Walpole GFW, Pacheco J, Chauhan N, Clark J, Anderson KE, Abbas YM, Brabant-Kirwan D, Montaño-Rendón F, Liu Z, Zhu H, Brumell JH, Deiters A, Stephens LR, Hawkins PT, Hammond GRV, Grinstein S, Fairn GD Signalling, Biological Chemistry

Despite their low abundance, phosphoinositides play a central role in membrane traffic and signalling. PtdIns(3,4,5)P and PtdIns(3,4)P are uniquely important, as they promote cell growth, survival and migration. Pathogenic organisms have developed means to subvert phosphoinositide metabolism to promote successful infection and their survival in host organisms. We demonstrate that PtdIns(3,4)P is a major product generated in host cells by the effectors of the enteropathogenic bacteria Salmonella and Shigella. Pharmacological, gene silencing and heterologous expression experiments revealed that, remarkably, the biosynthesis of PtdIns(3,4)P occurs independently of phosphoinositide 3-kinases. Instead, we found that the Salmonella effector SopB, heretofore believed to be a phosphatase, generates PtdIns(3,4)P de novo via a phosphotransferase/phosphoisomerase mechanism. Recombinant SopB is capable of generating PtdIns(3,4,5)P and PtdIns(3,4)P from PtdIns(4,5)P in a cell-free system. Through a remarkable instance of convergent evolution, bacterial effectors acquired the ability to synthesize 3-phosphorylated phosphoinositides by an ATP- and kinase-independent mechanism, thereby subverting host signalling to gain entry and even provoke oncogenic transformation.

+view abstract Nature cell biology, PMID: 35484249 May 2022

Hirschberger C, Sleight VA, Criswell KE, Clark SJ, Gillis JA Biological Chemistry

The origin of the jaw is a long-standing problem in vertebrate evolutionary biology. Classical hypotheses of serial homology propose that the upper and lower jaw evolved through modifications of dorsal and ventral gill arch skeletal elements, respectively. If the jaw and gill arches are derived members of a primitive branchial series, we predict that they would share common developmental patterning mechanisms. Using candidate and RNAseq/differential gene expression analyses, we find broad conservation of dorsoventral patterning mechanisms within the developing mandibular, hyoid and gill arches of a cartilaginous fish, the skate (Leucoraja erinacea). Shared features include expression of genes encoding members of the ventralising BMP and endothelin signalling pathways and their effectors, the joint markers nkx3.2 and gdf5 and pro-chondrogenic transcription factor barx1, and the dorsal territory marker pou3f3. Additionally, we find that mesenchymal expression of eya1/six1 is an ancestral feature of the mandibular arch of jawed vertebrates, while differences in notch signalling distinguish the mandibular and gill arches in skate. Comparative transcriptomic analyses of mandibular and gill arch tissues reveal additional genes differentially expressed along the dorsoventral axis of the pharyngeal arches, including scamp5 as a novel marker of the dorsal mandibular arch, as well as distinct transcriptional features of mandibular and gill arch muscle progenitors and developing gill buds. Taken together, our findings reveal conserved patterning mechanisms in the pharyngeal arches of jawed vertebrates, consistent with serial homology of their skeletal derivatives, as well as unique transcriptional features that may underpin distinct jaw and gill arch morphologies.

+view abstract Molecular biology and evolution, PMID: 33905525 27 Apr 2021

Chicanne G, Bertrand-Michel J, Viaud J, Hnia K, Clark J, Payrastre B Biological Chemistry

Our knowledge of the role and biology of the different phosphoinositides has greatly expanded over recent years. Reversible phosphorylation by specific kinases and phosphatases of positions 3, 4, and 5 on the inositol ring is a highly dynamic process playing a critical role in the regulation of the spatiotemporal recruitment and binding of effector proteins. The specific phosphoinositide kinases and phosphatases are key players in the control of many cellular functions, including proliferation, survival, intracellular trafficking, or cytoskeleton reorganization. Several of these enzymes are mutated in human diseases. The impact of the fatty acid composition of phosphoinositides in their function is much less understood. There is an important molecular diversity in the fatty acid side chains of PI. While stearic and arachidonic fatty acids are the major acyl species in PIP, PIP, and PIP, other fatty acid combinations are also found. The role of these different molecular species is still unknown, but it is important to quantify these different molecules and their potential changes during cell stimulation to better characterize this emerging field. Here, we describe a sensitive high-performance liquid chromatography-mass spectrometry method that we used for the first time to profile the changes in phosphoinositide molecular species (summed fatty acyl chain profiles) in human and mouse platelets under resting conditions and following stimulation. This method can be applied to other hematopoietic primary cells isolated from human or experimental animal models.

