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Introduction: Obesity affects nearly 1 in 4 European adults increasing their risk for mortality and physical and psychological morbidity. Obesity is a chronic relapsing disease characterized by abnormal or excessive adiposity with risks to health. Medical nutrition therapy based on the latest scientific evidence should be offered to all Europeans living with obesity as part of obesity treatment interventions. Methods: A systematic review was conducted to identify the latest evidence published in the November 2018–March 2021 period and to synthesize them in the European guidelines for medical nutrition therapy in adult obesity. Results: Medical nutrition therapy should be administered by trained dietitians as part of a multidisciplinary team and should aim to achieve positive health outcomes, not solely weight changes. A diverse range of nutrition interventions are shown to be effective in the treatment of obesity and its comorbidities, and dietitians should consider all options and deliver personalized interventions. Although caloric restriction-based interventions are effective in promoting weight reduction, long-term adherence to behavioural changes may be better supported via alternative interventions based on eating patterns, food quality, and mindfulness. The Mediterranean diet, vegetarian diets, the Dietary Approaches to Stop Hypertension, portfolio diet, Nordic, and low-carbohydrate diets have all been associated with improvement in metabolic health with or without changes in body weight. In the November 2018–March 2021 period, the latest evidence published focused around intermittent fasting and meal replacements as obesity treatment options. Although the role of meal replacements is further strengthened by the new evidence, for intermittent fasting no evidence of significant advantage over and above continuous energy restriction was found. Pulses, fruit and vegetables, nuts, whole grains, and dairy foods are also important elements in the medical nutrition therapy of adult obesity. Discussion: Any nutrition intervention should be based on a detailed nutritional assessment including an assessment of personal values, preferences, and social determinants of eating habits. Dietitians are expected to design interventions that are flexible and person centred. Approaches that avoid caloric restriction or detailed eating plans (non-dieting approaches) are also recommended for improvement of quality of life and body image perceptions.

In Ireland, the number of individuals living with and beyond cancer equates to almost 4% of the population, projected to increase. Little is known about how cancer changes views on food and nutrition among cancer survivors. A patient-centered perspective is imperative for developing future health interventions in this cohort. This study aimed to explore these perceptions and views amongst Irish cancer survivors. Participants living across Ireland and who had completed active cancer treatment at least six months ago were eligible for this study. Semi-structured focus groups were conducted online via Microsoft Teams. Data was analysed using Braun and Clarke's updated six-phase process for reflexive thematic analysis. Five focus group interviews were held with cancer survivors (n=20). The cohort was predominately female (n=17) with a mean age of 51.3 ± 11.9 years. Three core themes emerged (i) the role of nutrition, (ii) cancer as a catalyst for change and (iii) the overall lack of nutritional support in oncology. Findings suggest how a cancer diagnosis prompts a re-evaluation of health status and may prime survivors toward healthier dietary choices. The views of our participants indicate that survivors long for guidance from health professionals about their nutrition and the side effects of their cancer treatment and medication that impact dietary intake. This article is protected by copyright. All rights reserved.

BackgroundMalnutrition is a global emergency, creating an overlapping burden on individual, public and economic health. The double burden of malnutrition affects approximately 2.3 billion adults worldwide. Following 3 years of capacity building work in Kolkata, with assistance of local volunteers and organisations, we established an empowering nutrition education model in the form of a ‘mobile teaching kitchen (MTK)’ with the aim of creating culinary health educators from lay slum-dwelling women.AimsTo evaluate the piloting of a novel MTK nutrition education platform and its effects on the participants, alongside data collection feasibility.MethodsOver 6 months, marginalised (RG Kar and Chetla slums) women underwent nutrition training using the MTK supported by dietitians, doctors and volunteers. Preintervention and postintervention assessments of knowledge, attitudes and practices (KAP), as well as anthropometric and clinical nutritional status of both the women and their children were recorded. The education was delivered by a ‘See One, Do One, Teach One’ approach with a final assessment of teaching delivery performed in the final session.ResultsTwelve women were trained in total, six from each slum. Statistically significant improvements were noted in sections of KAP, with improvements in nutrition knowledge (+4.8) and practices (+0.8). In addition, statistically significant positive changes were seen in ‘understanding of healthy nutrition for their children’ (p=0.02), ‘sources of protein rich food’ (p=0.02) and ‘not skipping meals if a child is ill’ (p≤0.001).ConclusionThe MTK as a public health intervention managed to educate, empower and upskill two groups of lay marginalised women into MTK Champions from the urban slums of Kolkata, India. Improvements in their nutrition KAP demonstrate just some of the effects of this programme. By the provision of healthy meals and nutritional messages, the MTK Champions are key drivers nudging improvements in nutrition and health related awareness with a ripple effect across the communities that they serve. There is potential to upscale and adapt this programme to other settings, or developing into a microenterprise model, that can help future MTK Champions earn a stable income.

