Receptor activation regulates cortical, but not vesicular localization of NDP kinase

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.

Interaction of nucleoside diphosphate kinase B with heterotrimeric G protein βγ dimers: consequences on G protein activation and stability

“…In contrast, an increase in NDPK content was not observed when hypertrophy was induced with thyroid hormone. Consistent with recent reports showing NDPK translocation by stimulation of GPCRs (Gallagher et al 2003;Rochdi et al 2004), these data suggest that sustained stimulation of β-adrenergic receptors increases association of NDPK with the plasma membrane. Taking the recent findings in the zebrafish into account, the primary regulation might occur at the level of mRNA encoding the interacting G protein α subunit (Müller et al 1994).…”

NDP kinase moves into developing primary cilia

“…), specific for NDP kinase A and B [Gallagher et al, 2003], and two distinct polyclonal antibodies to frog NDP kinase, M1 and F1 [Yi et al, 1996;Otero, 1997]. Acetylated α-tubulin was detected with a monoclonal antibody (clone 6-11B-1, Piperno and Fuller [1985]) from Zymed.…”

“…The most abundant and ubiquitous members of this family of proteins, NDP kinases A and B (NM23-1 and 2), are hexamers of two highly homologous polypeptides of 18 kDa, with characteristics of cytosolic proteins. Nevertheless, both proteins are found in association with plasma membranes in stimulated cells [Gallagher et al, 2003], and in the nucleus [Lacombe et al, 2000;Munier et al, 2003]. In recent years, several other proteins expressed at lower levels in specific tissues or organelles were found to contain consensus sequences that define NDP kinases.…”

“…Thus, although NDP kinases A and B do not bind directly to purified tubulin [Gallagher et al, 2003], these proteins bind to microtubules during interphase [Pinon et al, 1999], are associated with membranes that co-pellet with taxol-stabilized microtubules, and co-localize with α-tubulin around cytoplasmic vesicles in fibroblasts [Gallagher et al, 2003].…”

Targeting Tumor Metastasis by Regulating Nm23 Gene Expression

“…These genes encode the A and B subunits respectively of NDPK. Nm23 family protein involves in multiple biological functions in cell adhesion, cell migration, cellular differentiation, microtubule polymerization, signal transduction pathway, histidine dependent phosphorylation, vascular invasion, endocytosis, tumor cell shape and in apoptosis (Kimura et al, 2000;Krishnan et al, 2001;Otsuki et al, 2001;Fournier et al, 2003;Gallagher et al, 2003;Narayanan and Ramaswami, 2003;Sirotkovic-Skerley et al, 2005;Jung and Seong, 2007). Scientific evidence suggests that Nm23 has a dual role in tumor progression: i) over expression in primary tumors at early stages, ii) the association between the loss of Nm23-H1 expression in later stages and tumor aggressiveness and metastatic potential.…”