 and Ser-101 residues on Sp1 became phosphorylated upon HSV-1 infection. Serine-to-alanine mutations at both sites on Sp1 considerably abolished hyperphosphorylation of Sp1 upon infection. Although ATM phosphorylated Ser-101 but not Ser-56 on Sp1 in vitro, phosphorylation of Sp1 at both sites was not detected at all upon infection in ATM-deficient cells, suggesting that cellular kinase(s) activated by ATM could be involved in phosphorylation at Ser-56. Upon viral infection, Sp1-dependent transcription in ATM expression-silenced cells was almost the same as that in ATM-intact cells, suggesting that ATM-dependent phosphorylation of Sp1 might hardly affect its transcriptional activity during the HSV-1 infection. ATM-dependent Sp1 phosphorylation appears to be a global response to various DNA damage stress including viral DNA replication.

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Figures

FIG. 1.
FIG. 1.
Hyperphosphorylation of Sp1 is induced upon HSV-1 infection. HeLa (A) and HFF2 cells (C) were infected with HSV-1 at MOIs of 10 and 5, respectively, and were harvested at the indicated times postinfection. Whole-cell lysates were prepared, and equal amounts of proteins from each sample (2.5 or 30 μg) were subjected to immunoblot analysis with anti-Sp1 antibody. (B and D) Whole-cell lysates obtained from HSV-1-infected HeLa and HFF2 cells at the indicated times postinfection were treated with (+) or without (−) CIAP for 30 min at 37°C. The samples were subjected to immunoblot analysis with anti-Sp1 antibody.
FIG. 2.
FIG. 2.
Hyperphosphorylation of Sp1 is induced even upon infection of HSV-1 mutants defective for viral PK, Us3, UL13 or UL39 gene product. HFF2 cells were infected with wild-type HSV-1 (wt HSV-1), Us3-defective virus (R7041), UL13-defective virus (R7356), or UL39-defective virus (ICP6Δ) at an MOI of 5. At 12 and 24 hpi, cells were harvested and whole-cell lysates were prepared. Equal amounts of proteins from each sample (15 μg) were subjected to immunoblot analysis with anti-Sp1, ICP4, and UL42 proteins specific antibodies. Mock, mock infection.
FIG. 3.
FIG. 3.
ATM is involved in hyperphosphorylation of Sp1 induced by HSV-1 infection. (A and B) 293T-ATM shRNA and 293T-Control vector cells (A) and ATM defective AT10S/T-n cells (B) were infected with HSV-1 at an MOI of 10 and harvested at the indicated times postinfection. Whole-cell lysates were prepared, and equal amounts of proteins from each sample (7 to 15 μg) were subjected to immunoblot analysis with the indicated antibodies. Each value at the bottom of the panel of Sp1 (“F/W: %”) represents the percentage of the level of the faster-migrating form to whole amounts of Sp1, calculated as described in Materials and Methods. M, mock infection. (C) Human glioma M059J (DNA-PKcs null and the lower level of ATM) and MJ-M6 (M059J cells expressing DNA-PKcs) cells were infected with HSV-1 at an MOI of 2.5 and harvested at the indicated times postinfection. Whole-cell lysates (15 μg) were subjected to immunoblot analysis for expression profiles of Sp1, ATM, ATM phosphorylated at Ser-1981, DNA-PKcs, ICP4, and GAPDH. Anti-GAPDH antibody was used to confirm equal protein loading.
FIG. 4.
FIG. 4.
(A) Hyperphosphorylation of Sp1 induced upon exposure to IR. HFF2 cells were exposed to gamma irradiation (IR) with 10 Gy and harvested at 15 min post-IR. Whole-cell lysates were prepared, and 30-μg aliquots of proteins from each sample were subjected to immunoblot analysis with anti-Sp1 and ATM S1981 antibodies. (B) Sp1 hyperphosphorylation induced upon IR is correlated with the activation of ATM. The 293T-ATM shRNA and 293T-Control vector cells were exposed to IR with 20 Gy and harvested at 15 min post-IR. Whole-cell lysates were prepared, and equal amounts of proteins from each sample were subjected to immunoblot analysis with anti-Sp1 and ATM S1981 antibodies. −, No IR.
FIG. 5.
FIG. 5.
Mapping of phosphorylation sites of Sp1 upon HSV-1 infection. (A) Schematic diagram of functional domains and putative ATM phosphorylation sites of Sp1. The 785 amino acids of Sp1 have two S/T-rich regions, two Q-rich regions, and three zinc fingers. The domains A, B, C, and D correspond to multiple transcriptional activation domains. The sign “−/+” represents a region of high charge density (14). Motifs of serine (S) or threonine (T), followed by glutamine (Q) (SQ and TQ) in Sp1, are putative phosphorylation sites for ATM. Sp1 contains 15 SQ and TQ sites. The amino acid sequences around Ser-56 and Ser-101 are shown in detail. (B) HeLa cells were transfected with pcDNA-RHF/Sp1 expressing wild-type (wt) Sp1 or a variety of expression vectors for mutated Sp1 containing the indicated serine or threonine residues replaced with alanine. Cells were infected with HSV-1 at an MOI of 10 at 24 h posttransfection and harvested at 24 hpi. Whole-cell lysates were prepared, and 25-μg portions of proteins from each sample were subjected to immunoblot analysis with anti-ICP4 antibody or anti-Flag antibody to detect exogenously expressed Sp1 proteins (Flag-Sp1). Each value at the bottom of the panel of Flag-Sp1 (“F/W: %”) represents the percentage of level of the faster-migrating form to whole amounts of Flag-Sp1 as described in Materials and Methods.