The present study aimed to describe the proportion of cancer patients and survivors who receive nutrition advice, the sources of that advice and to explore the nutrition advice this cohort would like to have received.A descriptive cross-sectional survey was completed by 211 cancer patients and survivors recruited through two hospitals in the North-West of Ireland. The survey consisted of open-ended and closed questions that aimed to explore the provision of and sources of nutrition advice and desired guidance in this cohort. Descriptive analysis was conducted using SPPS, while open-ended questions underwent thematic analysis.Respondents were mainly female (n = 133; 63%), aged 50-69 years old (n = 118; 56%), attending the outpatient department (n = 128; 60.7%) and5 years since diagnosis (n = 150; 71.7%). Breast (n = 69; 32.7%) was the most common cancer type. Respondents experienced a mean ± SD of 5 ± 3.1 nutrition impact symptoms. The most common was changes in taste/smell (n = 122; 57.8%). Although 53.6% (n = 113) reported being given advice, only 34.1% (n = 72) received this advice from a dietitian. The main sources of nutritional advice were friends and family. Respondents expressed their desire for nutrition advice from a dietitian and that this should be individualised, clear and practical. There was also a desire to avoid misinformation and uncertainty.The results of the present study can be used to help tailor nutrition support for this group. There is a need to ensure that patients and survivors consistently receive evidence-based advice tailored to their needs, cancer type and treatment stage.

Ascertaining cancer survivors' specific needs and preferences regarding nutrition information delivery is important in developing and designing future health interventions in oncology. The present study aimed to explore the nutrition information needs of cancer survivors and their preferences regarding intervention delivery.The present study is a mixed-methods study comprising a cross-sectional survey and focus groups. Participants were eligible for inclusion if they were aged ≥ 18 years, living in Ireland with a cancer diagnosis and had completed treatment at least 6 months previously. Cancer support centres promoted the online survey on social media. Focus groups were conducted with 20 individuals.The cohort (n = 56) was predominantly female (n = 50; 89.3%) breast cancer survivors (n = 42; 75%). Seven (12.5%) had received nutrition advice from a dietitian. Most were interested in receiving nutrition advice (n = 52; 92.9%), with variability in delivery mode. There was interest in improving sleep quality (n = 35; 62.5%), making positive lifestyle changes (n = 28; 50%) and accessing cancer-specific physical activity classes (n = 27; 48.2%). The best time to deliver information was throughout the cancer journey (n = 31; 55.4%). Time and motivation (n = 15; 26.8%) were the main barriers. Facilitators were keeping healthy (n = 42; 75%) and weight maintenance (n = 31; 55.4%). Four themes emerged from thematic analysis: lack of nutrition guidance, an abundance of misinformation, one size does not fit all and dietitians as the preferred source of advice.There is a desire for evidence-based nutrition advice and for dietitians to be integrated into cancer clinics and cancer survivorship care. This research guides future nutrition interventions to improve cancer survivorship outcomes.

