Len Stephens - Academia.edu (original) (raw)

Papers by Len Stephens

Science, 2013

Genetic mutations cause primary immunodeficiencies (PIDs) that predispose to infections. Here, we... more Genetic mutations cause primary immunodeficiencies (PIDs) that predispose to infections. Here, we describe activated PI3K-δ syndrome (APDS), a PID associated with a dominant gain-of-function mutation in which lysine replaced glutamic acid at residue 1021 (E1021K) in the p110δ protein, the catalytic subunit of phosphoinositide 3-kinase δ (PI3Kδ), encoded by the PIK3CD gene. We found E1021K in 17 patients from seven unrelated families, but not among 3346 healthy subjects. APDS was characterized by recurrent respiratory infections, progressive airway damage, lymphopenia, increased circulating transitional B cells, increased immunoglobulin M, and reduced immunoglobulin G2 levels in serum and impaired vaccine responses. The E1021K mutation enhanced membrane association and kinase activity of p110δ. Patient-derived lymphocytes had increased levels of phosphatidylinositol 3,4,5-trisphosphate and phosphorylated AKT protein and were prone to activation-induced cell death. Selective p110δ inhibitors IC87114 and GS-1101 reduced the activity of the mutant enzyme in vitro, which suggested a therapeutic approach for patients with APDS.

Nature, 2000

The PX domain, which until recently was an orphan domain, has emerged as the latest member of the... more The PX domain, which until recently was an orphan domain, has emerged as the latest member of the phosphoinositide-binding module superfamily. Structural studies have revealed that it has a novel fold and identified key residues that interact with the bound phosphoinositide, enabling some prediction of phosphoinositide-binding specificity. Specificity for PtdIns(3)P appears to be the most common, and several proteins containing PX domains localise to PtdIns(3)P-rich endosomal and vacuolar structures through their PX domains: these include the yeast t-SNARE Vam7p, mammalian sorting nexins (involved in membrane trafficking events) and the Ser/Thr kinase CISK, which is implicated in cell survival. Additionally, phosphoinositide binding to the PX domains of p40(phox) and p47(phox) appears to play a critical role in the active assembly of the neutrophil oxidase complex.

Through selective disruption of phosphoinositide 3-kinase (PI3K) activity and the use of green fl... more Through selective disruption of phosphoinositide 3-kinase (PI3K) activity and the use of green fluorescent protein tagged derivatives of domains capable of specifically binding the lipid products of PI3Ks in vivo, it has been shown that this family of signalling enzymes have vital and distinct roles in chemotaxis, phagocytosis and phagosome maturation in leukocytes.

FEBS Journal, 2006

Ribosomal protein S6 kinase (S6K) is activated by an array of mitogenic stimuli and is a key play... more Ribosomal protein S6 kinase (S6K) is activated by an array of mitogenic stimuli and is a key player in the regulation of cell growth. The activation process of S6 kinase involves a complex and sequential series of multiple Ser/Thr phosphorylations and is mainly mediated via phosphatidylinositol 3-kinase (PI3K)-3-phosphoinositide-dependent protein kinase-1 (PDK1) and mTor-dependent pathways. Upstream regulators of S6K, such as PDK1 and protein kinase B (PKB/Akt), are recruited to the membrane via their pleckstrin homology (PH) or protein-protein interaction domains. However, the mechanism of integration of S6K into a multi-enzyme complex around activated receptor tyrosine kinases is not clear. In the present study, we describe a specific interaction between S6K with receptor tyrosine kinases, such as platelet-derived growth factor receptor (PDGFR). The interaction with PDGFR is mediated via the kinase or the kinase extension domain of S6K. Complex formation is inducible by growth factors and leads to S6K tyrosine phosphorylation. Using PDGFR mutants, we have shown that the phosphorylation is exerted via a PDGFR-src pathway. Furthermore, src kinase phosphorylates and coimmunoprecipitates with S6K in vivo. Inhibitors towards tyrosine kinases, such as genistein and PP1, or src-specific SU6656, but not PI3K and mTor inhibitors, lead to a reduction in tyrosine phosphorylation of S6K. In addition, we mapped the sites of tyrosine phosphorylation in S6K1 and S6K2 to Y39 and Y45, respectively. Mutational and immunofluorescent analysis indicated that phosphorylation of S6Ks at these sites does not affect their activity or subcellular localization. Our data indicate that S6 kinase is recruited into a complex with RTKs and src and becomes phosphorylated on tyrosine/s in response to PDGF or serum.

Advances in Biological Regulation, 2014

The EMBO journal, 2014

Inositol phospholipids are critical regulators of membrane biology throughout eukaryotes. The gen... more 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...