FIG. 6.
FIG. 6.
Ser-56 and Ser-101 of Sp1 are phosphorylated upon HSV-1 infection. (A) HeLa cells were transfected with pcDNA-RHF/Sp1 (wt), pcDNA-RHF/Sp1-S56A, or pcDNA-RHF/Sp1-S101A and infected with HSV-1 at an MOI of 10 at 24 h posttransfection. At 24 hpi, cells were harvested and lysed. The whole-cell lysates were subjected to IP with anti-Flag M2 affinity resin, and the immunoprecipitated samples were subjected to immunoblot (IB) analysis with anti-Flag, α-Sp1 (pS56), and α-Sp1 (pS101) antibodies. (B) Whole-cell lysates obtained from HSV-1-infected HeLa cells at 12 hpi were treated with (+) or without (−) CIAP. The samples were applied for immunoblot analysis with the indicated antibodies. (C) HeLa cells were infected with HSV-1 at an MOI of 10 and harvested at the indicated times postinfection. Whole-cell lysates were prepared, and equal amounts of proteins from each sample were applied for immunoblot analysis with the indicated antibodies. (D) AT10S/T-n cells were infected with HSV-1 at an MOI of 10 and harvested at the indicated times postinfection. Whole-cell lysates were prepared and subjected to immunoblot analysis with the indicated antibodies. Whole-cell lysate from HSV-1-infected HeLa cells at 4 hpi was also applied as a positive control.
FIG. 7.
FIG. 7.
Sp1 is phosphorylated by ATM in vitro. The lysates of 293T cells transfected with plasmid expressing Flag-tagged wild-type ATM (Flag-wt ATM) or kinase-dead ATM (Flag-kd ATM) were immunoprecipitated with anti-Flag antibody. Each of immunocomplexes containing Flag-wt ATM or Flag-kd ATM protein were incubated with purified Sp1 or p53 as substrates in the presence of [γ-32P]ATP. Sp1 and p53 were resolved by SDS-10% PAGE, followed by autoradiography. The amounts of immunoprecipitated Flag-wt ATM and Flag-kd ATM were confirmed by immunoblot analysis with anti-ATM antibody.
FIG. 8.
FIG. 8.
ATM phosphorylates Sp1 at Ser-101 but not Ser-56 in vitro. (A) Schematic diagram of GST fusion proteins of truncated Sp1 and identical fragments with one or both of Ser-56 and Ser-101 mutated to alanine. (B) Lysates of 293T cells transfected with plasmid expressing Flag-tagged wild-type ATM (Flag-wt ATM) or kinase-dead ATM (Flag-kd ATM) were immunoprecipitated with anti-Flag antibody. Immunocomplexes of Flag-wt ATM or Flag-kd ATM were resolved on SDS-10% PAGE gel and subjected to immunoblot analysis with anti-ATM antibody. (C) IP-kinase assays. GST-Sp18-167, GST-Sp18-167-S56A, GST-Sp18-167-S101A, and GST-Sp18-167-S56/101A were expressed in E. coli, purified, and used as substrates for IP-kinase assays. Immunocomplexes containing Flag-wt ATM (Wt) or Flag-kd ATM (Kd) protein were each incubated with purified GST-Sp18-167, GST-Sp18-167-S56A, GST-Sp18-167-S101A, or GST-Sp18-167-S56/101A as substrates in the presence of [γ-32P]ATP. Samples were resolved by SDS-10% PAGE, followed by autoradiography. The amounts of each GST fusion protein were confirmed by Coomassie brilliant blue (CBB) staining.
FIG. 9.
FIG. 9.
ATM-dependent Sp1 phosphorylation does not affect Sp1-dependent transcription upon infection. (A and D) Schematic illustration of reporter vectors: p65F1CAT contains three Sp1-binding sites (GC-boxes) of the TATA less p65 promoter sequence (−575 to +38) (A), and pCAT TATA+Sp1(−55)+Sp1(−75) contains two GC-boxes and TATA consensus sequence of HCMV major immediate-early gene (D). (B and E) 293T-ATM shRNA and 293T-Control vector cells were transfected with either p65F1CAT (B) or pCAT TATA+Sp1(−55)+Sp1(−75) (E), infected with HSV-1 at 24 h posttransfection at an MOI of 5, and harvested at 12 hpi. CAT assays were performed as described in Materials and Methods. All transfection experiments were in triplicate. (C and F) Data from three independent experiments in panels B and E were plotted on the graph, respectively. The levels of activity (i.e., the conversion efficiency) were determined by calculating the percentage of the conversion of unacetylated [14C]chloramphenicol to the acetylated form. In order to confirm phosphorylation states and amounts of Sp1 in 293T-ATM shRNA and 293T-Control vector cells infected with HSV-1 at 12 hpi, equal amounts of proteins from each sample were subjected to immunoblot analysis with anti-Sp1 antibody (panels C and F, inset images).
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