Phosphorylation on Thr-55 by TAF1 mediates degradation of p53: a role for TAF1 in cell G1 progression

The largest subunit of TFIID, TAF1, possesses an intrinsic protein kinase activity and is important for cell G1 progression and apoptosis. Since p53 functions by inducing cell G1 arrest and apoptosis, we investigated the link between TAF1 and p53. We found that TAF1 induces G1 progression in a p53-dependent manner. TAF1 interacts with and phosphorylates p53 at Thr-55 in vivo. Substitution of Thr-55 with an alanine residue (T55A) stabilizes p53 and impairs the ability of TAF1 to induce G1 progression. Furthermore, both RNAi-mediated TAF1 ablation and apigenin-mediated inhibition of the kinase activity of TAF1 markedly reduced Thr-55 phosphorylation. Thus, phosphorylation and the resultant degradation of p53 provide a mechanism for regulation of the cell cycle by TAF1. Significantly, the Thr-55 phosphorylation was reduced following DNA damage, suggesting that this phosphorylation contributes to the stabilization of p53 in response to DNA damage.     

p16INK4a as a Second Effector of the Telomere Damage Pathway

Telomere damage resulting from telomere shortening can potentially suppress tumorigenesis by permanently arresting or eliminating incipient cancer cells. Dysfunctional telomeres activate the canonical DNA damage signaling pathway, resulting in a p53-mediated G1/S arrest and senescence or apoptosis. Experimental induction of telomere damage through inhibition of the telomeric protein TRF2 recapitulates aspects of telomere attrition, including a p53-mediated cell cycle arrest. Using this system, we have shown that telomere damage can also elicit a G1/S arrest through the RB-regulator p16INK4a, especially in cells lacking p53 function. Here we discuss the significance of p16INK4a as a second effector of the telomere damage response.     

Viral transport of DNA damage that mimics a stalled replication fork

Adeno-associated virus type 2 (AAV2) infection incites cells to arrest with 4N DNA content or die if the p53 pathway is defective. This arrest depends on AAV2 DNA, which is single stranded with inverted terminal repeats that serve as primers during viral DNA replication. Here, we show that AAV2 DNA triggers damage signaling that resembles the response to an aberrant cellular DNA replication fork. UV treatment of AAV2 enhances the G2 arrest by generating intrastrand DNA cross-links which persist in infected cells, disrupting viral DNA replication and maintaining the viral DNA in the single-stranded form. In cells, such DNA accumulates into nuclear foci with a signaling apparatus that involves DNA polymerase delta, ATR, TopBP1, RPA, and the Rad9/Rad1/Hus1 complex but not ATM or NBS1. Focus formation and damage signaling strictly depend on ATR and Chk1 functions. Activation of the Chk1 effector kinase leads to the virus-induced G2 arrest. AAV2 provides a novel way to study the cellular response to abnormal DNA replication without damaging cellular DNA. By using the AAV2 system, we show that in human cells activation of phosphorylation of Chk1 depends on TopBP1 and that it is a prerequisite for the appearance of DNA damage foci.     

ATR is not required for p53 activation but synergizes with p53 in the replication checkpoint.

ATR (ataxia telangiectasia and Rad-3-related) is a protein kinase required for survival after DNA damage. A critical role for ATR has been hypothesized to be the regulation of p53 and other cell cycle checkpoints. ATR has been shown to phosphorylate p53 at Ser(15), and this damage-induced phosphorylation is diminished by expression of a catalytically inactive (ATR-kd) mutant. p53 function could not be examined directly in prior studies of ATR, however, because p53 was mutant or because cells expressed the SV40 large T antigen that blocks p53 function. To test the interactions of ATR and p53 directly we generated human U2OS cell lines inducible for either wild-type or kinase-dead ATR that also have an intact p53 pathway. Indeed, ATR-kd expression sensitized these cells to DNA damage and caused a transient decrease in damage-induced serine 15 phosphorylation of p53. However, we found that the effects of ATR-kd expression do not result in blocking the response of p53 to DNA damage. Specifically, prior ATR-kd expression had no effect on DNA damage-induced p53 protein up-regulation, p53-DNA binding, p21 mRNA up-regulation, or G(1) arrest. Instead of promoting survival via p53 regulation, we found that ATR protects cells by delaying the generation of mitotic phosphoproteins and inhibiting premature chromatin condensation after DNA damage or hydroxyurea. Although p53 inhibition (by E6 or MDM2 expression) had little effect on premature chromatin condensation, when combined with ATR-kd expression there was a marked loss of the replication checkpoint. We conclude that ATR and p53 can function independently but that loss of both leads to synergistic disruption of the replication checkpoint.     

Poly(ADP-ribose) polymerase inhibition enhances p53-dependent and -independent DNA damage responses induced by DNA damaging agent

Targeting DNA repair with poly(ADP-ribose) polymerase (PARP) inhibitors has shown a broad range of anti-tumor activity in patients with advanced malignancies with and without BRCA deficiency. It remains unclear what role p53 plays in response to PARP inhibition in BRCA-proficient cancer cells treated with DNA damaging agents. Using gene expression microarray analysis, we find that DNA damage response (DDR) pathways elicited by veliparib (ABT-888), a PARP inhibitor, plus topotecan comprise the G1/S checkpoint, ATM, and p53 signaling pathways in p53-wildtype cancer cell lines and BRCA1, BRCA2 and ATR pathway in p53-mutant lines. In contrast, topotecan alone induces the G1/S checkpoint pathway in p53-wildtype lines and not in p53-mutant cells. These responses are coupled with G2/G1 checkpoint effectors p21CDKN1A upregulation, and Chk1 and Chk2 activation. The drug combination enhances G2 cell cycle arrest, apoptosis and a marked increase in cell death relative to topotecan alone in p53-wildtype and p53-mutant or -null cells. We also show that the checkpoint kinase inhibitor UCN-01 abolishes the G2 arrest induced by the veliparib and topotecan combination and further increases cell death in both p53-wildtype and -mutant cells. Collectively, PARP inhibition by veliparib enhances DDR and cell death in BRCA-proficient cancer cells in a p53-dependent and -independent fashion. Abrogating the cell-cycle arrest induced by PARP inhibition plus chemotherapeutics may be a strategy in the treatment of BRCA-proficient cancer.     

