Cell Death and Differentiation (2015) 22, 156–163 & 2015 Macmillan Publishers Limited All rights reserved 1350-9047/15 www.nature.com/cdd Cofactor Strap regulates oxidative phosphorylation and mitochondrial p53 activity through ATP synthase S Maniam1,4, AS Coutts1,4, MR Stratford2, J McGouran3, B Kessler3 and NB La Thangue*,1 Metabolic reprogramming is a hallmark of cancer cells. Strap (stress-responsive activator of p300) is a novel TPR motif OB-fold protein that contributes to p53 transcriptional activation. We show here that, in addition to its established transcriptional role, Strap is localised at mitochondria where one of its key interaction partners is ATP synthase. Significantly, the interaction between Strap and ATP synthase downregulates mitochondrial ATP production. Under glucose-limiting conditions, cancer cells are sensitised by mitochondrial Strap to apoptosis, which is rescued by supplementing cells with an extracellular source of ATP. Furthermore, Strap augments the apoptotic effects of mitochondrial p53. These findings define Strap as a dual regulator of cellular reprogramming: first as a nuclear transcription cofactor and second in the direct regulation of mitochondrial respiration. Cell Death and Differentiation (2015) 22, 156–163; doi:10.1038/cdd.2014.135; published online 29 August 2014 An established characteristic of tumour cells is their under- model whereby Strap unfolds to become more accessible lying metabolic changes.1 Early observations that during the DNA-damage response.12 tumour cells had persistently high glycolysis even under It has become increasingly evident that p53 is able to modify aerobic conditions led to the suggestion that this underlying a variety of metabolic pathways, including glycolysis and metabolic change was fundamental to the process of oxidative phosphorylation, enabling cells to respond to tumourigenesis.2 In normal cells, ATP production is primarily metabolic stress.13–17 Further, p53 is essential for cell survival produced in mitochondria by oxidative phosphorylation, under glucose deprivation and in tumour cells under metabolic driven by the TCA cycle in the presence of oxygen.3 When stress is able to augment apoptosis.13,16 Some studies have energy is needed rapidly or oxygen becomes limiting, cells even suggested that the ability of p53 to act as a tumour turn on glycolysis to meet the extra energy demands. In suppressor may be influenced by its role in metabolic contrast, tumour cells consume large amounts of glucose and regulation, rather than its ability to influence apoptosis, maintain high levels of glycolysis even in the presence of senescence or cell cycle arrest.18 oxygen.4 In addition to its established transcription-dependent p53 has a critical role in mediating cell cycle arrest and cell mechanisms, p53 also induces apoptosis in a transcription- death, which occurs via transcription-dependent and -inde- independent fashion through its localisation to mitochon- pendent mechanisms.5 As a transcription factor, p53 reg- dria.19 At mitochondria, p53 interacts with and regulates Bcl2 ulates a variety of target genes connected with cell fate (cell family members, such as Bax and Bak.20–22 This allows p53 to cycle arrest, apoptosis, autophagy and senescence, for influence the intrinsic pathway of apoptosis through the example) and metabolic reprogramming.5–7 Further, its ability release of soluble proteins, like cytochrome c, which then to function as a transcription factor is influenced by a complex initiate caspase activation in the cytosol.23 The release of array of posttranslational modifications and cofactors.6,8 Strap cytochrome c occurs as a consequence of compromised is one such cofactor that regulates p53 transcriptional activity integrity of the outer mitochondrial membrane, referred under a variety of cellular conditions.9 Upon DNA damage, for to as mitochondrial outer membrane permeabilisation example, it is phosphorylated by the DNA damage signalling (MOMP).20,21 Bax and Bak oligomerise at the mitochondrial ATM and Chk2 kinases, resulting in Strap stabilisation, altered membrane influencing MOMP and the subsequent release of cellular location and enhanced p53 activity.10,11 An analysis of pro-apoptotic factors, such as cytochrome c.24 the three-dimensional structure of Strap highlighted its In continuing the analysis of Strap and its role in regulating unusual domain organisation, being composed of tandem p53 biology, we have uncovered a new and hitherto TPR motifs together with an OB-fold, with both domains unexpected level of control. Remarkably, we have found that required for it to function in the p53 response.12 The location of Strap is, like p53, localised to mitochondria and identified one phosphorylated residues in Strap suggested a structural of its key interaction partners as mitochondrial ATP synthase. 