Artemis links ATM to G2/M checkpoint recovery via regulation of Cdk1-cyclin B - PubMed (original) (raw)

Liyi Geng et al. Mol Cell Biol. 2007 Apr.

Abstract

Artemis is a phospho-protein that has been shown to have roles in V(D)J recombination, nonhomologous end-joining of double-strand breaks, and regulation of the DNA damage-induced G(2)/M cell cycle checkpoint. Here, we have identified four sites in Artemis that are phosphorylated in response to ionizing radiation (IR) and show that ATM is the major kinase responsible for these modifications. Two of the sites, S534 and S538, show rapid phosphorylation and dephosphorylation, and the other two sites, S516 and S645, exhibit rapid and prolonged phosphorylation. Mutation of both of these latter two residues results in defective recovery from the G(2)/M cell cycle checkpoint. This defective recovery is due to promotion by mutant Artemis of an enhanced interaction between unphosphorylated cyclin B and Cdk1, which in turn promotes inhibitory phosphorylation of Cdk1 by the Wee1 kinase. In addition, we show that mutant Artemis prevents Cdk1-cyclin B activation by causing its retention in the centrosome and inhibition of its nuclear import during prophase. These findings show that ATM regulates G(2)/M checkpoint recovery through inhibitory phosphorylations of Artemis that occur soon after DNA damage, thus setting a molecular switch that, hours later upon completion of DNA repair, allows activation of the Cdk1-cyclin B complex. These findings thus establish a novel function of Artemis as a regulator of the cell cycle in response to DNA damage.

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Figures

FIG. 1.

FIG. 1.

The role of ATM and DNA-PK in the phosphorylation of Artemis at specific SQ sites after IR treatment is dose dependent. (A) Schematic of Artemis showing (S/T)Q motifs conserved between human and mouse proteins. (B) Specificity of Artemis phospho-specific peptide antibodies to pS516, pS534, pS538, and pS645 as shown by immunoblot analysis. GST fusions of wild-type and the indicated Artemis S-to-A mutants (GST-Artemis mt) were stably expressed in HEK293 cells and exposed to either IR or UV. Rows A, D, G, and J show the signal detected from wild-type and mutant Artemis using the phospho-specific antibodies; rows B, E, H, and K show the lack of signal when the phospho-specific antibodies were preincubated with the appropriate phospho-peptide; and rows C, F, I, and L were probed with an Artemis polyclonal antibody. (C) Phospho-specific antibodies were used to probe the modification of Artemis at either low-dose (2 Gy) or high-dose (10 Gy) IR in the presence or absence of caffeine or wortmannin (wort). (D) Cells were depleted of ATM or ATM and ATR by siRNA treatment, and lysates were probed with the indicated antibodies. (E) Cells were depleted of DNA-PKcs by siRNA treatment and exposed to either low-dose or high-dose IR. Cell lysates were subsequently probed with the indicated antibodies. UT, untreated cells.

FIG. 2.

FIG. 2.

Mutation of the S516 and S645 phosphorylation sites in Artemis causes a prolonged G2/M delay after IR. (A) Immunoblot showing kinetics of phosphorylation and dephosphorylation of GST-Artemis at the indicated sites after exposure to IR. (B) Immunoblot showing expression of the indicated Artemis wild-type and mutant proteins in HEK293 cells. (C) Cell cycle analysis after IR of HEK293 cells stably expressing wild-type Artemis or the S516/645A or S516/645D mutant. (D) Graphical analysis of results shown in panel C. (E) Phospho-histone H3 (P-H3) staining analysis of wild-type and mutant Artemis cell lines after exposure to IR. (F) Cell cycle analysis of Artemis-expressing cells pulse-labeled with BrdU and immediately exposed to IR. WT, wild type; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

FIG. 3.

FIG. 3.

The effects of Artemis dysfunction on the G2/M checkpoint are not due to a failure to repair DSBs. (A) Cell cycle analysis of HEK293 cells after exposure to IR upon depletion of Artemis by siRNA. (B) Immunoblot showing knockdown of Artemis in HEK293 cells by siRNA. (C) Cell cycle analysis of cells depleted of Artemis by siRNA, pulse-labeled with BrdU, and exposed to IR. (D) Phospho-histone H3 (P-H3) staining analysis of cells depleted of Artemis by siRNA and exposed to IR. (E and F) Levels of γ-H2AX after IR in HEK293 cells were determined in cells depleted of Artemis by siRNA or stably expressing the S516/645A or S516/645D mutants. WT, wild type; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

FIG. 4.

FIG. 4.

Artemis affects Cdk1 inhibitory phosphorylation through regulation of cyclin B after IR. (A) Immunoblot analysis showing Cdk1 isoforms in HEK293 cells stably expressing the S516/645A mutant compared to cells expressing wild-type Artemis or the S516/645D mutant. (B) Immunoblot showing Cdk1 and cyclin B after depletion of Artemis by siRNA. (C) Immunoprecipitations (IP) of cyclin B from the indicated cell lysates were blotted with antibodies to Cdk1 and Wee1. (D) Lysates from the indicated cell lines were fractionated into cytoplasmic (C) or nuclear (N) fractions and probed with antibodies to the indicated proteins. Cyclin B-P indicates the phosphorylated protein. (E) Immunoprecipitations of Artemis from the indicated cell lysates were blotted with cyclin B antibody. WT, wild type; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

FIG. 5.

FIG. 5.

Artemis regulates the nuclear import of Cdk1-cyclin B from the centrosome. Shown are immunohistochemical analyses of cells expressing wild-type Artemis or the S516/645A and S516/645D mutants using the indicated antibodies. All cells were fixed 18 h after IR exposure. Arrowheads indicate centrosomes in panels C and D.

FIG. 5.

FIG. 5.

Artemis regulates the nuclear import of Cdk1-cyclin B from the centrosome. Shown are immunohistochemical analyses of cells expressing wild-type Artemis or the S516/645A and S516/645D mutants using the indicated antibodies. All cells were fixed 18 h after IR exposure. Arrowheads indicate centrosomes in panels C and D.

FIG. 6.

FIG. 6.

A schematic model depicting the role of Artemis in the cellular response to IR. Dashed line indicates postulated pathway. See the text for additional details.

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