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An inducible translocation strategy to rapidly activate and inhibit small GTPase signaling pathways

Abstract

We made substantial advances in the implementation of a rapamycin-triggered heterodimerization strategy. Using molecular engineering of different targeting and enzymatic fusion constructs and a new rapamycin analog, Rho GTPases were directly activated or inactivated on a timescale of seconds, which was followed by pronounced cell morphological changes. As signaling processes often occur within minutes, such rapid perturbations provide a powerful tool to investigate the role, selectivity and timing of Rho GTPase–mediated signaling processes.

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Figure 1: Rapid binding of a cytoplasmic YFP-tagged FRB to a plasma membrane targeted FKBP construct by addition of a rapamycin analog.
Figure 2: Development of an effective hetero-oligomerization strategy for small GTPase activation.
Figure 3: Rapid effects on cell morphology and downstream effectors by heterodimerization.

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Acknowledgements

We thank ARIAD Pharmaceuticals for providing AP21967, as well as plasmids encoding FKBP and FRB. We also thank H. Sugimura and M. Fivaz for the generous gift of Tiam1 and Lyn11-CFP constructs, respectively, to F. Fernandez, M. Fivaz and A. Hahn for critical review of this manuscript, and to M. Fivaz and members of the M. Scott laboratory for technical advice on the pull-down assay. This study was supported in part by National Institutes of Health (GM063702 to T.M. and GM-068589 to T.J.W.). T.I. is a recipient of a fellowship from the Quantitative Chemical Biology program.

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Correspondence to Takanari Inoue.

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Inoue, T., Heo, W., Grimley, J. et al. An inducible translocation strategy to rapidly activate and inhibit small GTPase signaling pathways. Nat Methods 2, 415–418 (2005). https://doi.org/10.1038/nmeth763

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