The laboratory

Len Stephens and Phill Hawkins are jointly responsible for a lab currently comprising 5 post-docs, 4 PhD students and 1 research technician. The programmes of work in the laboratory are currently aimed at understanding the molecular mechanisms and physiological significance of intracellular signalling networks which involve phosphoinositide 3OH-kinases (PI3Ks).

Background to Research

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 (see Fig. 1).

Figure 1 (Click to enlarge)

Activation of Class I PI3Ks by cell surface receptors. Left: Activation of Class IA PI3Ks by protein tyrosine kinase-coupled receptors. Right: Activation of Class IB PI3Ks by G-protein coupled receptors.

PI3Ks are classified according the nomenclature of their catalytic subunits (p110α, -β, -γ and -δ). p110α, -β, and -δ associate with p85 or p50-p55 regulatory subunits and together they constitute the Class IA PI3Ks. Class IA PI3Ks are classically activated through protein tyrosine kinase-coupled receptors. p110γ; associates with p101 or p84 regulatory subunits and constitutes the Class IB PI3K. Class IB PI3K signals downstream of G-protein coupled receptors.

It is now known that a huge variety of receptors (e.g. including those for growth factors, antigens and various inflammatory stimuli) from different structural families and with differing signal transduction mechanisms, can activate Class I PI3Ks to synthesise the messenger phosphatidylinositol 3,4,5-trisphosphate (PIP3) in the inner leaflet of the plasma membrane. The PIP3 that is then made is thought to regulate the location and activity of several primary effectors by binding to conserved domains within their protein structure, the most clearly understood of which are a subgroup of pleckstrin homology (PH) domains. Thus, PIP3 is thought to co-ordinate the regulation of various downstream responses (Fig. 2).

Fig. 2. Major effectors of Class I PI3K signalling

PIP3 is a critical second messenger that is able to interact with the PH domains of a variety of different proteins. Recruitment and activation of these proteins enables the signal to be relayed to downstream targets, ultimately resulting in regulation of vital cellular functions.

Figure adapted from Hawkins et al., 2006.

Class III PI3Ks are thought to be involved in intracellular trafficking through the endosomal/lysosomal system. More recently, Class III PI3Ks have been shown to be regulated by starvation and, co-discovered by this laboratory, in the maturation of the phagosome. Class III PI3Ks synthesise phosphatidylinositol 3-phosphate (PI(3)P) and signal via the binding of PI(3)P to proteins which carry specific FYVE or PX domains.

Current projects

In recent years, the lab has increasingly focused on the role of PI3Ks in the signalling mechanisms which allow cell surface receptors on mammalian neutrophils 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.

Current projects can be split into two major categories. The first of these is work that is trying to understand the molecular mechanisms which allow different receptor types to activate PI3Ks. Historically the lab has had a particular interest in defining the mechanisms that allow soluble stimuli (e.g. fMLP, histamine) operating through G-protein coupled receptors to activate Class I PI3Ks and hence rapid synthesis of PIP3; this work led to the discovery of the PI3Kγ isoform and some understanding of how it is regulated by Gβγ subunits and Ras. We are also currently trying to understand how other Class I PI3K isoforms are involved in regulating the processes of adhesion (integrins) and phagocytosis (integrin, FcR and scavenger receptors).

The second major area of work is trying to understand the molecular mechanisms which allow the lipid products of activated PI3Ks to regulate key neutrophil responses. The two main PI3K-dependent responses we are currently focusing on are chemotaxis in gradients of soluble stimuli (Fig. 3), which is a crucial mechanism allowing recruitment of neutrophils to sites of inflammation and infection, and activation of the NADPH oxidase.

Figure 3 (Click to enlarge)



Chemotaxis of neutrophils towards a gradient of fMLP

Video of neutrophils chemotaxing towards fMLP released by a micropipette. The length of this image sequence is approximately 5 minutes, after which the neutrophils clearly demonstrate directional migration towards the pipette.

The activation of the NADPH oxidase, or 'respiratory burst', is an important weapon used by neutrophils and other phagocytic cells to kill pathogens engulfed by phagocytosis. Work from our lab and others has recently discovered that PI(3)P generation on phagosomal membranes (Fig. 4) is an important signal for recruitment of an active NADPH oxidase complex to this location and we are currently trying to understand how this PI(3)P signal is generated by activation of appropriate receptors and how it is used to regulate the oxidase complex.

Figure 4 (Click to enlarge)

PI(3)P accumulates around the phagosome of RAW 264.7 cells.

RAW 264.7 cells were stably transfected with the GFP-PX domain from p40^phox. This domain binds to PI(3)P with high affinity and specificity.
Upon ingestion of IgG-opsonised zymosan particles, the PX domain rapidly accumulates on the phagosome, indicating an increase of levels of PI(3)P in the phagosomal membrane.

