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Spatially Distinct Binding of Cdc42 to PAK1 and N-WASP in Breast Carcinoma Cells

Maddy Parsons,^1,* James Monypenny,^2, Simon M. Ameer-Beg,^3, Thomas H. Millard,⁴ Laura M. Machesky,⁴ Marion Peter,¹ Melanie D. Keppler,¹ Giampietro Schiavo,⁵ Rose Watson,⁶ Jonathan Chernoff,⁷ Daniel Zicha,² Borivoj Vojnovic,³ and Tony Ng¹

Randall Centre, King's College London, Guy's Medical School Campus,¹ Light Microscopy Laboratory,² Molecular NeuroPathoBiology Laboratory,⁵ Electron Microscopy Unit,⁶ Cancer Research UK London Research Institute, London, Gray Cancer Institute, Mount Vernon Hospital, Northwood, Middlesex,³ School of Biosciences, Division of Molecular and Cell Biology, University of Birmingham, Birmingham United Kingdom,⁴ Fox Chase Cancer Center, Philadelphia, Pennsylvania⁷

Received 3 June 2004/ Returned for modification 12 August 2004/ Accepted 26 November 2004

While a significant amount is known about the biochemical signaling pathways of the Rho family GTPase Cdc42, a better understanding of how these signaling networks are coordinated in cells is required. In particular, the predominant subcellular sites where GTP-bound Cdc42 binds to its effectors, such as p21-activated kinase 1 (PAK1) and N-WASP, a homolog of the Wiskott-Aldritch syndrome protein, are still undetermined. Recent fluorescence resonance energy transfer (FRET) imaging experiments using activity biosensors show inconsistencies between the site of local activity of PAK1 or N-WASP and the formation of specific membrane protrusion structures in the cell periphery. The data presented here demonstrate the localization of interactions by using multiphoton time-domain fluorescence lifetime imaging microscopy (FLIM). Our data here establish that activated Cdc42 interacts with PAK1 in a nucleotide-dependent manner in the cell periphery, leading to Thr-423 phosphorylation of PAK1, particularly along the lengths of cell protrusion structures. In contrast, the majority of GFP-N-WASP undergoing FRET with Cy3-Cdc42 is localized within a transferrin receptor- and Rab11-positive endosomal compartment in breast carcinoma cells. These data reveal for the first time distinct spatial association patterns between Cdc42 and its key effector proteins controlling cytoskeletal remodeling.

Supplemental material for this article may be found at http://mcb.asm.org/.

M.P., J.M., and S.M.A.-B. contributed equally to this study.

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