1Laboratory of Cancer Biology, Department of Oncology, Medical Science Division, University of Oxford, Oxford, UK; 2Department of Oncology, Medical Science Division, University of Oxford, Oxford, UK and 3Target Discovery Institute, Nuffield Department of Medicine, University of Oxford, Oxford, UK *Corresponding author: NB La Thangue, Department of Oncology, Medical Science Division, University of Oxford, Old Road Campus Research Building, Old Road Campus, Off Roosevelt Drive, Oxford OX3 7DQ, UK. Tel: þ 0044 1865 617090; Fax: þ 0044 1865 617092; Email: [email protected] 4These authors are joint first authors. Abbreviations: AMPK, AMP-activated protein kinase; ADP, adenosine diphosphate; ATM, ataxia telangiectasia mutated; ATP, adenosine triphosphate; Bcl2, B-cell lymphoma 2; Chk2, checkpoint kinase 2; COXIV, cytochrome c oxidase IV subunit; DAPI, 40,6-diamidino-2-phenylindole; GLS2, glutaminase 2; GLUT4, glucose transporter type 4; MOMP, mitochondrial outer membrane permeabilisation; NT, non-targeting; OB, oligonucleotide/oligosaccharide-binding fold; PARP, Poly [ADP- ribose] polymerase; PCNA, proliferating cell nuclear antigen; SCO2, SCO2 cytochrome c oxidase assembly protein; Strap, stress-responsive activator of p300; TCA, tricarboxylic acid cycle; TIGAR, TP53-inducible glycolysis and apoptosis regulator; TPR, tetratricopeptide repeat; UV, ultraviolet; WT, wild type Received 28.2.14; revised 16.6.14; accepted 22.7.14; Edited by G Melino; published online 29 August 2014 Cofactor Strap regulates oxidative phosphorylation S Maniam et al 157 This interaction causes reduced ATP synthesis which, in turn, We therefore considered the possibility that Strap may sensitises cells to mitochondrial-dependent apoptosis. Most interact with mitochondrial proteins. To facilitate an analysis of significantly, mitochondrial Strap augments apoptosis stimu- Strap’s mitochondrial role, we made a derivative of Strap that lated by the mitochondrial arm of the p53 response. These was exclusively directed to mitochondria using a targeting results highlight an unanticipated overlap and convergence in sequence taken from Bcl2 (referred to as L-Strap); as the pathways affected by Strap and p53 and provide further expected, L-Strap localised to mitochondria (Figure 1d) and support for their interplay in transcription and metabolism. lacked any transcription cofactor activity on p53 (Supplementary Figure S1e). We then immunoprecipitated Results L-Strap from cells, and any bound proteins were subsequently identified by mass spectrometry. The ATP synthase b-subunit Strap locates to mitochondria and regulates ATP was found to be a predominant interacting protein (Figure 1e). synthase. To further explore Strap’s subcellular localisation, The b-subunit is an essential component of ATP synthase, we prepared fractions from different cell types and measured which is a large molecular complex localised at the inner the level of Strap in total lysate, mitochondria and cytoplasm. mitochondrial membrane responsible for synthesising ATP by Surprisingly, we identified Strap in the mitochondrial fraction oxidative phosphorylation.3 We verified that the ATP synthase (Figure 1a), which was evident when mitochondria were b-subunit interacted with both wild-type and L-Strap by prepared according to different methods and in a variety of immunoprecipitation (Supplementary Figure S1b), which cell types (Figures 1a–c and Supplementary Figure S1a). was consistent with the co-localisation of L-Strap and ATP H1299 SAOS2 HeLa TL mit cyt TL mit cyt TL mit cyt Strap PCNA COXIV mit NT S : siRNA tot 12 Strap Strap Calnexin p53 PCNA COXIV COXIV HA-L-Strap cytochrome c merge i ii In IP -L -L Flag actin L-Strap ATP synthase β-subunit Figure 1 Strap is localised at the mitochondria and interacts with ATP synthase. (a) Endogenous Strap from various cell types was detected in total lysate (TL), mitochondrial (mit) and cytoplasmic (cyt) fractions. Cytochrome oxidase IV (COXIV) was used as a mitochondrial marker. PCNA was used as a control for nuclear contamination. The mitochondrial fractions were estimated to be 95% enriched. (b) U2OS cells were transfected with non-targeting (NT) or Strap (S) siRNA for 72 h. Mitochondrial fractions were prepared and immunoblotted with the indicated antibodies. COXIV served as a mitochondrial marker. (c) Endogenous Strap from U2OS cells was detected in mitochondrial (mit) fractions, prepared using different extraction techniques (1 and 2; see Materials and Methods). Nuclear and endoplasmic reticulum material was monitored using PCNA and calnexin antibodies respectively. COXIV served as a mitochondrial marker. Total cell lysate (tot) serves as a comparison. (d) U2OS cells