The purpose of this research was to explore the nutrition practices among post-treatment cancer survivors across Ireland. Cancer survivors aged 18+ years living across Ireland, who were not palliative and had completed active cancer treatment at least six months previous, were recruited to complete an online survey assessing dietary quality, food choice and satisfaction with food-related life as well as clinical and nutrition status. It was circulated by cancer support networks and on social media. Descriptive statistics are presented. The cohort (n = 170) was predominantly female (85.9%) and had breast cancer (64.7%). Mean age was 51.5 ± 10.9 years and 42.7% of the cohort were >five years post-treatment. Only 20% and 12% of the cohort had been assessed by a dietitian during and post-treatment, respectively. The mean dietary quality score was 10.3 ± 1.7, which was measured by the Leeds short-form food frequency questionnaire (SFFFQ). Using a 5-point Likert scale, the median satisfaction with food-related life score was 19 (3.3), which evaluates cognitive judgements on the person’s food-related life. The food choice questionnaire (FCQ) assesses the relative importance of a range of factors related to dietary choice to individuals. The primary determinant of food choice in this cohort was the natural content (31.7%) followed by health (24.7%). Vitamin and mineral supplement use was reported by 69.8% of the cohort; the most consumed was Vitamin D. Four themes emerged from an optional open-ended question: awareness of nutritional importance; desire for specific nutritional advice and dietetic referral; cancer and treatment nutrition impacts were highlighted; as well as struggles with weight gain. This research provides useful insight into the nutrition practices of Irish cancer survivors. A desire and need for individualised and specific advice are evident.

Processing bodies (PBs) are ribonucleoprotein granules important for cytokine mRNA decay that are targeted for disassembly by many viruses. Kaposi’s sarcoma-associated herpesvirus is the etiological agent of the inflammatory endothelial cancer, Kaposi’s sarcoma, and a PB-regulating virus. The virus encodes kaposin B (KapB), which induces actin stress fibers (SFs) and cell spindling as well as PB disassembly. We now show that KapB-mediated PB disassembly requires actin rearrangements, RhoA effectors, and the mechanoresponsive transcription activator, YAP. Moreover, ectopic expression of active YAP or exposure of ECs to mechanical forces caused PB disassembly in the absence of KapB. We propose that the viral protein KapB activates a mechanoresponsive signaling axis and links changes in cell shape and cytoskeletal structures to enhanced inflammatory molecule expression using PB disassembly. Our work implies that cytoskeletal changes in other pathologies may similarly impact the inflammatory environment.

The DNA-dependent protein kinase catalytic subunit (DNA-PKcs) has well-established roles in DNA double-strand break repair, and recently, nonrepair functions have also been reported. To better understand its cellular functions, we deleted DNA-PKcs from HeLa and A549 cells using CRISPR/Cas9. The resulting cells were radiation sensitive, had reduced expression of ataxia-telangiectasia mutated (ATM), and exhibited multiple mitotic defects. Mechanistically, nocodazole-induced upregulation of cyclin B1, anillin, and securin was decreased in DNA-PKcs-deficient cells, as were phosphorylation of Aurora A on threonine 288, phosphorylation of Polo-like kinase 1 (PLK1) on threonine 210, and phosphorylation of targeting protein for Xenopus Klp2 (TPX2) on serine 121., The DNA-dependent protein kinase catalytic subunit (DNA-PKcs) has well-established roles in DNA double-strand break repair, and recently, nonrepair functions have also been reported. To better understand its cellular functions, we deleted DNA-PKcs from HeLa and A549 cells using CRISPR/Cas9. The resulting cells were radiation sensitive, had reduced expression of ataxia-telangiectasia mutated (ATM), and exhibited multiple mitotic defects. Mechanistically, nocodazole-induced upregulation of cyclin B1, anillin, and securin was decreased in DNA-PKcs-deficient cells, as were phosphorylation of Aurora A on threonine 288, phosphorylation of Polo-like kinase 1 (PLK1) on threonine 210, and phosphorylation of targeting protein for Xenopus Klp2 (TPX2) on serine 121. Moreover, reduced nocodazole-induced expression of anillin, securin, and cyclin B1 and phosphorylation of PLK1, Aurora A, and TPX2 were rescued by inhibition of the anaphase-promoting complex/cyclosome (APC/C) by proTAME, which prevents binding of the APC/C-activating proteins Cdc20 and Cdh1 to the APC/C. Altogether, our studies suggest that loss of DNA-PKcs prevents inactivation of the APC/C in nocodazole-treated cells.