The work in our laboratory is 'question-led' and hence generally involves the use of several methods and techniques. These have included purification of proteins by catalytic activity, cloning and expression of gene products and the generation of several transgenic mice. Our work is supported by excellent mass spec, transgenic and imaging facilities at Babraham.

Selected publications

Hawkins PT, Stephens LR (2007) PI3Kγ is a key regulator of inflammatory responses and cardiovascular homeostasis.
Science 318 64-66
DOI link: http://dx.doi.org/10.1126/science.1145420
Abstract: Click here
Full text: Click here

Ferguson GJ, Milne L, Kulkarni S, Sasaki T, Walker S, Andrews S, Crabbe T, Finan P, Jones G, Jackson S, Camps M, Rommel C, Wymann M, Hirsch E, Hawkins PT, Stephens LR (2007) PI(3)Kγ has an important context-dependent role in neutrophil chemokinesis.
Nature Cell Biology 9 86-91
http://dx.doi.org/10.1038/ncb1517

Ellson CD, Davidson K, Ferguson GJ, O'Connor R, Stephens LR, Hawkins PT (2006) Neutrophils from p40phox-/- mice exhibit severe defects in NADPH oxidase regulation and oxidant-dependent bacterial killing.
Journal of Experimental Medicine 203 1927-1937
http://dx.doi.org/10.1084/jem.20052069

Suire S, Condliffe AM, Ferguson GJ, Ellson CD, Guillou H, Davidson K, Welch HCE, Coadwell WJ, Turner M, Chilvers ER, Hawkins PT, Stephens LR (2006) Gβγs and the Ras binding domain of p110γ are both important regulators of PI(3)Kγ signalling in neutrophils.
Nature Cell Biology 8 1303-1309
http://dx.doi.org/10.1038/ncb1494

Condliffe AM, Davidson K, Anderson KE, Ellson CD, Crabbe T, Okkenhaug K, Vanhaesebroeck B, Turner M, Webb LMC, Wymann MP, Hirsch E, Ruckle T, Camps M, Rommel C, Jackson SP, Chilvers ER, Stephens LR, Hawkins PT (2005) Sequential activation of Class IB and Class IA PI3K is important for the primed respiratory burst of human but not murine neutrophils.
Blood 106 1432-1440
http://dx.doi.org/10.1182/blood-2005-03-0944

Suire S, Coadwell WJ, Ferguson GJ, Davidson K, Hawkins PT, Stephens LR (2005) p84, a new Gβγ-activated regulatory subunit of the type IB phosphoinositide 3-kinase p110γ.
Current Biology 15 566-570
http://dx.doi.org/10.1016/j.cub.2005.02.020

Krugmann S, Anderson KE, Ridley SH, Risso N, McGregor AH, Coadwell WJ, Davidson K, Eguinoa A, Ellson CD, Lipp P, Manifava M, Ktistakis NT, Painter G, Thuring JW, Cooper MA, Lim Z-Y, Holmes AB, Dove SK, Michell RH, Grewal A, Nazarian A, Erdjument-Bromage H, Tempst P, Stephens LR, Hawkins PT (2002) Identification of ARAP3, a novel PI3K effector regulating both Arf and Rho GTPases, by selective capture on phosphoinositide affinity matrices.
Molecular Cell 9 95-108
http://dx.doi.org/10.1016/S1097-2765(02)00434-3

Welch HCE, Coadwell WJ, Ellson CD, Ferguson GJ, Andrews SR, Erdjument-Bromage H, Tempst P, Hawkins PT, Stephens LR (2002) P-Rex1, a PtdIns(3,4,5)P3- and Gβγ-regulated guanine-nucleotide exchange factor for Rac.
Cell 108 809-821
http://dx.doi.org/10.1016/S0092-8674(02)00663-3

Ellson CD, Anderson KE, Morgan G, Chilvers ER, Lipp P, Stephens LR, Hawkins PT (2001) Phosphatidylinositol 3-phosphate is generated in phagosomal membranes.
Current Biology 11 1631-1635
http://dx.doi.org/10.1016/S0960-9822(01)00447-X

Ellson CD, Gobert-Gosse S, Anderson KE, Davidson K, Erdjument-Bromage H, Tempst P, Thuring JW, Cooper MA, Lim Z-Y, Holmes AB, Gaffney PRJ, Coadwell WJ, Chilvers ER, Hawkins PT, Stephens LR (2001) PtdIns(3)P regulates the neutrophil oxidase complex by binding to the PX domain of p40phox.
Nature Cell Biology 3 679-682
http://dx.doi.org/10.1038/35083076

Pacold ME, Suire S, Perisic O, Lara-Gonzalez S, Davis CT, Walker EH, Hawkins PT, Stephens LR, Eccleston JF, Williams RL (2000) Crystal structure and functional analysis of Ras binding to its effector phosphoinositide 3-kinase γ.
Cell 103 931-943
http://dx.doi.org/10.1016/S0092-8674(00)00196-3