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 The Vermeren
 Group

Research


The Laboratory

We are a young, small laboratory (see also group members). We are interested in the cross-talk of small GTPases during complex cellular processes, and in particular in the roles of GTPase activating proteins (GAPs). We are part of the larger Inositide Laboratory and share space in open plan lab and bits of equipment. Moreover, we have weekly, shared lab meetings with Len Stephens and Phill Hawkins’, Heidi Welch’s and Nick Ktistakis’ groups.

Background to Research

Small GTPases of the Ras superfamily regulate a host of cellular functions including cell motility, adhesion, vesicular transport, transcription, cell division and survival to name but a few. The superfamily is grouped into the Ras, Arf, Rho families of small GTPases. All of these have in common, that they are cycling between an active, GTP-bound and an inactive, GDP-bound form, effectively acting as molecular switches. Intrinsic hydrolysis of GTP to GDP is in some cases very slow and is enhanced by GTPase activating proteins (GAPs), whilst guanine nucleotide exchange factors (GEFs) catalyse exchange of GDP for GTP (Fig 1).

Fig. 1 Fig. 1 Figure 1. Cycling of a small GTPase. Only when GTP-bound can the small GTPase bind to and activate an effector. GAPs aid hydrolysis of GTP for GDP whilst GEFs catalyse exchange of GDP for GTP. © Babraham Institute 2009

    Fig. 1 (hover to enlarge)

Only the GTP-bound form of the small GTPase can bind to,and activate, effector proteins. GAPs, like effectors bind to the GTP-bound form of small GTPases. In some cases, a GAP can act as an effector and as a regulator of a small GTPase. Altogether our knowledge of GAPs is still limited. Large numbers of GAPs have been identified but only in few cases do we understand their substrate specificity, mode of regulation and physiological function fully.

We recently identified ARAP3 in a screen for phosphoinositide 3-OH kinase (PI3K) effectors, by virtue of its binding to phosphatidylinositol-(3,4,5)P3 (PtdIns(3,4,5)P3), the lipid second messenger that PI3K produces. ARAP3 is a dual Arf and Rho GAP, it is part of the ARAP family of dual GAPs. The acronym stands for Arf and Rho GAP with Ankyrin repeats and PH domains. All ARAP3 share their unusual domain structure, comprising an N-terminal SAM domain, five PH domains, an Arf GAP and a Rho GAP domain as well as a poorly conserved Ras association domain (Fig 2).

Fig. 2 Fig. 2 Figure 2. The ARAP family of Arf and Rho GAPs. All ARAP proteins share their domain structure, but the reported substrate specificities are not identical. © Babraham Institute 2009

    Fig. 2 (hover to enlarge)

Analysis of ARAP3’s catalytic activities identified it as a PtdIns(3,4,5)P3-dependent Arf6 GAP and a PI3K and Rap-GTP activated RhoA GAP (Fig 3). Ectopic expression of ARAP3 caused dramatic retractions instead of lamellipodium formation of overexpressing cells when PI3K is activated, indicating ARAP3 might be involved in cellular adhesion, and tightly controlled by PI3K.

Fig. 3 Fig. 3 Figure 3. Mode of regulation of ARAP3. PI3K is the main regulator, causing a plasma membrane recruitment of ARAP3. The Arf GAP activity depends of PtdIns(3,4,5)P3 even in vitro; Rap-GTP binds to ARAP3’s Ras association domain and activates its Rho GAP activity. © Babraham Institute 2009

    Fig. 3 (hover to enlarge)

Similarly, knock-down of ARAP3 caused cells to be “stuck” in position, and again interfered with lamellipodium formation (Fig 4). ARAP3 knock-down also caused cells to have a reduced ability to polarise when they become motile, indicating ARAP3 might be involved in the control of one, or more, aspects of cellular motility.

Fig. 4 Fig. 4 Figure 4. ARAP3 is important for lamellipodium formation. F-actin of control pig aortic endothelial cells (top), ARAP3 overexpressing cells (middle) and ARAP3 knock-down cells (bottom) are shown starved (left) or stimulated with PDGF for 5 minutes (right). Overexpression and reduction of ARAP3 interfere with lamellipodium formation in different ways. © Babraham Institute 2009

    Fig. 4 (hover to enlarge)

 

Current projects

We continue to analyse ARAP3 using biochemical methods and transient transfection based systems to try and understand why ARAP3 has five PH domains. However, the main focus of our work has shifted to try and understand more physiological aspects.

We are very excited about the possibility that ARAP3 might be involved in the control of cell motility, and are attempting to study this in two systems at present. Our first model relies on endothelial cells. Endothelial  cells are the initial building blocks of blood vessels, and we found ARAP3 is highly expressed in endothelial cells. Our knock-down model, where reduction of ARAP3 caused a striking phenotype, was performed is a porcine endothelial cell line. We now wish to address whether ARAP3 might be important for vessel formation (angiogenesis), and if so, whether it might be important for tumour progression?

Our second system to be studied are neutrophils, where we identified ARAP3 and where it is very highly expressed. Neutrophils are highly specialised immune cells which are involved in the non-specific immune response, and which are characterised by their ability to chemotax (move to the source of a chemoattractant) faster than the other cells in our immune system. Once activated, Neutrophils can produce reactive oxygen species, which are intended to combat invaders – but cause inflammation too. Finally, neutrophils phagocytose and kill bacteria and large particles. We are currently using a model system to study the role of ARAP3 in neutrophil function.