Processing bodies (PBs) are ribonucleoprotein granules that suppress cytokine mRNA translation that are targeted for disassembly by many viruses. Kaposi’s sarcoma-associated herpesvirus is the etiological agent of the inflammatory endothelial cancer, Kaposi’s sarcoma, and a PB-regulating virus. The virus encodes Kaposin B (KapB), which induces actin stress fibres (SFs) and cell spindling as well as PB disassembly. We now show that KapB-mediated PB disassembly requires actin rearrangements, RhoA effectors and the mechanoresponsive transcription activator, YAP. Moreover, ectopic expression of active YAP or exposure of ECs to mechanical forces caused PB disassembly in the absence of KapB and mechanoresponsive PB disassembly also required YAP. Using the viral protein KapB, we identified a new consequence of the exposure of cells to mechanical forces that alter actin dynamics and activate YAP, namely the disassembly of PBs.ImportanceFor the first time, we demonstrate that processing bodies (PBs), cytoplasmic sites of RNA decay, are regulated by mechanical signaling events that alter actin dynamics and that this requires the mechanoresponsive transcription factor, YAP. Using the overexpression of a viral protein called KapB, known previously to mediate PB disassembly, we show that actin stress fibers (SFs) and the mechanoresponsive transcription factor, YAP, are required for PB loss. We also show that other established mechanical signals (shear stress or stiff extracellular matrix) that lead to the formation of SFs and activate YAP also cause PB disassembly. This is important because it means that KapB activates, from the inside out, a pathway that links cell shape to post-transcriptional gene regulation via cytoplasmic PBs.

Centrosomal protein of 192 kDa (CEP192) is a scaffolding protein that recruits the mitotic protein kinases Aurora A and PLK1 to the centrosome. Here we demonstrate that CEP192 also recruits the type one protein phosphatase (PP1) via a highly conserved KHVTF docking motif. The threonine of the KHVTF motif is phosphorylated during mitosis and protein kinase inhibition studies suggest this to be a PLK1-dependent process.

Up to 50% of the adult population fail to meet the recommended total water intake despite the adverse health outcomes associated with chronic low fluid intake. Total fluid intake plays an important role in the energy and nutrient intake of individuals. Dietitians’ role is to advise and guide the population toward a healthier diet. However, based on existing evidence, dietitians’ current knowledge and practices regarding healthy hydration is an area for improvement. The European Healthy Hydration Awareness Campaign (EuHHAC) is an initiative between the European Federation of the Associations of Dietitians (EFAD) and the Hydration and Health Department of Danone Research, which aims to increase knowledge and awareness of healthy hydration and provide dietitians with evidence-based resources to use in practice. Since 2016, a series of tasks were undertaken by the EuHHAC group targeting dietitians across Europe. These tasks included an online survey addressing gaps in hydration knowledge, a webinar providing information on the terminology and impact of hydration on health, an interactive session addressing facilitators and barriers to healthy hydration, and a tutorial summarizing key hydration information and take-home messages. Dietitians were actively engaged in each aspect. Based on the feedback received, awareness of hydration was increased among dietitians. To further enhance understanding, an online Hydration Resource Center, including the resources of EuHHAC, will be created and uploaded on the EFAD website. Once completed an evaluation study will be undertaken to assess the usefulness of the resources in practice. The deliverables will be cascaded across Europe and worldwide via social media, conferences, and publications.

Community pharmacist is one of the most prominent and accessible healthcare professions. The community pharmacists&rsquo, role in healthcare is evolving, with opportunities being taken to reduce pressure on primary care services. However, the question remains of how well community pharmacists are equipped for this changing role. This was a sequentially designed study using a mix of methods to explore nutrition education among community pharmacists in Northern Ireland. It consisted of two phases. Phase 1 was a cross-sectional exploration to map the attitudes and practice of Northern Ireland (NI) pharmacists towards diet-related health promotion and disease prevention. An online questionnaire with open and closed questions to gain both quantitative and qualitative responses was developed and distributed to community pharmacists practising in NI. A total of 91% considered nutrition important in reducing the global burden of disease. While the majority (89%) believed patients would value nutritional advice from a pharmacist, 74% were not confident in providing advice to a patient with diabetes. From the consensus gained in Phase 1 a nutrition education intervention (Phase 2) for pre-registration pharmacists was developed using the Hardens 10 question system. The training programme was advertised to pre-registration pharmacy students in NI. It was delivered by nutrition experts who have education qualifications. The intervention was evaluated using a before and after questionnaire that assessed knowledge, attitudes, and practice (KAP). Phase 2 did find sustained improvement from the baseline in KAP but there was a decline from immediately post-training to three months post-training. This suggests the need to further embed nutrition education. The education programme was found to be effective for the target population and sets the stage for the development of an implementation strategy for a wider roll-out with evaluation.