Current Biology, 2002

Background: Type I phosphoinositide 3-kinases are responsible for the hormone-sensitive synthesis... more Background: Type I phosphoinositide 3-kinases are responsible for the hormone-sensitive synthesis of the lipid messenger phosphatidylinositol(3,4,5)-trisphosphate. Type IA and IB subfamily members contain a Ras binding domain and are stimulated by activated Ras proteins both in vivo and in vitro. The mechanism of Ras activation of type I PI3Ks is unknown, in part because no robust in vitro assay of

Nature Cell Biology, 2001

The production of reactive oxygen species (ROS) by neutrophils has a vital role in defence agains... more The production of reactive oxygen species (ROS) by neutrophils has a vital role in defence against a range of infectious agents, and is driven by the assembly of a multi-protein complex containing a minimal core of five proteins: the two membrane-bound subunits of cytochrome b 558 (gp91 phox and p22 phox ) and three soluble factors (GTP-Rac, p47 phox and p67 phox (refs 1, 2). This minimal complex can reconstitute ROS formation in vitro in the presence of non-physiological amphiphiles such as SDS. p40 phox has subsequently been discovered as a binding partner for p67 phox (ref.

PLOS One, 2011

The EMBO Journal, 2006

The production of reactive oxygen species by the NADPH oxidase complex of phagocytes plays a crit... more The production of reactive oxygen species by the NADPH oxidase complex of phagocytes plays a critical role in our defence against bacterial and fungal infections. The PX domains of two oxidase components, p47(phox) and p40(phox), are known to bind phosphoinositide products of PI3Ks but the physiological roles of these interactions are unclear. We have created mice which carry an R58A mutation in the PX domain of their p40(phox) gene, which selectively prevents binding to PtdIns3P. p40(phoxR58A/R58A) embryos do not develop normally but p40(phoxR58A/-) mice are viable and neutrophils from these animals exhibit significantly reduced oxidase responses compared to those from their p40(phox+/-) siblings (e.g. 60% reduced in response to phagocytosis of Staphylococcus aureus). Wortmannin inhibition of the S. aureus oxidase response correlates with inhibition of phagosomal PtdIns3P accumulation and overlaps with the reduction in this response caused by the R58A mutation, suggesting PI3K regulation of this response is substantially dependent on PtdIns3P-binding to p40(phox). p40(phoxR58A/-) mice are significantly compromised in their ability to kill S. aureus in vivo, defining the physiological importance of this interaction.

Science, 1998

Protein kinase B (PKB) is activated in response to phosphoinositide 3-kinases and their lipid pro... more Protein kinase B (PKB) is activated in response to phosphoinositide 3-kinases and their lipid products phosphatidylinositol 3,4, 5-trisphosphate [PtdIns(3,4,5)P3] and PtdIns(3,4)P2 in the signaling pathways used by a wide variety of growth factors, antigens, and inflammatory stimuli. PKB is a direct target of these lipids, but this regulation is complex. The lipids can bind to the pleckstrin homologous domain of PKB, causing its translocation to the membrane, and also enable upstream, Thr308-directed kinases to phosphorylate and activate PKB. Four isoforms of these PKB kinases were purified from sheep brain. They bound PtdIns(3,4,5)P3 and associated with lipid vesicles containing it. These kinases contain an NH2-terminal catalytic domain and a COOH-terminal pleckstrin homologous domain, and their heterologous expression augments receptor activation of PKB, which suggests they are the primary signal transducers that enable PtdIns(3,4,5)P3 or PtdIns- (3,4)P2 to activate PKB and hence to control signaling pathways regulating cell survival, glucose uptake, and glycogen metabolism.

PLoS ONE, 2011

Short chain fatty acids (SCFAs) have recently attracted attention as potential mediators of the e... more Short chain fatty acids (SCFAs) have recently attracted attention as potential mediators of the effects of gut microbiota on intestinal inflammation. Some of these effects have been suggested to occur through the direct actions of SCFAs on the GPR43 receptor in neutrophils, though the precise role of this receptor in neutrophil activation is still unclear. We show that mouse bone marrow derived neutrophils (BMNs) can chemotax effectively through polycarbonate filters towards a source of acetate, propionate or butyrate. Moreover, we show that BMNs move with good speed and directionality towards a source of propionate in an EZ-Taxiscan chamber coated with fibrinogen. These effects of SCFAs were mimicked by low concentrations of the synthetic GPR43 agonist phenylacetamide-1 and were abolished in GPR43 2/2 BMNs. SCFAs and phenylacetamide-1 also elicited GPR43-dependent activation of PKB, p38 and ERK and these responses were sensitive to pertussis toxin, indicating a role for Gi proteins. Phenylacetamide-1 also elicited rapid and transient activation of Rac1/2 GTPases and phosphorylation of ribosomal protein S6. Genetic and pharmacological intervention identified important roles for PI3Kc, Rac2, p38 and ERK, but not mTOR, in GPR43-dependent chemotaxis. These results identify GPR43 as a bona fide chemotactic receptor for neutrophils in vitro and start to define important elements in its signal transduction pathways.

