The human homologs of checkpoint kinases Chk1 and Cds1 (Chk2) phosphorylate p53 at multiple DNA damage-inducible sites - PubMed (original) (raw)

. 2000 Feb 1;14(3):289-300.

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The human homologs of checkpoint kinases Chk1 and Cds1 (Chk2) phosphorylate p53 at multiple DNA damage-inducible sites

S Y Shieh et al. Genes Dev. 2000.

Erratum in

Abstract

Upon DNA damage, the amino terminus of p53 is phosphorylated at a number of serine residues including S20, a site that is particularly important in regulating stability and function of the protein. Because no known kinase has been identified that can modify this site, HeLa nuclear extracts were fractionated and S20 phosphorylation was followed. We discovered that a S20 kinase activity copurifies with the human homolog of the Schizosaccharomyces pombe checkpoint kinase, Chk1 (hCHK1). We confirmed that recombinant hCHK1, but not a kinase-defective version of hCHK1, can phosphorylate p53 in vitro at S20. Additional inducible amino- and carboxy-terminal sites in p53 are also phosphorylated by hCHK1, indicating that this is an unusually versatile protein kinase. It is interesting that hCHK1 strongly prefers tetrameric to monomeric p53 in vitro, consistent with our observation that phosphorylation of amino-terminal sites in vivo requires that p53 be oligomeric. Regulation of the levels and activity of hCHK1 in transfected cells is directly correlated with the levels of p53; expression of either a kinase-defective hCHK1 or antisense hCHK1 leads to reduced levels of cotransfected p53, whereas overexpression of wild-type hCHK1 or the kinase domain of hCHK1 results in increased levels of expressed p53 protein. The human homolog of the second S. pombe checkpoint kinase, Cds1 (CHK2/hCds1), phosphorylates tetrameric p53 but not monomeric p53 in vitro at sites similar to those phosphorylated by hCHK1 kinase, suggesting that both checkpoint kinases can play roles in regulating p53 after DNA damage.

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Figures

Figure 1

Figure 1

p53 S20 kinase cofractionates with hCHK1. HeLa cell nuclear extracts were fractionated first through a phosphocellulose P11 column. Aliquots from each fraction were taken and used in kinase assays followed by Western analyses with anti-phosphoserine antibodies directed against p53 phosphorylated at S15, S20, S33, and S37 to monitor the specific kinase activity. The P11 0.85 fraction was then passed through Mono-Q Sepharose and fractions that contained the S20 kinase activity were pooled and further fractionated through a heparin column. (Inset) (Western blot) shows the coelution of S20 kinase activity with hCHK1 in fractions eluting from the heparin column.

Figure 2

Figure 2

Recombinant hCHK1 phosphorylates p53 in vitro. (A) hCHK1 phosphorylates p53 at both S15 and S20, whereas DNA–PK targets S15 but not S20. His-tagged p53 prepared from bacteria was incubated alone, or with purified DNA–PK in the presence or absence of DNA as indicated, or with GST–hCHK1 in the absence of DNA. Phosphorylation of p53 was determined by Western blotting with respective anti-phospho-S15 and anti-phospho-S20 antibodies. The level of p53 in each reaction was then determined by stripping the blot and reprobing with an anti-His-tag antibody. (B) The kinase activity of hCHK1 is not affected by DNA or wortmannin. Kinase assays were carried out as described above in the presence or absence of 100 ng of DNA, and/or 0.5 μ

m

(lanes 4,7), 1 μ

m

(lane 8), or 2 μ

m

(lane 9) wortmannin. The mixtures were analyzed by SDS-PAGE, and the gel was dried and autoradiographed. (C) S37, but not S33 or S392, is phosphorylated by hCHK1. His-tagged p53 was incubated with wild-type (WT) or the kinase-defective mutant (D130A) of GST–hCHK1. The reactions were then analyzed by Western blotting with either anti-phospho-S33, S37, or S392 antibodies. Equal loading was demonstrated by reprobing the membrane with anti-His antibody, which recognizes p53, and anti-GST antibody, which recognizes hCHK1.