Proper hydration and nutrition have been found to be contributing factors to a better recovery in patients after stroke. A better knowledge of factors influencing fluid and energy intake may contribute to a better care of the acute stroke patient. Aim of this study was to describe hydration status, fluid intake, and energy intake of stroke patients during acute hospitalization in a stroke unit. A retrospective descriptive study was conducted in stroke patients admitted to a stroke unit. All patients received neurological evaluation and urinary osmolality (uOsm) measure at admission and discharge. During stroke unit stay patients received a day-by-day diary to record data about beverages, food, and therapy. Water and energy content were then reported based on a national nutritional database. Ninety-five patients were included in the analysis. uOsm did not differ between patients with or without dysphagia, with uOsm > 500 mosm/kg in 58.1% at admission and 57.8% at discharge. Inadequate fluid intake was found in 41.2% of the sample with no difference between groups. Insufficient energy intake was found in 95.6% of the sample, with no patients with dysphagia reaching the minimum suggested amount. Our results suggest that a relevant proportion of stroke patients with and without dysphagia may not improve their hydration status during the first days after admission. Diet and therapy were insufficient to achieve recommended fluid intake and energy intake in a large proportion of patients, suggesting a more careful monitoring of hydration and nutrition needs.

Background The NNEdPro Core Team, Bhavishya Shakti Researchers and Inner Wheel Club of Greater Calcutta launched Mobile Teaching Kitchens as a nutritional education tool in two Indian slums in February 2018 to improve awareness of diet diversity and disease prevention by using locally sourced foods and cooking skills. Local volunteers, trained in healthy cooking, transferred core nutritional principals by cooking sustainable, nutritional and affordable meals. They followed a “See one, Do one and Teach one” model to transfer knowledge to their peers where markers of malnutrition were assessed before and after this programme. Objective To evaluate the efficacy of using mobile teaching kitchens to provide nutrition education through cooking, teaching healthy eating to a disadvantaged community in the urban slums of India. Study Design, Settings, Participants Longitudinal evaluation of mothers and children in RG Kar Canal Slum and Chetla Slum in Kolkata, India from 2018 to 2019. Measurable Outcome/Analysis Anthropometric and clinical signs of the children were assessed in person, alongside assessment of knowledge, attitudes and practice of the mothers through questionnaires. Results Preliminary measurements post-analysis from both slums (n = 14) showed that overall anthropometric measures of health in the mothers remained static. There was a mean change of 2.75cm growth in height in children. Other markers of nutritional status such as weight and mid-upper arm circumference did not significantly change. Statistically significant changes were seen in self-perceived nutritional knowledge about dietary protein sources, where the median response increased from 2 to 3 out of 5 (5 indicating excellent understanding) (P Conclusion Overall improvement in clinical status and nutritional knowledge was seen using Mobile Teaching Kitchens as an educational tool. Further evaluation of this particular teaching method is warranted due to the small sample size and heterogeneous results. Funding British Medical Association (BMA), Economic and Social Research Council (ESRC), Global Open Data for Agriculture and Nutrition (GODAN), The British Dietetics Association (BDA), University of Cambridge.

Aim. The aim of this study was to investigate dietitians’ knowledge, attitudes, and practices (KAP) regarding hydration and patient care. Methods. A cross-sectional online survey was administered to UK dietitians via the British Dietetic Association monthly newsletter and included 18 items on hydration knowledge (n=8), attitudes (n=4), and practices (n=6). KAP scores were calculated by adding the total number of correct knowledge responses and by ranking attitude and practice responses on a Likert scale. Results. 97 dietitians completed the online survey and displayed varying levels of KAP regarding hydration and patient care. The mean unweighted scores were knowledge 5.0 (±1.3) out of 8; attitude 13.9 (±1.3) out of 16; practice 14.9 (±2.6) out of 24. Dietitians appeared to be guided by clinical reasoning and priorities for nutrition care. Conclusions. There may be scope to further assess and potentially enhance the KAP of dietitians regarding hydration and patient care. Innovative approaches to hydration promotion are warranted and may include focusing on dietitians’ personal hydration status, increasing communication with other healthcare professionals, and partnering with patients to take a proactive role in hydration monitoring.

