title: Spatially distinct binding of Cdc42 to PAK1 and N-WASP in breast carcinoma cells.
abstract: 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.
authors.affiliation: Randall Centre, King's College London, 3rd Floor, New Hunt's House, Guy's Medical School Campus, London SE1 1UL, United Kingdom. maddy.parsons@kcl.ac.uk
authors.forename: Maddy
authors.initials: M
authors.lastname: Parsons
authors.forename: James
authors.initials: J
authors.lastname: Monypenny
authors.forename: Simon M
authors.initials: SM
authors.lastname: Ameer-Beg
authors.forename: Thomas H
authors.initials: TH
authors.lastname: Millard
authors.forename: Laura M
authors.initials: LM
authors.lastname: Machesky
authors.forename: Marion
authors.initials: M
authors.lastname: Peter
authors.forename: Melanie D
authors.initials: MD
authors.lastname: Keppler
authors.forename: Giampietro
authors.initials: G
authors.lastname: Schiavo
authors.forename: Rose
authors.initials: R
authors.lastname: Watson
authors.forename: Jonathan
authors.initials: J
authors.lastname: Chernoff
authors.forename: Daniel
authors.initials: D
authors.lastname: Zicha
authors.forename: Borivoj
authors.initials: B
authors.lastname: Vojnovic
authors.forename: Tony
authors.initials: T
authors.lastname: Ng
mesh_terms: D001665:Binding Sites; D001943:Breast Neoplasms / Q000737:chemistry / Q000378:metabolism*; D002277:Carcinoma / Q000737:chemistry / Q000378:metabolism*; D018797:Cell Cycle Proteins / Q000378:metabolism; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism; D022163:Clathrin-Coated Vesicles / Q000737:chemistry / Q000378:metabolism; D011992:Endosomes / Q000737:chemistry / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer; D049452:Green Fluorescent Proteins / Q000032:analysis; D020662:Guanine Nucleotide Exchange Factors / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D006801:Humans; D009419:Nerve Tissue Proteins / Q000032:analysis / Q000378:metabolism*; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000032:analysis / Q000378:metabolism*; D064067:Rho Guanine Nucleotide Exchange Factors; D051317:Wiskott-Aldrich Syndrome Protein, Neuronal; D020764:cdc42 GTP-Binding Protein / Q000032:analysis / Q000378:metabolism*; D054462:p21-Activated Kinases
chemical_list: D018797:Cell Cycle Proteins; D020662:Guanine Nucleotide Exchange Factors; D009419:Nerve Tissue Proteins; D064067:Rho Guanine Nucleotide Exchange Factors; C495771:WASL protein, human; D051317:Wiskott-Aldrich Syndrome Protein, Neuronal; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D006160:Guanosine Triphosphate; C515903:PAK1 protein, human; D017346:Protein Serine-Threonine Kinases; D054462:p21-Activated Kinases; D020764:cdc42 GTP-Binding Protein