+view abstract Methods in molecular biology, PMID: 33481230 2021

Rynkiewicz NK, Anderson KE, Suire S, Collins DM, Karanasios E, Vadas O, Williams R, Oxley D, Clark J, Stephens LR, Hawkins PT Signalling, Mass Spectrometry, Biological Chemistry

The PI3Kγ isoform is activated by Gi-coupled GPCRs in myeloid cells, but the extent to which the two endogenous complexes of PI3Kγ, p101/p110γ and p84/p110γ, receive direct regulation through Gβγ or indirect regulation through RAS and the sufficiency of those inputs is controversial or unclear. We generated mice with point mutations that prevent Gβγ binding to p110γ (RK552DD) or to p101 (VVKR777AAAA) and investigated the effects of these mutations in primary neutrophils and in mouse models of neutrophilic inflammation. Loss of Gβγ binding to p110γ substantially reduced the activation of both p101/p110γ and p84/p110γ in neutrophils by various GPCR agonists. Loss of Gβγ binding to p101 caused more variable effects, depending on both the agonist and cellular response, with the biggest reductions seen in PIP production by primary neutrophils in response to LTB4 and MIP-2 and in the migration of neutrophils during thioglycolate-induced peritonitis or MIP2-induced ear pouch inflammation. We also observed that p101 neutrophils showed enhanced p84-dependent ROS responses to MLP and C5a, suggesting that competition may exist between p101/p110γ and p84/p110γ for Gβγ subunits downstream of GPCR activation. GPCRs did not activate p110γ in neutrophils from mice lacking both the p101 and p84 regulatory subunits, indicating that RAS binding to p110γ is insufficient to support GPCR activation in this cell type. These findings define a direct role for Gβγ subunits in activating both of the endogenous PI3Kγ complexes and indicate that the regulatory PI3Kγ subunit biases activation toward different GPCRs.

+view abstract Science signaling, PMID: 33144519 03 Nov 2020

Streeter MD, Rowan S, Ray J, McDonald DM, Volkin J, Clark J, Taylor A, Spiegel DA Biological Chemistry

Although there is ample evidence that the advanced glycation end-product (AGE) glucosepane contributes to age-related morbidities and diabetic complications, the impact of glucosepane modifications on proteins has not been extensively explored due to the lack of sufficient analytical tools. Here, we report the development of the first polyclonal anti-glucosepane antibodies using a synthetic immunogen that contains the core bicyclic ring structure of glucosepane. We investigate the recognition properties of these antibodies through ELISAs involving an array of synthetic AGE derivatives and determine them to be both high-affinity and selective in binding glucosepane. We then employ these antibodies to image glucosepane in aging mouse retinae via immunohistochemistry. Our studies demonstrate for the first time accumulation of glucosepane within the retinal pigment epithelium, Bruch's membrane, and choroid: all regions of the eye impacted by age-related macular degeneration. Co-localization studies further suggest that glucosepane colocalizes with lipofuscin, which has previously been associated with lysosomal dysfunction and has been implicated in the development of age-related macular degeneration, among other diseases. We believe that the anti-glucosepane antibodies described in this study will prove highly useful for examining the role of glycation in human health and disease.