Scaffold attachment factor A (SAF-A), also called heterogenous nuclear ribonuclear protein U (hnRNP-U), is phosphorylated on serine 59 by the DNA-dependent protein kinase (DNA-PK) in response to DNA damage. Since SAF-A, DNA-PK catalytic subunit (DNA-PKcs), and protein phosphatase 6 (PP6), which interacts with DNA-PKcs, have all been shown to have roles in mitosis, we asked whether DNA-PKcs phosphorylates SAF-A in mitosis. We show that SAF-A is phosphorylated on serine 59 in mitosis, that phosphorylation requires polo-like kinase 1 (PLK1) rather than DNA-PKcs, that SAF-A interacts with PLK1 in nocodazole-treated cells, and that serine 59 is dephosphorylated by protein phosphatase 2A (PP2A) in mitosis. Moreover, cells expressing SAF-A in which serine 59 is mutated to alanine have multiple characteristics of aberrant mitoses, including misaligned chromosomes, lagging chromosomes, polylobed nuclei, and delayed passage through mitosis. Our findings identify serine 59 of SAF-A as a new target of both PLK1 and PP2A in mitosis and reveal that both phosphorylation and dephosphorylation of SAF-A serine 59 by PLK1 and PP2A, respectively, are required for accurate and timely exit from mitosis.

Many patients leave hospital in poor nutritional states, yet little is known about the post-discharge nutrition care in which patients are engaged. This study describes the nutrition-care activities 30-days post-discharge reported by patients and what covariates are associated with these activities. Quasi-randomly selected patients recruited from 5 medical units across Canada (n = 513) consented to 30-days post-discharge data collection with 48.5% (n = 249) completing the telephone interview. Use of nutrition care post-discharge was reported and bivariate analysis completed with relevant covariates for the two most frequently reported activities, following recommendations post-discharge or use of oral nutritional supplements (ONS). A total of 42% (n = 110) received nutrition recommendations at hospital discharge, with 65% (n = 71/110) of these participants following those recommendations; 26.5% (n = 66) were taking ONS after hospitalization. Participants who followed recommendations were more likely to report following a special diet (p = 0.002), different from before their hospitalization (p = 0.008), compared to those who received recommendations, but reported not following them. Patients taking ONS were more likely to be at nutrition risk (p < 0.0001), malnourished (p = 0.0006), taking ONS in hospital (p = 0.01), had a lower HGS (p = 0.0013; males only), and less likely to believe they were eating enough to meet their body’s needs (p = 0.005). This analysis provides new insights on nutrition-care post-discharge.

Staff play key roles in the prevention, detection, and treatment of hospital malnutrition. Understanding staff knowledge, attitudes, and practices (KAP) is important for developing and evaluating change management strategies.The More-2-Eat project improved nutrition care in 5 Canadian hospitals by implementing the Integrated Nutrition Pathway for Acute Care (INPAC). To understand staff views before (T1) and after 1 year of implementation (T2), a reliable KAP questionnaire, based on INPAC, was administered. T2 included questions about involvement in implementation. The mean difference between T2 and T1 responses was calculated, and t tests were used for comparisons.The questionnaire was completed at T1 (n = 189) and T2 (n = 147) (unpaired); 57 staff completed both questionnaires (paired). A significant increase in total score was seen in unpaired results at T2 (from 93.6/128 [range, 51-124] to 99.5/128 [range, 54-119]; t = 5.97, P.0001), with an increase in knowledge/attitudes (KA) (t = 2.4, P = .016) and practice (t = 3.57, P.0001) components. There were no statistically significant changes in paired responses. Seventy percent (n = 102/147) noticed positive changes in practices, 12% (n = 18) noticed positive/negative changes, 1% (n = 1) noticed negative change, and 17% (n = 25) noticed no change. Fifty-nine percent (n = 86) felt involved in the change, and these staff had higher KA and KAP scores than those who did not feel involved.Staff involvement is important in the implementation process for improving nutrition care.

