Spatially distinct binding of Cdc42 to PAK1 and N-WASP in breast carcinoma cells.
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.
Parsons
Maddy
M
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
Monypenny
James
J
Ameer-Beg
Simon M
SM
Millard
Thomas H
TH
Machesky
Laura M
LM
Peter
Marion
M
Keppler
Melanie D
MD
Schiavo
Giampietro
G
Watson
Rose
R
Chernoff
Jonathan
J
Zicha
Daniel
D
Vojnovic
Borivoj
B
Ng
Tony
T
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
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