Sonja Vermeren (née Krugmann)
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Regulation of cross-talk between small G-proteins by ARAP proteins
The Ras superfamily of small GTPases contains amongst others the families of Rho , Ras and Arf small G proteins. Between them, they control a host of cellular functions including cytoskeletal remodelling, vesicular transport, cell motility, adhesion, cell division and survival, transcription and translation. All small G proteins cycle between an inactive, GDP-bound form and an active, GTP-bound form. Only the latter causes the activation of effector proteins. Intrinsic hydrolysis of GTP to GDP is often slow and is catalysed by GTPase activating proteins (GAPs) whilst exchange of GDP for GTP is catalysed by guanine nucleotide exchange factors (GEFs).
Figure 1 (Click to enlarge)
Domain structure and activities of ARAP3. The N-terminal PH domain mediates binding to PtdIns(3,4,5)P3. The GAP domains are specific for Arf6 and RhoA, respectively. PtdIns(3,4,5)P3 is required for Arf6 GAP activity and activates RhoA GAP activity. Rap-GTP binds to the Ras binding domain and activates RhoA GAP activity.
We have recently identified the dual GAP ARAP3 in a screen for phosphatidylinositol-(3,4,5)-P3 (PtdIns(3,4,5)P
3 ) binding proteins from porcine neutrophils [1]. This 180kDa protein contains an unusual domain structure with a SAM domain, five PH domains, an Arf-GAP and a Rho-GAP domain as well as a poorly conserved Ras binding domain. ARAP3 is broadly expressed, but shows highest expression in neutrophils and cells of epithelial origin. Analysis of ARAP3 lipid binding showed ARAP3 binds both in vitro and in vivo very tightly to PtdIns(3,4,5)P
3 using its most N-terminal PH domain. Analysis of the catalytic activities showed ARAP3 to be a PtdIns(3,4,5)P
3 –dependent Arf6 GAP both in vitro and in vivo [1] and a Rap-GTP and PtdIns(3,4,5)P
3 –activated RhoA GAP [2]. Two further ARAP family members exist, which display identical domain structures and overlapping expression profiles.
We used siRNA to knock-down ARAP3 expression in an endothelial cell model [3]. In contrast to control cells, ARAP3-deficient cells were unable to form lamellipodia after stimulation with growth factors. In a motility assay, ARAP3-deficient cells polarised more poorly than control counterparts. Further, ARAP3-deficient cells displayed increased activities of RhoA and Arf6 and interestingly also showed faster and steeper activation of Rac after challenge with growth factor. We conclude, ARAP3 is an ideally suited candidate protein to mediate cross-talk between different small G proteins. It is likely ARAP3 is an important regulator of complicated biological processes such as neutrophil chemotaxis. Current studies are aimed at probing this hypothesis.
Figure 2 (Click to enlarge)
ARAP3 deficient cells have a striking phenotype. Control RNAi cells undergo a characteristic shape change, loose their stress fibres and form lamellipodia when activated with PDGF. ARAP3 RNAi cells are more rounded and contain more stress fibres. When stimulated with PDGF, their shape does not change, they keep their stress fibres and they do not form lamellipodia.
Recent, selected publications
Raaijmakers JH, Deneubourg L, Rehmann H, de Koning J, Zhang Z, Krugmann S, Erneux C, Bos JL (2007) The PI3K effector Arap3 interacts with the PI(3,4,5)P3 phosphatase SHIP2 in a SAM domain-dependent manner.
Cellular Signalling 19 1249-1257
http://dx.doi.org/10.1016/j.cellsig.2006.12.015
Krugmann S, Andrews SR, Stephens LR, Hawkins PT (2006) ARAP3 is essential for formulation of lamellipodia after growth factor stimulation.
Journal of Cell Science 119 425-432
http://dx.doi.org/10.1242/jcs.02755
Krugmann S, Williams R, Stephens LR, Hawkins PT (2004) ARAP3 is a PI3K- and Rap-regulated GAP for RhoA.
Current Biology 14 1380-1384
http://dx.doi.org/10.1016/j.cub.2004.07.058
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
Krugmann S, Cooper MA, Williams DH, Hawkins PT, Stephens LR (2002) Mechanism of the regulation of type IB phosphoinositide 3OH-kinase by G-protein βγ subunits.
Biochemical Journal 362 725-731
http://www.biochemj.org/bj/362/bj3620725.htm
Krugmann S, Jordens I, Gevaert K, Driessens M, Vandekerckhove J, Hall A (2001) Cdc42 induces filopodia by promoting the formation of an IRSp53:Mena complex.
Current Biology 11 1645-1655
http://dx.doi.org/ 10.1016/S0960-9822(01)00506-1
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Sonja Vermeren (Project Leader)
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Bethany Hughes (Research Assistant)
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Helen Craig (PhD student)
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Susann Voigt (Leonardo da Vinci student)
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Laure Gambardella (Post-doc)
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