+view abstract ACS chemical biology, PMID: 32975399 07 Oct 2020

Ohashi Y, Tremel S, Masson GR, McGinney L, Boulanger J, Rostislavleva K, Johnson CM, Niewczas I, Clark J, Williams RL Biological Chemistry

The lipid kinase VPS34 orchestrates diverse processes, including autophagy, endocytic sorting, phagocytosis, anabolic responses and cell division. VPS34 forms various complexes that help adapt it to specific pathways, with complexes I and II being the most prominent ones. We found that physicochemical properties of membranes strongly modulate VPS34 activity. Greater unsaturation of both substrate and non-substrate lipids, negative charge and curvature activate VPS34 complexes, adapting them to their cellular compartments. Hydrogen/deuterium exchange mass spectrometry (HDX-MS) of complexes I and II on membranes elucidated structural determinants that enable them to bind membranes. Among these are the Barkor/ATG14L autophagosome targeting sequence (BATS), which makes autophagy-specific complex I more active than the endocytic complex II, and the Beclin1 BARA domain. Interestingly, even though Beclin1 BARA is common to both complexes, its membrane-interacting loops are critical for complex II, but have only a minor role for complex I.

+view abstract eLife, PMID: 32602837 30 Jun 2020

Stammers M, Niewczas IS, Segonds-Pichon A, Clark J Biological Chemistry

Collagen I is a major tendon protein whose polypeptide chains are linked by covalent cross-links. It is unknown how the cross-linking contributes to the mechanical properties of tendon or whether cross-linking changes in response to stretching or relaxation. Since their discovery, imine bonds within collagen have been recognized as being important in both cross-link formation and collagen structure. They are often described as acidic or thermally labile, but no evidence is available from direct measurements of cross-link levels whether these bonds contribute to the mechanical properties of collagen. Here, we used MS to analyze these imine bonds after reduction with sodium borohydride while under tension and found that their levels are altered in stretched tendon. We studied the changes in cross-link bonding in tail tendon from 11-week-old C57Bl/6 mice at 4% physical strain, at 10% strain, and at breaking point. The cross-links hydroxy-lysino-norleucine (HLNL), dihydroxy-lysino-norleucine (DHLNL), and lysino-norleucine (LNL) increased or decreased depending on the specific cross-link and amount of mechanical strain. We also noted a decrease in glycated lysine residues in collagen, indicating that the imine formed between circulating glucose and lysine is also stress-labile. We also carried out mechanical testing, including cyclic testing at 4% strain, stress relaxation tests, and stress-strain profiles taken at breaking point, both with and without sodium borohydride reduction. The results from both the MS studies and mechanical testing provide insights into the chemical changes during tendon stretching and directly link these chemical changes to functional collagen properties.

+view abstract The Journal of biological chemistry, PMID: 32546479 16 Jun 2020

Stammers M, Ivanova IM, Niewczas IS, Segonds-Pichon A, Streeter M, Spiegel DA, Clark J Biological Chemistry

Collagen is a structural protein whose internal cross-linking critically determines the properties and functions of connective tissue. Knowing how the cross-linking of collagen changes with age is key to understanding why the mechanical properties of tissues change over a lifetime. The current scientific consensus is that collagen cross-linking increases with age and that this increase leads to tendon stiffening. Here, we show that this view should be reconsidered. Using MS-based analyses, we demonstrate that during aging of healthy C57BL/6 mice, the overall levels of collagen cross-linking in tail tendon decrease with age. However, the levels of lysine glycation in collagen, which is not considered a cross-link, increased dramatically with age. We found that in 16-week-old diabetic db/db mice, glycation reaches levels similar to those observed in 98-week-old C57BL/6 mice, while the other cross-links typical of tendon collagen either decreased or remained the same as those observed in 20 week old WT mice. These results, combined with findings from mechanical testing of tendons from these mice, indicate that overall collagen cross-linking in mouse tendon decreases with age. Our findings also reveal that lysine glycation appears to be an important factor that contributes to tendon stiffening with age and in diabetes.

+view abstract The Journal of biological chemistry, PMID: 32381510 07 May 2020

Coulon D, Faure L, Grison M, Pascal S, Wattelet-Boyer V, Clark J, Guedard ML, Testet E, Bessoule JJ Biological Chemistry

