Life Sciences Research for Lifelong Health

Len Stephens

Research Summary

The programmes of work in the laboratory are currently aimed at understanding the molecular mechanisms and physiological significance of intracellular signalling networks which involve a family of enzymes called phosphoinositide 3OH-kinases (PI3Ks).

PI3Ks are now accepted to be critical regulators of numerous important and complex cell responses, including cell growth, division, survival and movement.

PI3Ks catalyse the formation of one or more critical phospholipid messenger molecules, which signal information by binding to specific domains in target proteins. Currently the best understood pathway involves the activation of Class I PI3Ks by cell surface receptors.

In recent years, the laboratory has increasingly focused on the role of PI3Ks in the signalling mechanisms which allow receptors on neutrophils (white blood cells) to control various aspects of neutrophil function.

Neutrophils are key players in the front line of our immune system, responsible primarily for the recognition and destruction of bacterial and fungal pathogens. However, they are also involved in the amplification cascades that underlie various inflammatory pathologies, e.g. Acute Respiratory Distress Syndrome (ARDS) and rheumatoid arthritis.

Latest Publications

Frontline Science: TNF-α and GM-CSF1 priming augments the role of SOS1/2 in driving activation of Ras, PI3K-γ, and neutrophil proinflammatory responses.
Suire S, Baltanas FC, Segonds-Pichon A, Davidson K, Santos E, Hawkins PT, Stephens LR

Circulating neutrophils are, by necessity, quiescent and relatively unresponsive to acute stimuli. In regions of inflammation, mediators can prime neutrophils to react to acute stimuli with stronger proinflammatory, pathogen-killing responses. In neutrophils G protein-coupled receptor (GPCR)-driven proinflammatory responses, such as reactive oxygen species (ROS) formation and accumulation of the key intracellular messenger phosphatidylinositol (3,4,5)-trisphosphate (PIP ), are highly dependent on PI3K-γ, a Ras-GTP, and Gβγ coincidence detector. In unprimed cells, the major GPCR-triggered activator of Ras is the Ras guanine nucleotide exchange factor (GEF), Ras guanine nucleotide releasing protein 4 (RasGRP4). Although priming is known to increase GPCR-PIP signaling, the mechanisms underlying this augmentation remain unclear. We used genetically modified mice to address the role of the 2 RasGEFs, RasGRP4 and son of sevenless (SOS)1/2, in neutrophil priming. We found that following GM-CSF/TNFα priming, RasGRP4 had only a minor role in the enhanced responses. In contrast, SOS1/2 acquired a substantial role in ROS formation, PIP accumulation, and ERK activation in primed cells. These results suggest that SOS1/2 signaling plays a key role in determining the responsiveness of neutrophils in regions of inflammation.

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Journal of leukocyte biology, , 1938-3673, , 2019

PMID: 30720883

Quantitation of class IA PI3Ks in mice reveals p110-free-p85s and isoform-selective subunit associations and recruitment to receptors.
Tsolakos N, Durrant TN, Chessa T, Suire SM, Oxley D, Kulkarni S, Downward J, Perisic O, Williams RL, Stephens L, Hawkins PT

Class IA PI3Ks have many roles in health and disease. The rules that govern intersubunit and receptor associations, however, remain unclear. We engineered mouse lines in which individual endogenous class IA PI3K subunits were C-terminally tagged with 17aa that could be biotinylated in vivo. Using these tools we quantified PI3K subunits in streptavidin or PDGFR pull-downs and cell lysates. This revealed that p85α and β bound equivalently to p110α or p110β but p85α bound preferentially to p110δ. p85s were found in molar-excess over p110s in a number of contexts including MEFs (p85β, 20%) and liver (p85α, 30%). In serum-starved MEFs, p110-free-p85s were preferentially, compared with heterodimeric p85s, bound to PDGFRs, consistent with in vitro assays that demonstrated they bound PDGFR-based tyrosine-phosphorylated peptides with higher affinity and co-operativity; suggesting they may act to tune a PI3K activation threshold. p110α-heterodimers were recruited 5-6× more efficiently than p110β-heterodimers to activated PDGFRs in MEFs or to PDGFR-based tyrosine-phosphorylated peptides in MEF-lysates. This suggests that PI3Kα has a higher affinity for relevant tyrosine-phosphorylated motifs than PI3Kβ. Nevertheless, PI3Kβ contributes substantially to acute PDGF-stimulation of PIP and PKB in MEFs because it is synergistically, and possibly sequentially, activated by receptor-recruitment and small GTPases (Rac/CDC42) via its RBD, whereas parallel activation of PI3Kα is independent of its RBD. These results begin to provide molecular clarity to the rules of engagement between class IA PI3K subunits in vivo and past work describing "excess p85," p85α as a tumor suppressor, and differential receptor activation of PI3Kα and PI3Kβ.

