DNA-PK phosphorylation sites on Oct-1 promote cell survival following DNA damage

The Ku heterodimer (Ku70/Ku80) is a main component of the nonhomologous end-joining (NHEJ) pathway that repairs DNA double-strand breaks (DSBs). Ku binds the broken DNA end and recruits other proteins to facilitate the processing and ligation of the broken end. While Ku interacts with many proteins involved in DNA damage/repair-related functions, few interactions have been mapped to the N-terminal von Willebrand A (vWA) domain, a predicted protein interaction domain. The mutagenesis of Ku70 vWA domain S155/D156 unexpectedly increased cell survival following ionizing radiation (IR) treatment. DNA repair appeared unaffected, but defects in the activation of apoptosis and alterations in the DNA damage signaling response were identified. In particular, Ku70 S155A/D156A affected the IR-induced transcriptional response of several activating transcription factor 2 (ATF2)-regulated genes involved in apoptosis regulation. ATF2 phosphorylation and recruitment to DNA damage-induced foci was increased in Ku70-deficient cells, suggesting that Ku represses ATF2 activation. Ku70 S155A/D156A substitutions further enhanced this repression. S155A substitution alone was sufficient to confer enhanced survival, whereas alteration to a phosphomimetic residue (S155D) reversed this effect, suggesting that S155 is a phosphorylation site. Thus, these findings infer that Ku links signals from the DNA repair machinery to DNA damage signaling regulators that control apoptotic pathways.

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“…Whole-cell extracts were prepared as described previously (64). Nuclear extracts were prepared as described previously (2).…”

Section: Methodsmentioning

confidence: 99%

Ku Regulates Signaling to DNA Damage Response Pathways through the Ku70 von Willebrand A Domain

Fell

Schild-Poulter

2012

Molecular and Cellular Biology

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“…Briefly, InR1-G9 cells were incubated with 8-orthophosphate (H3 32 PO 4 , 1 mCi per 10-cm plate) in the presence or absence of the indicated reagents for 2 h. Cell lysates were collected for immunoprecipitation against Oct-1. The amount of 32 P-labeled Oct-1 in the immunoprecipitation eluate was then assessed by SDS-PAGE, followed by x-ray film exposure (40).…”

Section: Methodsmentioning

confidence: 99%

POU Homeodomain Protein Oct-1 Functions as a Sensor for Cyclic AMP

Wang

Sun

et al. 2009

Journal of Biological Chemistry

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Cyclic AMP is a fundamentally important second messenger for numerous peptide hormones and neurotransmitters that control gene expression, cell proliferation, and metabolic homeostasis. Here we show that cAMP works with the POU homeodomain protein Oct-1 to regulate gene expression in pancreatic and intestinal endocrine cells. This ubiquitously expressed transcription factor is known as a stress sensor. We found that it also functions as a repressor of Cdx-2, a proglucagon gene activator. Through a mechanism that involves the activation of exchange protein activated by cyclic AMP, elevation of cAMP leads to enhanced phosphorylation and nuclear exclusion of Oct-1 and reduced interactions between Oct-1 or nuclear co-repressors and the Cdx-2 gene promoter, detected by chromatin immunoprecipitation. In rat primary pancreatic islet cells, cAMP elevation also reduces nuclear Oct-1 content, which causes increased proglucagon and proinsulin mRNA expression. Our study therefore identifies a novel mechanism by which cAMP regulates hormone-gene expression and suggests that ubiquitously expressed Oct-1 may play a role in metabolic homeostasis by functioning as a sensor for cAMP.Many peptide hormones and neurotransmitters use the second messengers, such as cyclic AMP (cAMP), to exert their biological functions, including regulation of gene expression and metabolic homeostasis (1-7). Extensive studies have shown that in addition to the activation of protein kinase A (PKA), 5 cAMP is able to trigger intracellular signaling events via other mechanisms, including Epac (the activation of exchangeprotein directly activated by cAMP) (8 -13). As nonkinase effectors of cAMP, Epac molecules are evidently involved in regulating gene expression, cell adhesion, and pancreatic peptide hormone secretion (4,10,12,14). In pancreatic islet and intestinal endocrine L cells, cAMP elevation is associated with increased expression of proglucagon (gcg) or proinsulin genes (15, 16). Expression of these two hormone-encoding genes is also controlled by transcriptional activators, including certain homeodomain proteins such as the caudal homeodomain protein Cdx-2 (17-22). We have demonstrated previously that in both PKA-active and PKA-deficient pancreatic and intestinal proglucagon-producing endocrine cell lines, cAMP elevation leads to increased Cdx-2 expression (23). Furthermore, Cdx-2 expression in a PKA-deficient pancreatic islet InR1-G9 cell line can be activated by an Epac pathway-specific cAMP analogue 8-pMeOPT-2Ј-O-Me-cAMP (23). More recently, we have observed expression of Epac2 in pancreatic and intestinal proglucagon-producing cells and demonstrated that Epac signaling serves as the mediator of cAMP in regulating the expression of gcg (14).In this study, we further explored mechanistically how cAMP-Epac signaling activates Cdx-2 expression in pancreatic and intestinal endocrine cells. Our observations suggest the existence of a novel mechanism by which cAMP regulates pancreatic and intestinal hormone-gene expression. It is likely that this r...

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“…As a result, the expression of Histone H2B and U2 RNA is decreased in order for cells to survive. [8][9][10] It was identified that there are 13 Ser/Thr potential phosphorylation sites in the Oct-1 molecule by DNA-PK. Substitution of Ser/ Thr by Ala eliminated Oct-1 phosphorylation in response to IR.…”

Section: Oct-1 Is a Stress Sensormentioning

confidence: 99%