-lipid acyltransferases are enzymes involved in various processes such as lipid synthesis and remodelling. Here, we characterized the activity of an acyltransferase from (LPIAT). In vitro, this protein, expressed in membrane, displayed a 2--phosphatidylinositol acyltransferase activity with a specificity towards saturated long chain acyl CoAs (C16:0- and C18:0-CoAs), allowing the remodelling of phosphatidylinositol. , gene was expressed in mature seeds and very transiently during seed imbibition, mostly in aleurone-like layer cells. Whereas the disruption of this gene did not alter the lipid composition of seed, its overexpression in leaves promoted a strong increase in the phosphatidylinositol phosphates (PIP) level without affecting the PIP2 content. The spatial and temporal narrow expression of this gene as well as the modification of PIP metabolism led us to investigate its role in the control of seed germination. Seeds from the mutant germinated faster and were less sensitive to abscisic acid (ABA) than wild-type or overexpressing lines. We also showed that the protective effect of ABA on young seedlings against dryness was reduced for line. In addition, germination of mutant seeds was more sensitive to hyperosmotic stress. All these results suggest a link between phosphoinositides and ABA signalling in the control of seed germination.

+view abstract International journal of molecular sciences, PMID: 32121266 28 Feb 2020

Bansode S, Bashtanova U, Li R, Clark J, Müller KH, Puszkarska A, Goldberga I, Chetwood HH, Reid DG, Colwell LJ, Skepper JN, Shanahan CM, Schitter G, Mesquida P, Duer MJ Biological Chemistry

Collagen fibrils are central to the molecular organization of the extracellular matrix (ECM) and to defining the cellular microenvironment. Glycation of collagen fibrils is known to impact on cell adhesion and migration in the context of cancer and in model studies, glycation of collagen molecules has been shown to affect the binding of other ECM components to collagen. Here we use TEM to show that ribose-5-phosphate (R5P) glycation of collagen fibrils - potentially important in the microenvironment of actively dividing cells, such as cancer cells - disrupts the longitudinal ordering of the molecules in collagen fibrils and, using KFM and FLiM, that R5P-glycated collagen fibrils have a more negative surface charge than unglycated fibrils. Altered molecular arrangement can be expected to impact on the accessibility of cell adhesion sites and altered fibril surface charge on the integrity of the extracellular matrix structure surrounding glycated collagen fibrils. Both effects are highly relevant for cell adhesion and migration within the tumour microenvironment.

+view abstract Scientific reports, PMID: 32099005 25 Feb 2020

Swan DJ, Aschenbrenner D, Lamb CA, Chakraborty K, Clark J, Pandey S, Engelhardt KR, Chen R, Cavounidis A, Ding Y, Krasnogor N, Carey CD, Acres M, Needham S, Cant AJ, Arkwright PD, Chandra A, Okkenhaug K, Uhlig HH, Hambleton S Immunology, Biological Chemistry

+view abstract Haematologica, PMID: 31073077 2019

Begg M, Edwards CD, Hamblin JN, Pifani E, Wilson R, Gilbert J, Vitulli G, Mallett D, Morrell J, Hingle MI, Uddin S, Ehtesham F, Marotti M, Harell A, Newman C, Fernando D, Clark J, Cahn A, Hessel EM , Biological Chemistry

This study describes the pharmacokinetic (PK) and pharmacodynamic (PD) profile of GSK2292767A, a novel low solubility inhaled PI3Kδ inhibitor developed as an alternative to nemiralisib, which is a highly soluble inhaled inhibitor of PI3Kδ with a lung profile consistent with once-daily dosing. GSK2292767A has a similar in vitro cellular profile to nemiralisib and reduces eosinophilia in a murine PD model by 63% (n=5, p<0.05). To explore whether a low soluble compound results in effective PI3Kδ inhibition in humans, a first time in human study was conducted with GSK2292767A in healthy volunteers who smoke. GSK2292767A was generally well tolerated with headache being the most common reported adverse event. PD changes in induced sputum were measured in combination with drug concentrations in plasma from single (0.05-2 mg, n=37), and 14-day repeat (2 mg, n=12) doses of GSK2292767A. Trough bronchoalveolar lavage (BAL) for PK was taken after 14 days repeat dosing. GSK2292767A displayed a linear increase in plasma exposure with dose, with marginal accumulation after 14 days. Induced sputum showed a 27% (90% CI 15, 37) reduction in phosphatidylinositol-trisphosphate (PIP3, the product of PI3K activation) 3 h after a single dose. Reduction was not maintained 24 h after single or repeat dosing. BAL analysis confirmed presence of GSK2292767A in lung at 24 h, consistent with the preclinical lung retention profile. Despite good lung retention, target engagement was only present at 3 h. This exposure-response disconnect is an important observation for future inhaled drug design strategies considering low solubility to drive lung retention.