hMSH2 recruits ATR to DNA damage sites for activation during DNA damage-induced apoptosis

DNA damage response (DDR) activates a complex signaling network that triggers DNA repair, cell cycle arrest, and/or cell death. Depending on the type and severity of DNA lesion, DDR is controlled by "master" regulators including ATM and ATR protein kinases. Cisplatin, a major chemotherapy drug that cross-links DNA, induces ATR-dependent DDR, resulting in apoptosis. However, it is unclear how ATR is activated. To identify the key regulators of ATR, we analyzed the proteins that associate with ATR after cisplatin treatment by blue native-PAGE and co-immunoprecipitation. The mismatch repair protein hMSH2 was found to be a major ATR-binding protein. Functionally, ATR activation and its recruitment to nuclear foci during cisplatin treatment were attenuated, and DNA damage signaling, involving Chk2, p53, and PUMA-α, was suppressed in hMSH2-deficient cells. ATR activation induced by the DNA methylating agent N-methyl-N-nitrosourea was also shown to be hMSH2-dependent. Intriguingly, hMSH2-mediated ATR recruitment and activation appeared independent of replication protein A, Rad17, and the Rad9-Hus1-Rad1 protein complex. Together the results support a hMSH2-dependent pathway of ATR activation and downstream Chk2/p53 signaling.     

Role and regulation of p53 during an ultraviolet radiation-induced G1 cell cycle arrest.

p53 can play a key role in response to DNA damage by activating a G1 cell cycle arrest. However, the importance of p53 in the cell cycle response to UV radiation is unclear. In this study, we used normal and repair-deficient cells to examine the role and regulation of p53 in response to UV radiation. A dose-dependent G1 arrest was observed in normal and repair-deficient cells exposed to UV. Expression of HPV16-E6, or a dominant-negative p53 mutant that inactivates wildtype p53, caused cells to become resistant to this UV-induced G1 arrest. However, a G1 to S-phase delay was still observed after UV treatment of cells in which p53 was inactivated. These results indicate that UV can inhibit G1 to S-phase progression through p53-dependent and independent mechanisms. Cells deficient in the repair of UV-induced DNA damage were more susceptible to a G1 arrest after UV treatment than cells with normal repair capacity. Moreover, no G1 arrest was observed in cells that had completed DNA repair prior to monitoring their movement from G1 into S-phase. Finally, p53 was stabilized under conditions of a UV-induced G1 arrest and unstable when cells had completed DNA repair and progressed from G1 into S-phase. These results suggest that unrepaired DNA damage is the signal for the stabilization of p53, and a subsequent G1 phase cell cycle arrest in UV-irradiated cells.     

Telomere shortening triggers senescence of human cells through a pathway involving ATM, p53, and p21(CIP1), but not p16(INK4a)

Cellular senescence can be triggered by telomere shortening as well as a variety of stresses and signaling imbalances. We used multiparameter single-cell detection methods to investigate upstream signaling pathways and ensuing cell cycle checkpoint responses in human fibroblasts. Telomeric foci containing multiple DNA damage response factors were assembled in a subset of senescent cells and signaled through ATM to p53, upregulating p21 and causing G1 phase arrest. Inhibition of ATM expression or activity resulted in cell cycle reentry, indicating that stable arrest requires continuous signaling. ATR kinase appears to play a minor role in normal cells but in the absence of ATM elicited a delayed G2 phase arrest. These pathways do not affect expression of p16, which was upregulated in a telomere- and DNA damage-independent manner in a subset of cells. Distinct senescence programs can thus progress in parallel, resulting in mosaic cultures as well as individual cells responding to multiple signals.     

Distinct roles for DNA-PK,ATM and ATR in RPA phosphorylation and checkpoint activation in response to replication stress

DNA damage encountered by DNA replication forks poses risks of genome destabilization, a precursor to carcinogenesis. Damage checkpoint systems cause cell cycle arrest, promote repair and induce programed cell death when damage is severe. Checkpoints are critical parts of the DNA damage response network that act to suppress cancer. DNA damage and perturbation of replication machinery causes replication stress, characterized by accumulation of single-stranded DNA bound by replication protein A (RPA), which triggers activation of ataxia telangiectasia and Rad3 related (ATR) and phosphorylation of the RPA32, subunit of RPA, leading to Chk1 activation and arrest. DNA-dependent protein kinase catalytic subunit (DNA-PKcs) [a kinase related to ataxia telangiectasia mutated (ATM) and ATR] has well characterized roles in DNA double-strand break repair, but poorly understood roles in replication stress-induced RPA phosphorylation. We show that DNA-PKcs mutant cells fail to arrest replication following stress, and mutations in RPA32 phosphorylation sites targeted by DNA-PKcs increase the proportion of cells in mitosis, impair ATR signaling to Chk1 and confer a G2/M arrest defect. Inhibition of ATR and DNA-PK (but not ATM), mimic the defects observed in cells expressing mutant RPA32. Cells expressing mutant RPA32 or DNA-PKcs show sustained H2AX phosphorylation in response to replication stress that persists in cells entering mitosis, indicating inappropriate mitotic entry with unrepaired damage.