Receptor activation regulates cortical, but not vesicular localization of NDP kinase.
We used immunofluorescence techniques to determine the localization of nucleoside diphosphate (NDP) kinase in NIH-3T3 fibroblasts. We found that cytoplasmic NDP kinase can be separated into two populations according to subcellular localization and response to extracellular stimuli. Specifically, within minutes of stimulation of resting fibroblasts with serum, growth factors or bombesin, a portion of NDP kinase becomes associated with membrane ruffles and lamellipodia. Another pool of NDP kinase accumulates independently of stimulation around intracellular vesicles. Transfection of cells with activated Rac mimics, whereas expression of dominant negative Rac inhibits, the effects of extracellular stimulation on the translocation of NDP kinase to the cell cortex. Neither Rac mutant affects the vesicle-associated pool. Association of NDP kinase with vesicles depends on microtubule integrity and is disrupted by nocodazole. In cell-free assays NDP kinase binds tightly to membrane vesicles associated with taxol-stabilized microtubules. Binding of NDP kinase to this fraction is reduced by ATP and abolished by GTP, as well as guanine nucleotides that are NDP kinase substrates. Thus, the localization of the two NDP kinase pools identified here is regulated independently by distinct cellular components: the appearance of cortical NDP kinase is a consequence of Rac activation, whereas vesicular NDP kinase is responsive to microtubule dynamics and nucleotides, in particular GTP. These results suggest that in fibroblasts NDP kinase participates in Rac-related cortical events and in GTP-dependent processes linked to intracellular vesicle trafficking.
Gallagher
Betty C
BC
Department of Molecular Physiology and Biological Physics, University of Virginia Medical School, Charlottesville, VA 22908, USA.
Parrott
Kimberly A
KA
Szabo
Gabor
G
de S  Otero
Angela
A
D000818:Animals; D001692:Biological Transport; D002451:Cell Compartmentation; D002458:Cell Fractionation; D002462:Cell Membrane / Q000201:enzymology; D022162:Cytoplasmic Vesicles / Q000201:enzymology; D005455:Fluorescent Antibody Technique; D051379:Mice; D008869:Microtubule-Associated Proteins / Q000378:metabolism; D009154:Mutation; D041681:NIH 3T3 Cells; D009701:Nucleoside-Diphosphate Kinase / Q000378:metabolism*; D020830:rac1 GTP-Binding Protein / Q000235:genetics / Q000378:metabolism
D008869:Microtubule-Associated Proteins; D009701:Nucleoside-Diphosphate Kinase; D020830:rac1 GTP-Binding Protein