+view abstract The Journal of pharmacology and experimental therapeutics, PMID: 30940692 2019

Begg M, Wilson R, Hamblin JN, Montembault M, Green J, Deans A, Amour A, Worsley S, Fantom K, Cui Y, Dear G, Ahmad S, Kielkowska A, Clark J, Boyce M, Cahn A, Hessel EM , Biological Chemistry

Nemiralisib (GSK2269557) is a potent inhaled inhibitor of phosphoinositide 3-kinase delta (PI3Kδ) which is being developed for the treatment of respiratory disorders including COPD (Chronic Obstructive Pulmonary Disease). Determining the pharmacokinetic (PK) and pharmacodynamic (PD) responses of inhaled drugs early during drug development is key to informing the appropriate dose and preferred dose regimen in patients. We set out to measure PD changes in induced sputum in combination with drug concentrations in plasma and bronchoalveolar lavage (BAL) taken from healthy smokers (n=56) treated for up to 14 days with increasing doses of inhaled nemiralisib (0.1 mg to 6.4 mg). Induced sputum analysis demonstrated a dose-dependent reduction in phosphatidylinositol-trisphosphate (PIP3, the product of PI3K activation), with a maximum placebo-corrected reduction of 23% (90% CI 11-34%) and 36% (90% CI 11-64%) following single dose or 14 days of treatment with nemiralisib respectively (2 mg, once daily). Plasma analysis suggested a linear PK relationship with an observed accumulation of ~3-4.5-fold (peak vs. trough) in plasma exposure following 14 days of nemiralisib treatment. BAL analysis at trough confirmed higher levels of drug in lung vs. plasma (32-fold in the BAL fluid component, and 214-fold in the BAL cellular fraction). Comparison of drug levels in plasma and reductions in sputum PIP3 show a direct relationship between exposure and PIP3 reduction. In conclusion, these results demonstrate target engagement upon treatment with inhaled nemiralisib and provide confidence for a once-daily dosing regimen.

+view abstract The Journal of pharmacology and experimental therapeutics, PMID: 30886125 2019

Chow WY, Li R, Goldberga I, Reid DG, Rajan R, Clark J, Oschkinat H, Duer MJ, Hayward R, Shanahan CM Biological Chemistry

The sparse but functionally essential post-translational collagen modification 5-hydroxylysine can undergo further transformations, including crosslinking, O-glycosylation, and glycation. Dynamic nuclear polarization (DNP) and stable isotope enriched lysine incorporation provide sufficient solid-state NMR sensitivity to identify these adducts directly in skin and vascular smooth muscle cell extracellular matrix (ECM), without extraction procedures, by comparison with chemical shifts of model compounds. Thus, DNP provides access to the elucidation of structural consequences of collagen modifications in intact tissue.

+view abstract Chemical communications (Cambridge, England), PMID: 30299444 06 Nov 2018

Stark AK, Chandra A, Chakraborty K, Alam R, Carbonaro V, Clark J, Sriskantharajah S, Bradley G, Richter AG, Banham-Hall E, Clatworthy MR, Nejentsev S, Hamblin JN, Hessel EM, Condliffe AM, Okkenhaug K Immunology, Biological Chemistry

Streptococcus pneumoniae is a major cause of pneumonia and a leading cause of death world-wide. Antibody-mediated immune responses can confer protection against repeated exposure to S. pneumoniae, yet vaccines offer only partial protection. Patients with Activated PI3Kδ Syndrome (APDS) are highly susceptible to S. pneumoniae. We generated a conditional knock-in mouse model of this disease and identify a CD19B220 B cell subset that is induced by PI3Kδ signaling, resides in the lungs, and is correlated with increased susceptibility to S. pneumoniae during early phases of infection via an antibody-independent mechanism. We show that an inhaled PI3Kδ inhibitor improves survival rates following S. pneumoniae infection in wild-type mice and in mice with activated PI3Kδ. These results suggest that a subset of B cells in the lung can promote the severity of S. pneumoniae infection, representing a potential therapeutic target.

