title: A targeted siRNA screen identifies regulators of Cdc42 activity at the natural killer cell immunological synapse.
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.
authors: Richard Dimbleby Department of Cancer Research, King's College London, London SE1 1UL, UK.
authors: Leo M
authors: LM
authors: Carlin
authors: Rachel
authors: R
authors: Evans
authors: Hanna
authors: H
authors: Milewicz
authors: Luis
authors: L
authors: Fernandes
authors: Daniel R
authors: DR
authors: Matthews
authors: Michela
authors: M
authors: Perani
authors: James
authors: J
authors: Levitt
authors: Melanie D
authors: MD
authors: Keppler
authors: James
authors: J
authors: Monypenny
authors: Ton
authors: T
authors: Coolen
authors: Paul R
authors: PR
authors: Barber
authors: Borivoj
authors: B
authors: Vojnovic
authors: Klaus
authors: K
authors: Suhling
authors: Franca
authors: F
authors: Fraternali
authors: Simon
authors: S
authors: Ameer-Beg
authors: Peter J
authors: PJ
authors: Parker
authors: N Shaun B
authors: NS
authors: Thomas
authors: Tony
authors: T
authors: Ng
mesh_terms: D001683:Biological Clocks / Q000235:genetics / Q000276:immunology; D002461:Cell Line, Transformed; D064987:Cell- and Tissue-Based Therapy / Q000379:methods; D058543:Class Ia Phosphatidylinositol 3-Kinase / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D003599:Cytoskeleton / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D004789:Enzyme Activation / Q000235:genetics / Q000276:immunology; D020662:Guanine Nucleotide Exchange Factors / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D006801:Humans; D007111:Immunity, Cellular / Q000235:genetics; D007113:Immunity, Innate / Q000235:genetics; D054992:Immunological Synapses / Q000201:enzymology / Q000235:genetics / Q000276:immunology*; D007694:Killer Cells, Natural / Q000201:enzymology / Q000276:immunology*; D009369:Neoplasms / Q000201:enzymology / Q000235:genetics / Q000276:immunology / Q000628:therapy; D051057:Proto-Oncogene Proteins c-akt; D034741:RNA, Small Interfering; D064067:Rho Guanine Nucleotide Exchange Factors; D020764:cdc42 GTP-Binding Protein / Q000235:genetics / Q000276:immunology* / Q000378:metabolism
chemical_list: D020662:Guanine Nucleotide Exchange Factors; D034741:RNA, Small Interfering; D064067:Rho Guanine Nucleotide Exchange Factors; D058543:Class Ia Phosphatidylinositol 3-Kinase; D051057:Proto-Oncogene Proteins c-akt; D020764:cdc42 GTP-Binding Protein