The catalytic subunit of the DNA-dependent protein kinase (DNA-PKcs) plays a major role in the repair of DNA double-strand breaks (DSBs) by nonhomologous end joining (NHEJ). We have previously shown that DNA-PKcs is autophosphorylated in response to ionizing radiation (IR) and that dephosphorylation by a protein phosphatase 2A (PP2A)-like protein phosphatase (PP2A, PP4, or PP6) regulates the protein kinase activity of DNA-PKcs. Here we report that DNA-PKcs interacts with the catalytic subunits of PP6 (PP6c) and PP2A (PP2Ac), as well as with the PP6 regulatory subunits PP6R1, PP6R2, and PP6R3. Consistent with a role in the DNA damage response, silencing of PP6c by small interfering RNA (siRNA) induced sensitivity to IR and delayed release from the G(2)/M checkpoint. Furthermore, siRNA silencing of either PP6c or PP6R1 led to sustained phosphorylation of histone H2AX on serine 139 (gamma-H2AX) after IR. In contrast, silencing of PP6c did not affect the autophosphorylation of DNA-PKcs on serine 2056 or that of the ataxia-telangiectasia mutated (ATM) protein on serine 1981. We propose that a novel function of DNA-PKcs is to recruit PP6 to sites of DNA damage and that PP6 contributes to the dephosphorylation of gamma-H2AX, the dissolution of IR-induced foci, and release from the G(2)/M checkpoint in vivo.

Recent studies have established that DNA-dependent protein kinase (DNA-PK) undergoes a series of autophosphorylation events that facilitate successful completion of nonhomologous DNA end joining. Autophosphorylation at sites in two distinct clusters regulates DNA end access to DNA end-processing factors and to other DNA repair pathways. Autophosphorylation within the kinase's activation loop regulates kinase activity. Additional autophosphorylation events (as yet undefined) occur that mediate kinase dissociation. Here we provide the first evidence that autophosphorylation within the two major clusters (regulating end access) occurs in trans. Further, both UV-induced and double-strand break (DSB)-induced phosphorylation in the two major clusters is predominately autophosphorylation. Finally, we show that while autophosphorylation in trans on one of two synapsed DNA-PK complexes facilitates appropriate end processing, this is not sufficient to promote efficient end joining. This suggests that end joining in living cells requires additional phosphorylation events that either occur in cis or that occur on both sides of the DNA-PK synapse. These data support an emerging consensus that, via a series of autophosphorylation events, DNA-PK undergoes a sequence of conformational changes that promote efficient and appropriate repair of DSBs.

The protein kinase activity of the DNA-dependent protein kinase (DNA-PK) is required for the repair of DNA double-strand breaks (DSBs) via the process of nonhomologous end joining (NHEJ). However, to date, the only target shown to be functionally relevant for the enzymatic role of DNA-PK in NHEJ is the large catalytic subunit DNA-PKcs itself. In vitro, autophosphorylation of DNA-PKcs induces kinase inactivation and dissociation of DNA-PKcs from the DNA end-binding component Ku70/Ku80. Phosphorylation within the two previously identified clusters of phosphorylation sites does not mediate inactivation of the assembled complex and only partially regulates kinase disassembly, suggesting that additional autophosphorylation sites may be important for DNA-PK function. Here, we show that DNA-PKcs contains a highly conserved amino acid (threonine 3950) in a region similar to the activation loop or t-loop found in the protein kinase domain of members of the typical eukaryotic protein kinase family. We demonstrate that threonine 3950 is an in vitro autophosphorylation site and that this residue, as well as other previously identified sites in the ABCDE cluster, is phosphorylated in vivo in irradiated cells. Moreover, we show that mutation of threonine 3950 to the phosphomimic aspartic acid abrogates V(D)J recombination and leads to radiation sensitivity. Together, these data suggest that threonine 3950 is a functionally important, DNA damage-inducible phosphorylation site and that phosphorylation of this site regulates the activity of DNA-PKcs.