+view abstract Nature communications, PMID: 30093657 2018

Riento K, Zhang Q, Clark J, Begum F, Stephens E, Wakelam MJ, Nichols BJ Signalling, Biological Chemistry

Sphingosine-1-phosphate (S1P) is an important lipid signalling molecule. S1P is produced via intracellular phosphorylation of sphingosine (Sph). As a lipid with a single fatty alkyl chain, Sph may diffuse rapidly between cellular membranes and through the aqueous phase. Here, we show that the absence of microdomains generated by multimeric assemblies of flotillin proteins results in reduced S1P levels. Cellular phenotypes of flotillin knockout mice, including changes in histone acetylation and expression of Isg15, are recapitulated when S1P synthesis is perturbed. Flotillins bind to Sph in vitro and increase recruitment of Sph to membranes in cells. Ectopic re-localisation of flotillins within the cell causes concomitant redistribution of Sph. The data suggest that flotillins may directly or indirectly regulate cellular sphingolipid distribution and signalling.

+view abstract PloS one, PMID: 29787576 2018

Fellows R, Denizot J, Stellato C, Cuomo A, Jain P, Stoyanova E, Balázsi S, Hajnády Z, Liebert A, Kazakevych J, Blackburn H, Corrêa RO, Fachi JL, Sato FT, Ribeiro WR, Ferreira CM, Perée H, Spagnuolo M, Mattiuz R, Matolcsi C, Guedes J, Clark J, Veldhoen M, Bonaldi T, Vinolo MAR, Varga-Weisz P , Biological Chemistry

The recently discovered histone post-translational modification crotonylation connects cellular metabolism to gene regulation. Its regulation and tissue-specific functions are poorly understood. We characterize histone crotonylation in intestinal epithelia and find that histone H3 crotonylation at lysine 18 is a surprisingly abundant modification in the small intestine crypt and colon, and is linked to gene regulation. We show that this modification is highly dynamic and regulated during the cell cycle. We identify class I histone deacetylases, HDAC1, HDAC2, and HDAC3, as major executors of histone decrotonylation. We show that known HDAC inhibitors, including the gut microbiota-derived butyrate, affect histone decrotonylation. Consistent with this, we find that depletion of the gut microbiota leads to a global change in histone crotonylation in the colon. Our results suggest that histone crotonylation connects chromatin to the gut microbiota, at least in part, via short-chain fatty acids and HDACs.

+view abstract Nature communications, PMID: 29317660 2018

Clark DE, Waszkowycz B, Wong M, Lockey PM, Adalbert R, Gilley J, Clark J, Coleman MP Signalling, Biological Chemistry

NAMPT may represent a novel target for drug discovery in various therapeutic areas, including oncology and inflammation. Additionally, recent work has suggested that targeting NAMPT has potential in treating axon degeneration. In this work, publicly available X-ray co-crystal structures of NAMPT and the structures of two known NAMPT inhibitors were used as the basis for a structure- and ligand-based virtual screening campaign. From this, two novel series of NAMPT inhibitors were identified, one of which showed a statistically significant protective effect when tested in a cellular model of axon degeneration.

+view abstract Bioorganic & medicinal chemistry letters, PMID: 27158141 2016

Lochhead PA, Clark J, Wang LZ, Gilmour L, Squires M, Gilley R, Foxton C, Newell DR, Wedge SR, Cook SJ Signalling, Biological Chemistry

ERK5, encoded by MAPK7, has been proposed to play a role in cell proliferation, thus attracting interest as a cancer therapeutic target. While oncogenic RAS or BRAF cause sustained activation of the MEK1/2-ERK1/2 pathway, ERK5 is directly activated by MEK5. It has been proposed that RAS and RAF proteins can also promote ERK5 activation. Here we investigated the interplay between RAS-RAF-MEK-ERK and ERK5 signaling and studied the role of ERK5 in tumor cell proliferation in 2 disease-relevant cell models. We demonstrate that although an inducible form of CRAF (CRAF:ER*) can activate ERK5 in fibroblasts, the response is delayed and reflects feed-forward signaling. Additionally, oncogenic KRAS and BRAF do not activate ERK5 in epithelial cells. Although KRAS and BRAF do not couple directly to MEK5-ERK5, ERK5 signaling might still be permissive for proliferation. However, neither the selective MEK5 inhibitor BIX02189 or ERK5 siRNA inhibited proliferation of colorectal cancer cells harbouring KRAS(G12C/G13D) or BRAF(V600E). Furthermore, there was no additive or synergistic effect observed when BIX02189 was combined with the MEK1/2 inhibitor Selumetinib (AZD6244), suggesting that ERK5 was neither required for proliferation nor a driver of innate resistance to MEK1/2 inhibitors. Finally, even cancer cells with MAPK7 amplification were resistant to BIX02189 and ERK5 siRNA, showing that ERK5 amplification does not confer addiction to ERK5 for cell proliferation. Thus ERK5 signaling is unlikely to play a role in tumor cell proliferation downstream of KRAS or BRAF or in tumor cells with ERK5 amplification. These results have important implications for the role of ERK5 as an anti-cancer drug target.

