Abstract
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.
Highlights
The NM23/nucleoside diphosphate (NDP) kinase gene family encodes a group of eight homologous proteins with conserved structure
We examined the ability of NDP kinases A and B to respond to activation of receptor tyrosine kinases and G-protein-coupled receptors (GPCRs) by monitoring their spatial distribution in NIH-3T3 fibroblasts
NDP kinase forms ring-like structures, and phase-contrast microscopy (Fig. 1b) shows that most of these rings correspond to phase-bright vesicles of various sizes that are scattered through the cytoplasm, around the nucleus
Summary
The NM23/NDP kinase gene family encodes a group of eight homologous proteins with conserved structure. In higher organisms NDP kinases A and B are the most abundant members of the group and they are the best characterized to date (Lacombe et al, 2000). It has become apparent that changes in the expression levels or modifications in the structure of NDP kinases alter functions as diverse as development, cell migration and differentiation in unexpected ways, leading to the suggestion that NDP kinases are multifunctional proteins (Otero, 2000; Kimura et al, 2000; Postel et al, 2002). Members of this family were reported to inhibit growth-factor-induced cell motility of breast cancer cells (Kantor et al, 1993), attenuate the desensitization of muscarinic-activated atrial K+ channels (Xu et al, 1996; Otero et al, 1999) and regulate synaptic vesicle internalization in Drosophila (Krishnan et al, 2001)
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