Protein kinases and the response to DNA damage

doi:10.1006/scel.1994.1050

Seminars in Cell Biology

Volume 5, Issue 6, December 1994, Pages 427-436

https://doi.org/10.1006/scel.1994.1050 Get rights and content

Abstract

Exposure of eukaryotic cells to DNA damaging agents induces gene expression and inhibits cell cycle progression. The first response to damage that has been measured is an increase in protein kinase activity. Several protein kinases are activated in cells exposed to DNA damaging agents, and other kinases that correct damage hypersensitivity mutants have been identified genetically. Current challenges include determining which responses result from damage to DNA and identifying the immediate detectors of damaged DNA that initiate signal transduction.

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Cited by (34)

Nitric oxide prevents UV-induced phosphorylation of the p53 tumor-suppressor protein at serine 46: A possible role in inhibition of apoptosis
2003, Biochemical and Biophysical Research Communications
Citation Excerpt :
This inhibitor strongly suppressed the UV-induced phosphorylation of the kinase substrates (ATM/ATR substrates and akt) (Fig. 3B). Since in UV-irradiated cells only ATR, but not ATM, plays a major role in p53 phosphorylation [25–28], increased phosphorylation of ATM/ATR kinase substrate upon UV irradiation is considered to be mediated by increased activity of ATR kinase. Treatment with L-JNK1, the JNK specific inhibitor, did not affect the UV-induced p53 phosphorylation at Ser-15 and Ser-46 (Fig. 3C).
Ser-46 of p53 is phosphorylated in response to DNA-damage in vivo, and it plays a pivotal role for apoptotic signaling by p53 through regulating the transcriptional activation of genes involved in apoptosis. We found that the presence of the nitric oxide (NO) donor S-nitroso-N-acetyl-d,l-penicillamine (200–800 μM) during UV-irradiation of MCF-7 cells resulted in a significant reduction in the Ser-46 phosphorylation, compared to the UV-irradiated cells without NO. This reduction occurred independently of cyclic GMP generation and without affecting activities of p53 kinases such as the PI3K family, p38 MAPK, and HIPK2. The presence of NO was found to protect HCT116 human colon tumor cells containing wild-type p53 from UV-induced apoptosis, whereas no apparent inhibitory effect of NO on UV-induced apoptosis was observed in those deficient in p53. Our results suggest that NO-mediated protection of apoptosis is p53-dependent, occurring at least partly through NO-inhibition of phosphorylation of p53 on Ser-46.
Cellular responses to the DNA strand-scission enediyne C-1027 can be independent of ATM, ATR, and DNA-PK kinases
2002, Journal of Biological Chemistry
The current paradigm based upon ionizing radiation (IR) studies states that cells deficient in either ataxia-telangiectasia-mutated kinase (ATM) or related phosphatidylinositol 3 (PI 3) -kinases (ATR and DNA-PK) are hypersensitive to DNA strand breaks because they are unable to rapidly activate downstream effectors such as p53. Here we have contrasted cell responses to IR and C-1027, a radiomimetic antibiotic that induces DNA strand breaks. At equal levels of DNA double strand breaks, cell lines with inactive ATM or other phosphatidylinositol 3-kinases displayed classical hypersensitivity to IR but not to C-1027. Moreover, phosphorylation of p53 Ser-15 induced by C-1027 was independent of ATM, ATR, or DNA-PK function. We have concluded that the model based on IR studies cannot always be directly applied to DNA damage induced by other strand-scission agents.
Transcriptional effects of the potent enediyne anti-cancer agent calicheamicin γ<inf>1</inf><sup>I</sup>
2002, Chemistry and Biology
We have investigated the mode of action of calicheamicin in living cells by using oligonucleotide microarrays to monitor its effects on gene expression across the entire yeast genome. Transcriptional effects were observed as early as 2 min into drug exposure. Among these effects were the upregulation of two nuclear proteins encoding a Y′-helicase (a subtelomerically encoded protein whose function is to maintain telomeres) and a suppressor of rpc10 and rpb40 mutations (both rpc10 and rpb40 encode RNA polymerase subunits). With longer calicheamicin exposure, genes involved in chromatin arrangement, DNA repair and/or oxidative damage, DNA synthesis and cell cycle checkpoint control as well as other nuclear proteins were all differentially expressed. Additionally, ribosomal proteins and a variety of metabolic, biosynthetic, and stress response genes were also altered in their expression.
Human p53 is phosphorylated on serines 6 and 9 in response to DNA damage-inducing agents
2000, Journal of Biological Chemistry
Citation Excerpt :