+view abstract Cell cycle (Georgetown, Tex.), PMID: 26959608 2016

Anderson KE, Juvin V, Clark J, Stephens LR, Hawkins PT Signalling, Biological Chemistry

Phosphoinositides in primary mammalian tissue are highly enriched in a stearoyl/arachidonyl (C38:4) diacylgycerol backbone. However, mammalian cells grown in culture typically contain more diverse molecular species of phosphoinositides, characterised by a reduction in arachidonyl content in the sn-2 position. We have analysed the phosphoinositide species in MCF10a cells grown in culture by mass spectrometry. Under either serum or serum starved conditions the most abundant species of PI, PIP, PIP2 and PIP3 had masses which corresponded to C36:2, C38:4, C38:3, C38:2 and C36:1 diacylglycerol backbones and the relative proportions of each molecular species were broadly similar between each phosphoinositide class (approx. 50%, 25%, 10%, 10% and 10% respectively, for the species listed above). Supplementing the culture medium with BSA-loaded arachidonic acid promoted a rapid increase in the proportion of the C38:4 species in all phosphoinositide classes (from approx. 25%-60% of total species within 24 h), but the total amount of all combined species for each class remained remarkably constant. Stimulation of cells, cultured in either normal or arachidonate-enriched conditions, with 2 ng/ml EGF for 90 s caused substantial activation of Class I PI3K and accumulation of PIP3. Despite the increased proportion of C38:4 PIP3 under the arachidonate-supplemented conditions, the total amount of all combined PIP3 species accumulating in response to EGF was the same, with or without arachidonate supplementation; there were however small but significant preferences for the conversion of some PIP2 species to PIP3, with the polyunsaturated C38:4 and C38:3 species being more favoured over other species. These results suggest the enzymes which interconvert phosphoinositides are able to act on several different molecular species and homoeostatic mechanisms are in place to deliver similar phosphoinositide pool sizes under quite different conditions of arachidonate availability. They also suggest enzymes regulating PIP3 levels downstream of growth factor stimulation (i.e. PI3Ks and PIP3-phosphatases) show some acyl selectivity and further work should be directed at assessing whether different acyl species of PIP3 exhibit differing signalling potential.

+view abstract Advances in biological regulation, PMID: 26639089 2015

Clark J,Kay RR,Kielkowska A,Niewczas I,Fets L,Oxley D,Stephens LR,Hawkins PT Signalling, Biological Chemistry

Inositol phospholipids are critical regulators of membrane biology throughout eukaryotes. The general principle by which they perform these roles is conserved across species and involves binding of differentially phosphorylated inositol head groups to specific protein domains. This interaction serves to both recruit and regulate the activity of several different classes of protein which act on membrane surfaces. In mammalian cells, these phosphorylated inositol head groups are predominantly borne by a C38:4 diacylglycerol backbone. We show here that the inositol phospholipids of Dictyostelium are different, being highly enriched in an unusual C34:1e lipid backbone, 1-hexadecyl-2-(11Z-octadecenoyl)-sn-glycero-3-phospho-(1'-myo-inositol), in which the sn-1 position contains an ether-linked C16:0 chain; they are thus plasmanylinositols. These plasmanylinositols respond acutely to stimulation of cells with chemoattractants, and their levels are regulated by PIPKs, PI3Ks and PTEN. In mammals and now in Dictyostelium, the hydrocarbon chains of inositol phospholipids are a highly selected subset of those available to other phospholipids, suggesting that different molecular selectors are at play in these organisms but serve a common, evolutionarily conserved purpose.