Figure 2

Figure 2

Recombinant hCHK1 phosphorylates p53 in vitro. (A) hCHK1 phosphorylates p53 at both S15 and S20, whereas DNA–PK targets S15 but not S20. His-tagged p53 prepared from bacteria was incubated alone, or with purified DNA–PK in the presence or absence of DNA as indicated, or with GST–hCHK1 in the absence of DNA. Phosphorylation of p53 was determined by Western blotting with respective anti-phospho-S15 and anti-phospho-S20 antibodies. The level of p53 in each reaction was then determined by stripping the blot and reprobing with an anti-His-tag antibody. (B) The kinase activity of hCHK1 is not affected by DNA or wortmannin. Kinase assays were carried out as described above in the presence or absence of 100 ng of DNA, and/or 0.5 μ

m

(lanes 4,7), 1 μ

m

(lane 8), or 2 μ

m

(lane 9) wortmannin. The mixtures were analyzed by SDS-PAGE, and the gel was dried and autoradiographed. (C) S37, but not S33 or S392, is phosphorylated by hCHK1. His-tagged p53 was incubated with wild-type (WT) or the kinase-defective mutant (D130A) of GST–hCHK1. The reactions were then analyzed by Western blotting with either anti-phospho-S33, S37, or S392 antibodies. Equal loading was demonstrated by reprobing the membrane with anti-His antibody, which recognizes p53, and anti-GST antibody, which recognizes hCHK1.

Figure 2

Figure 2

Recombinant hCHK1 phosphorylates p53 in vitro. (A) hCHK1 phosphorylates p53 at both S15 and S20, whereas DNA–PK targets S15 but not S20. His-tagged p53 prepared from bacteria was incubated alone, or with purified DNA–PK in the presence or absence of DNA as indicated, or with GST–hCHK1 in the absence of DNA. Phosphorylation of p53 was determined by Western blotting with respective anti-phospho-S15 and anti-phospho-S20 antibodies. The level of p53 in each reaction was then determined by stripping the blot and reprobing with an anti-His-tag antibody. (B) The kinase activity of hCHK1 is not affected by DNA or wortmannin. Kinase assays were carried out as described above in the presence or absence of 100 ng of DNA, and/or 0.5 μ

m

(lanes 4,7), 1 μ

m

(lane 8), or 2 μ

m

(lane 9) wortmannin. The mixtures were analyzed by SDS-PAGE, and the gel was dried and autoradiographed. (C) S37, but not S33 or S392, is phosphorylated by hCHK1. His-tagged p53 was incubated with wild-type (WT) or the kinase-defective mutant (D130A) of GST–hCHK1. The reactions were then analyzed by Western blotting with either anti-phospho-S33, S37, or S392 antibodies. Equal loading was demonstrated by reprobing the membrane with anti-His antibody, which recognizes p53, and anti-GST antibody, which recognizes hCHK1.

Figure 3

Figure 3

Efficient phosphorylation of the p53 amino terminus by hCHK1 requires the tetramerization domain. (A) GST–hCHK1 was incubated with either His-tagged wild-type (WT, lane 2) or truncated 1–363 (lane 3), 1–307 (lane 4) p53 proteins prepared from bacteria. Phosphorylation was visualized either by autoradiography or by Western blotting with anti-phospho-S15 or anti-phospho-S20 antibodies. Levels of p53 proteins were detected by reprobing the blot with the p53 monoclonal antibody PAb1801. (B) HA-tagged wild-type (WT) p53 or a deletion mutant dlTD, which carries an internal deletion in the tetramerization domain, was prepared from baculovirus-infected insect cells and incubated alone (lanes 1,2) or with GST–hCHK1 (lanes 4,5). Phosphorylation was then determined as described in A.

Figure 4

Figure 4

Modulation of hCHK1 affects p53 levels in vivo. (A) Down-regulation of hChk-1 p53 decreases p53 levels in vivo. Constructs expressing p53 and either wild- type hCHK1 (chk1), the kinase-dead hCHK1 mutant D130A, or antisense CHK1 (chk1AS) were transfected into p53-null H1299 cells. Cells were collected and lysed 36–40 hr after transfection and levels of p53 and phosphorylation at S15 were determined by Western blotting with PAb1801 and anti-phospho-S15 antibodies, respectively. (B) Overexpression of hCHK1 increases p53 levels in vivo. MCF7 cells were transfected with p53 together with either empty vector, a short form of hCHK1 that contains the amino-terminal kinase domain alone (chk1-s), or full-length hCHK1. Cells were treated 24 hr after transfection with either γ (7 Gy) or UV (50 J/m2) irradiation, and harvested 2 hr and 5 hr later, respectively. Levels of p53 were determined by Western blotting with antibodies PAb421 and PAb1801. A nonspecific (NS) band detected during the probing was used as a loading control. (C) Relative levels of p53 shown in B (Exp 1) as well as two other experiments (Exp 2 and Exp 3) were quantified by densitometry.