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Proceedings of the National Academy of Sciences of the United States of America, 115, 1091-6490, 12176-12181, 2018

PMID: 30442661

Genome organization and chromatin analysis identify transcriptional downregulation of insulin-like growth factor signaling as a hallmark of aging in developing B cells.
Koohy H, Bolland DJ, Matheson LS, Schoenfelder S, Stellato C, Dimond A, Várnai C, Chovanec P, Chessa T, Denizot J, Manzano Garcia R, Wingett SW, Freire-Pritchett P, Nagano T, Hawkins P, Stephens L, Elderkin S, Spivakov M, Fraser P, Corcoran AE, Varga-Weisz PD

Aging is characterized by loss of function of the adaptive immune system, but the underlying causes are poorly understood. To assess the molecular effects of aging on B cell development, we profiled gene expression and chromatin features genome-wide, including histone modifications and chromosome conformation, in bone marrow pro-B and pre-B cells from young and aged mice.

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Genome biology, 19, 1474-760X, 126, 2018

PMID: 30180872

Group Members

Latest Publications

Quantitation of class IA PI3Ks in mice reveals p110-free-p85s and isoform-selective subunit associations and recruitment to receptors.

Tsolakos N, Durrant TN, Chessa T

Proceedings of the National Academy of Sciences of the United States of America
115 1091-6490:12176-12181 (2018)

PMID: 30442661

Profiling of phosphoinositide molecular species in human and mouse platelets identifies new species increasing following stimulation.

Mujalli A, Chicanne G, Bertrand-Michel J

Biochimica et biophysica acta
0006-3002: (2018)

PMID: 29902570

In-depth PtdIns(3,4,5)P3 signalosome analysis identifies DAPP1 as a negative regulator of GPVI-driven platelet function.

Durrant TN, Hutchinson JL, Heesom KJ

Blood advances
1 2473-9529:918-932 (2017)

PMID: 29242851

PTEN Regulates PI(3,4)P2 Signaling Downstream of Class I PI3K.

Malek M, Kielkowska A, Chessa T

Molecular cell
1097-4164: (2017)

PMID: 29056325

Class (I) Phosphoinositide 3-Kinases in the Tumor Microenvironment.

Gyori D, Chessa T, Hawkins PT

Cancers
9 : (2017)

PMID: 28273837

cAMP Signaling of Adenylate Cyclase Toxin Blocks the Oxidative Burst of Neutrophils through Epac-Mediated Inhibition of Phospholipase C Activity.

Cerny O, Anderson KE, Stephens LR

Journal of immunology (Baltimore, Md. : 1950)
198 1550-6606:1285-1296 (2017)

PMID: 28039302

In B cells, phosphatidylinositol 5-phosphate 4-kinase-α synthesizes PI(4,5)P2 to impact mTORC2 and Akt signaling.

Bulley SJ, Droubi A, Clarke JH

Proceedings of the National Academy of Sciences of the United States of America
1091-6490: (2016)

PMID: 27601656

Coincident signals from GPCRs and receptor tyrosine kinases are uniquely transduced by PI3Kβ in myeloid cells.

Houslay DM, Anderson KE, Chessa T

Science signaling
9 1937-9145:ra82 (2016)

PMID: 27531651