However, the peptide p53(Ac-1–12)(6P), was readily phosphorylated by CK1-δ. The response of mammalian cells to DNA damage is complex (reviewed in Refs. 24-26). Cell cycle progression is controlled by several checkpoints that are activated by DNA damage, by other stresses, and by mechanisms that ensure the orderly progression of cell cycle events.
To characterize the sites in human p53 that become phosphorylated in response to DNA damage, we have developed polyclonal antibodies that recognize p53 only when it is phosphorylated at specific sites. Several attempts to generate an antibody to p53 phosphorylated at Ser⁶ using a phosphoserine-containing peptide as an immunogen were unsuccessful; however, phosphorylation-specific antibodies were produced by using the phosphoserine mimetic,l-2-amino-4-phosphono-4,4-difluorobutanoic acid (F₂Pab), in place of phosphoserine. Fmoc-F₂Pab was prepared by an improved synthesis and chemically incorporated using solid phase peptide synthesis. Affinity-purified antibodies elicited by immunizing rabbits with an F₂Pab peptide coupled to keyhole limpet hemocyanin recognized a p53(1–39) peptide phosphorylated only at Ser⁶ but not the unphosphorylated peptide or the same peptide phosphorylated at Ser⁹, Ser¹⁵, Ser²⁰, Ser³³, or Ser³⁷. Untreated A549 cells exhibited a background of constitutive phosphorylation at Ser⁶ that increased approximately 10-fold upon exposure to either ionizing radiation or UV light. Similar results were obtained for Ser⁹ using antibodies raised against a conventional phosphopeptide. Ser⁹ was phosphorylated by casein kinase 1in vitro in a phosphoserine 6-dependent manner. Our data identify two additional DNA damage-induced phosphorylations in human p53 and show that F₂Pab-derivatized peptides can be used to develop phosphorylation site-specific polyclonal antibodies.
Post-translational modification of p53 protein in response to ionizing radiation analyzed by mass spectrometry
2000, Journal of Molecular Biology
The p53 tumor suppressor protein promotes cell cycle arrest or apoptosis in response to DNA damage and other forms of stress. p53 protein functions as a transcription factor by binding to specific DNA sequences and regulating the transcription of target genes. This activity of p53 is reported to be regulated by phosphorylation and acetylation occuring at various sites on the molecule. Here, we have used a direct and non-radioactive approach involving mass spectrometric analysis of p53 protein to identify sites that are covalently modified in vivo, either constitutively or in response to ionizing radiation. Following partial purification by immuno-affinity chromatography and enzymatic in-gel digestion, the resulting p53 peptides were analyzed by MALDI-TOF and nanoelectrospray mass spectrometry. Mass spectrometry analyses identified four sites at the N terminus that were phosphorylated in response to irradiation, a single constitutive phosphorylation site at serine 315 and several acetylation sites.
Multisite phosphorylation and the integration of stress signals at p53
1998, Cellular Signalling
The p53 tumour suppressor protein is a potent transcription factor that plays a major role in the defence against tumour development. p53 exists in a latent form that can be activated by a range of stresses including DNA damage, hypoxia, cytokines, metabolic changes, viral infection, and activated oncogenes. Activation of p53 can lead to cellular growth arrest prior to entry into either S phase or mitosis or can trigger cell death through apoptosis. The modification of p53 by multisite phosphorylation provides a potential link between stress signalling and the regulation of p53 activity, and there is now striking evidence that agents that activate p53 can lead to selective changes in its phosphorylation status. Topologically, the phosphorylation sites in p53 fall into two discrete functional domains. Four phosphorylation events take place within the N-terminal 83 amino acids containing the transactivation domain and a region involved in transcription-independent growth suppression. At least three of these modifications occur in response to agents that cause cellular stress such as DNA damage. At the C-terminus, there are three phosphorylation events, each of which can independently regulate the specific DNA-binding function of p53, suggesting convergent control by different signalling pathways. The multiplicity of these covalent modifications and their responsiveness to a wide range of signals suggest that p53 activity is tightly and coordinately controlled in response to stresses and changes in the cellular environment.

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Regular ArticleProtein kinases and the response to DNA damage

Abstract

Regular Article
Protein kinases and the response to DNA damage