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

Gβγ is a direct regulator of endogenous p101/p110γ and p84/p110γ PI3Kγ complexes in mouse neutrophils.
Rynkiewicz NK, Anderson KE, Suire S, Collins DM, Karanasios E, Vadas O, Williams R, Oxley D, Clark J, Stephens LR, Hawkins PT

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, 13, 656, 03 Nov 2020

PMID: 33144519

How is the acyl chain composition of phosphoinositides created and does it matter?
Barneda D, Cosulich S, Stephens L, Hawkins P

The phosphoinositide (PIPn) family of signalling phospholipids are central regulators in membrane cell biology. Their varied functions are based on the phosphorylation pattern of their inositol ring, which can be recognized by selective binding domains in their effector proteins and be modified by a series of specific PIPn kinases and phosphatases, which control their interconversion in a spatial and temporal manner. Yet, a unique feature of PIPns remains largely unexplored: their unusually uniform acyl chain composition. Indeed, while most phospholipids present a range of molecular species comprising acyl chains of diverse length and saturation, PIPns in several organisms and tissues show the predominance of a single hydrophobic backbone, which in mammals is composed of arachidonoyl and stearoyl chains. Despite evolution having favoured this specific PIPn configuration, little is known regarding the mechanisms and functions behind it. In this review, we explore the metabolic pathways that could control the acyl chain composition of PIPns as well as the potential roles of this selective enrichment. While our understanding of this phenomenon has been constrained largely by the technical limitations in the methods traditionally employed in the PIPn field, we believe that the latest developments in PIPn analysis should shed light onto this old question.

+ View Abstract

Biochemical Society transactions, 47, 5, 31 10 2019

DOI: 10.1042/BST20190205

PMID: 31657437

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.

+ View Abstract

Journal of leukocyte biology, , 1938-3673, 2019

PMID: 30720883