Nature Cell Biology, 2007

Phophoinositide 3-kinases (PI(3)Ks) are thought to have critical roles within the gradient-sensin... more Phophoinositide 3-kinases (PI(3)Ks) are thought to have critical roles within the gradient-sensing machinery of a variety of highly motile cells 1,2 , such as mammalian phagocytes 3 , allowing these cells to respond quickly and efficiently to shallow gradients of soluble stimuli. Our analysis of mammalian neutrophil migration towards ligands such as fMLP shows that, although PtdIns(3,4)P 2 and PtdIns(3,4,5)P 3 accumulate in a PI(3)Kγ-dependent fashion at the up-gradient leading-edge, this signal is not required for efficient gradientsensing and gradient-biased movement. PI(3)Kγ activity is however, a critical determinant of the proportion of cells that can move, that is, respond chemokinetically, in reaction to fMLP. Furthermore, this dependence of chemokinesis on PI(3)Kγ activity is context dependent, both with respect to the state of priming of the neutrophils and the type of surface on which they are migrating. We propose this effect of PI(3)Kγ is through roles in the regulation of some aspects of neutrophil polarization that are relevant to movement, such as integrinbased adhesion and the accumulation of polymerized (F)-actin at the leading-edge.

Nature Cell Biology, 2006

Through their ability to regulate production of the key lipid messenger PtdIns(3,4,5)P(3), the cl... more Through their ability to regulate production of the key lipid messenger PtdIns(3,4,5)P(3), the class I phosphatidylinositol-3-OH kinases (PI(3)Ks) support many critical cell responses. They, in turn, can be regulated by cell-surface receptors through signals acting on either their adaptor subunits (for example, through phosphotyrosine or Gbetagammas) or their catalytic subunits (for example, through GTP-Ras). The relative significance of these controlling inputs is undefined in vivo. Here, we have studied the roles of Gbetagammas, the adaptor p101, Ras and the Ras binding domain (RBD) in the control of the class I PI(3)K, PI(3)Kgamma, in mouse neutrophils. Loss of p101 leads to major reductions in the accumulation of PtdIns(3,4,5)P(3), activation of protein kinase B (PKB) and in migration towards G-protein activating ligands in vitro, and to an aseptically inflamed peritoneum in vivo. Loss of sensitivity of PI(3)Kgamma to Ras unexpectedly caused similar reductions, but additionally caused a substantial loss in production of reactive oxygen species (ROS). We conclude that Gbetagammas, p101 and the Ras-RBD interaction all have important roles in the regulation of PI(3)Kgamma in vivo and that they can simultaneously, but differentially, control distinct PI(3)Kgamma effectors.

Nature, 2003

... PI(3)K-mediated activation of the cell survival kinase PKB/Akt, and negative regulation of PI... more ... PI(3)K-mediated activation of the cell survival kinase PKB/Akt, and negative regulation of PI(3)K signalling by the tumour suppressor PTEN (refs 3, 4) are key regulatory events in tumorigenesis. Thus, a model has arisen that PI(3)Ks promote development of cancers. ...

Molecular Cell, 2000

IA and IB, with each subtype having a different regula-Hills Road, Cambridge CB2 2QH tory subunit... more IA and IB, with each subtype having a different regula-Hills Road, Cambridge CB2 2QH tory subunit. The class IA enzymes (␣, ␤, and ␦) have a United Kingdom p85 regulatory subunit containing two SH2 domains, † Department of Signalling which are essential for their activation by tyrosine kinase Inositide Laboratory receptors. The class IB PI3K␥ has a p101 subunit, which Babraham Institute is required for maximal G␤␥-stimulated formation of PIP 3 Cambridge CB2 4AT (Stephens et al., 1997; Krugmann et al., 1999; Maier et United Kingdom al., 1999). ‡ Institute of Biochemistry The prominent roles that PI3Ks play in a variety of University of Fribourg diseases have fueled intense efforts at developing iso-CH-1700 Fribourg type-specific inhibitors of PI3Ks. Wortmannin and its Switzerland analogs were shown to inhibit phagocytocis-induced respiratory burst (Baggiolini et al., 1987). Subsequent work established that this is due to specific inhibition Summary of PI3K activity. Recent reports of PI3K␥ knockout mice have shown that this isozyme plays a crucial role in The specific phosphoinositide 3-kinase (PI3K) inhibiinflammation (Hirsch et al., 2000; Li et al., 2000; Sasaki tors wortmannin and LY294002 have been invaluable et al., 2000)