The Stephens/Hawkins Laboratory
Len Stephens and Phill Hawkins are jointly responsible for a laboratory 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).
Fig 1 Activation of Class I PI3Ks by cell surface receptors. (click to enlarge)
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 (click to enlarge)
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 laboratory 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.
Fig. 3 Chemotaxis of neutrophils towards a gradient of fMLP
(click to open in YouTube)
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 laboratory 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 PI(3)P accumulates around the phagosome of RAW 264.7 cells.
(click to open in You Tube)
RAW 264.7 cells were stably transfected with the GFP-PX domain from p40phox. 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.
Updated 7 January, 2013