+view abstract The EMBO journal, PMID: 25180230 2014

X Zhang, O Vadas, O Perisic, KE Anderson, J Clark, PT Hawkins, LR Stephens, RL Williams Signalling, Biological Chemistry

Phosphoinositide 3-kinases (PI3Ks) are essential for cell growth, migration, and survival. The structure of a p110β/p85β complex identifies an inhibitory function for the C-terminal SH2 domain (cSH2) of the p85 regulatory subunit. Mutagenesis of a cSH2 contact residue activates downstream signaling in cells. This inhibitory contact ties up the C-terminal region of the p110β catalytic subunit, which is essential for lipid kinase activity. In vitro, p110β basal activity is tightly restrained by contacts with three p85 domains: the cSH2, nSH2, and iSH2. RTK phosphopeptides relieve inhibition by nSH2 and cSH2 using completely different mechanisms. The binding site for the RTK's pYXXM motif is exposed on the cSH2, requiring an extended RTK motif to reach and disrupt the inhibitory contact with p110β. This contrasts with the nSH2 where the pY-binding site itself forms the inhibitory contact. This establishes an unusual mechanism by which p85 SH2 domains contribute to RTK signaling specificities.

+view abstract Molecular cell, PMID: 21362552 2011

J Clark, KE Anderson, V Juvin, TS Smith, F Karpe, MJ Wakelam, LR Stephens, PT Hawkins Signalling, Biological Chemistry

Class I phosphoinositide-3-kinase (PI3K) isoforms generate the intracellular signaling lipid, phosphatidylinositol(3,4,5)trisphosphate (PtdIns(3,4,5)P(3)). PtdIns(3,4,5)P(3) regulates major aspects of cellular behavior, and the use of both genetic and pharmacological intervention has revealed important isoform-specific roles for PI3Ks in health and disease. Despite this interest, current methods for measuring PtdIns(3,4,5)P(3) have major limitations, including insensitivity, reliance on radiolabeling, low throughput and an inability to resolve different fatty-acyl species. We introduce a methodology based on phosphate methylation coupled to high-performance liquid chromatography-mass spectrometry (HPLC-MS) to solve many of these problems and describe an integrated approach to quantify PtdIns(3,4,5)P(3) and related phosphoinositides (regio-isomers of PtdInsP and PtdInsP(2) are not resolved). This methodology can be used to quantify multiple fatty-acyl species of PtdIns(3,4,5)P(3) in unstimulated mouse and human cells (≥10(5)) or tissues (≥0.1 mg) and their increase upon appropriate stimulation.

+view abstract Nature methods, PMID: 21278744 2011

DR Soond, E Bjørgo, K Moltu, VQ Dale, DT Patton, KM Torgersen, F Galleway, B Twomey, J Clark, JS Gaston, K Taskén, P Bunyard, K Okkenhaug Immunology, Biological Chemistry

We have previously described critical and nonredundant roles for the phosphoinositide 3-kinase p110delta during the activation and differentiation of naive T cells, and p110delta inhibitors are currently being developed for clinical use. However, to effectively treat established inflammatory or autoimmune diseases, it is important to be able to inhibit previously activated or memory T cells. In this study, using the isoform-selective inhibitor IC87114, we show that sustained p110delta activity is required for interferon-gamma production. Moreover, acute inhibition of p110delta inhibits cytokine production and reduces hypersensitivity responses in mice. Whether p110delta played a similar role in human T cells was unknown. Here we show that IC87114 potently blocked T-cell receptor-induced phosphoinositide 3-kinase signaling by both naive and effector/memory human T cells. Importantly, IC87114 reduced cytokine production by memory T cells from healthy and allergic donors and from inflammatory arthritis patients. These studies establish that previously activated memory T cells are at least as sensitive to p110delta inhibition as naive T cells and show that mouse models accurately predict p110delta function in human T cells. There is therefore a strong rationale for p110delta inhibitors to be considered for therapeutic use in T-cell-mediated autoimmune and inflammatory diseases.

+view abstract Blood, PMID: 20081091 2010