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Sci. Signal., 29 November 2011
Vol. 4, Issue 201, p. ra81
[DOI: 10.1126/scisignal.2001729]


A Targeted siRNA Screen Identifies Regulators of Cdc42 Activity at the Natural Killer Cell Immunological Synapse

Leo M. Carlin1,2*{dagger}{ddagger}, Rachel Evans1,2*, Hanna Milewicz3, Luis Fernandes2, Daniel R. Matthews1,2,4, Michela Perani4, James Levitt5, Melanie D. Keppler1,2, James Monypenny1,2,6, Ton Coolen2,7, Paul R. Barber2,8, Borivoj Vojnovic2,8, Klaus Suhling5, Franca Fraternali2, Simon Ameer-Beg1,2,4, Peter J. Parker4,9, N. Shaun B. Thomas3, and Tony Ng1,2,4,6{ddagger}

1 Richard Dimbleby Department of Cancer Research, King’s College London, London SE1 1UL, UK.
2 Randall Division of Cellular and Molecular Biophysics, King’s College London, London SE1 1UL, UK.
3 Cell Cycle and Epigenetics Laboratory, Department of Haematological Medicine, The Rayne Institute, King’s College London, London SE5 9NU, UK.
4 Division of Cancer Studies, King’s College London, London SE1 1UL, UK.
5 Department of Physics, King’s College London, London WC2R 2LS, UK.
6 Breakthrough Breast Cancer Research Unit, Research Oncology, King’s College London, Guy’s Medical School Campus, London SE1 1UL, UK.
7 Department of Mathematics, King’s College London, London WC2R 2LS, UK.
8 Gray Institute for Radiation Oncology and Biology, University of Oxford, Old Road Campus Research Building, Roosevelt Drive, Oxford OX3 7DQ, UK.
9 Cancer Research UK London Research Institute, Lincoln’s Inn Fields, London WC2A 3LY, UK.

* These authors contributed equally to this work.

{dagger} Present address: Centre for Molecular and Cellular Biology of Inflammation, King’s College London, London SE1 1UL, UK.

Abstract: Natural killer (NK) cells kill tumor cells and virally infected cells, and an effective NK cell response requires processes, such as motility, recognition, and directional secretion, that rely on cytoskeletal rearrangement. The Rho guanosine triphosphatase (GTPase) Cdc42 coordinates cytoskeletal reorganization downstream of many receptors. The Rho-related GTPase from plants 1 (ROP1) exhibits oscillatory activation behavior at the apical plasma membrane of growing pollen tubes; however, a similar oscillation in Rho GTPase activity has so far not been demonstrated in mammalian cells. We hypothesized that oscillations in Cdc42 activity might occur within NK cells as they interact with target cells. Through fluorescence lifetime imaging of a Cdc42 biosensor, we observed that in live NK cells forming immunological synapses with target cells, Cdc42 activity oscillated after exhibiting an initial increase. We used protein-protein interaction networks and structural databases to identify candidate proteins that controlled Cdc42 activity, leading to the design of a targeted short interfering RNA screen. The guanine nucleotide exchange factors RhoGEF6 and RhoGEF7 were necessary for Cdc42 activation within the NK cell immunological synapse. In addition, the kinase Akt and the p85α subunit of phosphoinositide 3-kinase (PI3K) were required for Cdc42 activation, the periodicity of the oscillation in Cdc42 activity, and the subsequent polarization of cytotoxic vesicles toward target cells. Given that PI3Ks are targets of tumor therapies, our findings suggest the need to monitor innate immune function during the course of targeted therapy against these enzymes.

{ddagger} To whom correspondence should be addressed. E-mail: leo.carlin{at} (L.M.C.);{at} (T.N.)

Citation: L. M. Carlin, R. Evans, H. Milewicz, L. Fernandes, D. R. Matthews, M. Perani, J. Levitt, M. D. Keppler, J. Monypenny, T. Coolen, P. R. Barber, B. Vojnovic, K. Suhling, F. Fraternali, S. Ameer-Beg, P. J. Parker, N. S. B. Thomas, T. Ng, A Targeted siRNA Screen Identifies Regulators of Cdc42 Activity at the Natural Killer Cell Immunological Synapse. Sci. Signal. 4, ra81 (2011).

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