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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.

Original publication

DOI

10.1128/MCB.25.5.1680-1695.2005

Type

Journal article

Journal

Mol Cell Biol

Publication Date

03/2005

Volume

25

Pages

1680 - 1695

Keywords

Binding Sites, Breast Neoplasms, Carcinoma, Cell Cycle Proteins, Cell Line, Tumor, Cell Membrane, Clathrin-Coated Vesicles, Endosomes, Fluorescence Resonance Energy Transfer, Green Fluorescent Proteins, Guanine Nucleotide Exchange Factors, Guanosine Triphosphate, Humans, Nerve Tissue Proteins, Phosphorylation, Protein-Serine-Threonine Kinases, Rho Guanine Nucleotide Exchange Factors, Wiskott-Aldrich Syndrome Protein, Neuronal, cdc42 GTP-Binding Protein, p21-Activated Kinases