Figure 4

Figure 4

Modulation of hCHK1 affects p53 levels in vivo. (A) Down-regulation of hChk-1 p53 decreases p53 levels in vivo. Constructs expressing p53 and either wild- type hCHK1 (chk1), the kinase-dead hCHK1 mutant D130A, or antisense CHK1 (chk1AS) were transfected into p53-null H1299 cells. Cells were collected and lysed 36–40 hr after transfection and levels of p53 and phosphorylation at S15 were determined by Western blotting with PAb1801 and anti-phospho-S15 antibodies, respectively. (B) Overexpression of hCHK1 increases p53 levels in vivo. MCF7 cells were transfected with p53 together with either empty vector, a short form of hCHK1 that contains the amino-terminal kinase domain alone (chk1-s), or full-length hCHK1. Cells were treated 24 hr after transfection with either γ (7 Gy) or UV (50 J/m2) irradiation, and harvested 2 hr and 5 hr later, respectively. Levels of p53 were determined by Western blotting with antibodies PAb421 and PAb1801. A nonspecific (NS) band detected during the probing was used as a loading control. (C) Relative levels of p53 shown in B (Exp 1) as well as two other experiments (Exp 2 and Exp 3) were quantified by densitometry.

Figure 5

Figure 5

Additional hCHK1 phosphorylation site(s) in the p53 carboxy-terminal domain (A) GST–hCHK1 or kinase-defective GST–CHK1 proteins were incubated with either WT p53 or truncated p53 proteins as indicated. Phosphorylation was detected by autoradiography (top). The proteins were also transferred to a nitrocellulose membrane and detected by Western blotting with anti-GST antibody for detecting hCHK1 and anti-His for detecting p53 proteins (bottom). (B) Autoradiograph of a kinase assay done similarly as in A with GST–CHK1 and 300 ng of the indicated p53 proteins. (dlN96) Amino acids 97–393; (dlN96dlC30) amino acids 97–363; (core) amino acids 97–305; (T+B) amino acids 311–393; (N159) amino acids 1–159.

Figure 6

Figure 6

CHK2/hCds1 phosphorylates p53 tetramers in vitro. (A) Phosphorylation of the p53 amino-terminal domain by CHK2 at S15 and S20 in vitro. His- and Flag-tagged CHK2/hCds1 prepared from bacteria or Flag-tagged CHK2/Cds1 prepared from baculovirus-infected insect cells was incubated alone or with His–p53 and the resulting phosphorylation was assessed by autoradiography or by Western blotting with anti-phosphoserine-specific antibodies. (B) CHK2/hCds1 phosphorylates additional sites in the amino terminus of p53. Wild-type Flag–CHK2/hCds1 or a kinase-dead mutant (D347A) prepared from baculovirus-infected insect cells was used to phosphorylate p53, and the phosphorylation status of p53 was analyzed as described above. (C) CHK2/hCds1 phosphorylates p53 tetramers but not monomers at S15 and S20. Flag–CHK2/Cds1 was incubated with either His-tagged wild-type (WT, lane 2), truncated 1–363 (lane 3), or 1–307 (lane 4) p53 proteins prepared from bacteria. Phosphorylation was visualized either by autoradiography (32P labeling) or by Western blotting with anti-phospho-S15 or anti-phospho-S20 antibodies. Levels of p53 proteins were detected by reprobing the blot with the p53 monoclonal antibody PAb1801. (D) HA-tagged wild-type (WT) p53 or the deletion mutant dlTD that lacks a functional tetramerization domain were incubated with bacterially expressed his–hCHK2 . Phosphorylation was then determined as described in A and C.

Figure 6

Figure 6

CHK2/hCds1 phosphorylates p53 tetramers in vitro. (A) Phosphorylation of the p53 amino-terminal domain by CHK2 at S15 and S20 in vitro. His- and Flag-tagged CHK2/hCds1 prepared from bacteria or Flag-tagged CHK2/Cds1 prepared from baculovirus-infected insect cells was incubated alone or with His–p53 and the resulting phosphorylation was assessed by autoradiography or by Western blotting with anti-phosphoserine-specific antibodies. (B) CHK2/hCds1 phosphorylates additional sites in the amino terminus of p53. Wild-type Flag–CHK2/hCds1 or a kinase-dead mutant (D347A) prepared from baculovirus-infected insect cells was used to phosphorylate p53, and the phosphorylation status of p53 was analyzed as described above. (C) CHK2/hCds1 phosphorylates p53 tetramers but not monomers at S15 and S20. Flag–CHK2/Cds1 was incubated with either His-tagged wild-type (WT, lane 2), truncated 1–363 (lane 3), or 1–307 (lane 4) p53 proteins prepared from bacteria. Phosphorylation was visualized either by autoradiography (32P labeling) or by Western blotting with anti-phospho-S15 or anti-phospho-S20 antibodies. Levels of p53 proteins were detected by reprobing the blot with the p53 monoclonal antibody PAb1801. (D) HA-tagged wild-type (WT) p53 or the deletion mutant dlTD that lacks a functional tetramerization domain were incubated with bacterially expressed his–hCHK2 . Phosphorylation was then determined as described in A and C.

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