Copurification of Vimentin, Energy Metabolism Enzymes, and a MER5 Homolog with Nucleoside Diphosphate Kinase

Otero, Angela de S.

doi:10.1074/jbc.272.23.14690

Cited by 49 publications

(27 citation statements)

References 33 publications

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“…4). The identities of these proteins are unknown, but NDPKs from other sources have also been shown to copurify with a number of other proteins (10,28).…”

Section: Discussionmentioning

confidence: 99%

Secreted Variant of Nucleoside Diphosphate Kinase from the Intracellular Parasitic Nematode Trichinella spiralis

et al. 2001

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The molecular components involved in the survival of the parasitic nematode Trichinella spiralis in an intracellular environment are poorly characterized. Here we demonstrate that infective larvae secrete a nucleoside diphosphate kinase when maintained in vitro. The secreted enzyme forms a phosphohistidine intermediate and shows broad specificity in that it readily accepts ␥-phosphate from both ATP and GTP and donates it to all nucleoside and deoxynucleoside diphosphate acceptors tested. The enzyme was partially purified from culture medium by ATP affinity chromatography and identified as a 17-kDa protein by autophosphorylation and reactivity with an antibody to a plant-derived homologue. Secreted nucleoside diphosphate kinases have previously been identified only in prokaryotic organisms, all of them bacterial pathogens. The identification of a secreted variant of this enzyme from a multicellular eukaryote is very unusual and is suggestive of a role in modulating host cell function.Nucleoside diphosphate kinases (NDPKs) play a key role in the maintenance of intracellular pools of deoxynucleoside triphosphates (dNTPs) and NTPs via the transfer of phosphate from an NTP donor to an NDP acceptor. In addition, certain variants of these enzymes are involved in a variety of cellular processes unrelated to their catalytic activity, such as differentiation, proliferation, and suppression of tumor metastasis (8).In particular, nm23-H2/NDPK B has been identified as a DNA-binding protein and transcriptional activator of the human c-myc gene, previously known as PuF (29,31). NDPKs are typically intracellular enzymes, although recently an ectoenzyme has been detected on the surface of mammalian cells (19,20), and NDPKs have been reported to be secreted by the prokaryotic pathogens Mycobacterium bovis, Pseudomonas aeruginosa, and Vibrio cholerae (32,38,39).Trichinella spiralis is a ubiquitous nematode parasite of a wide variety of mammalian species, including humans, and is remarkable among multicellular parasites in adopting an intracellular habitat, both in the systemic phase of infection in skeletal muscle cells and in the enteral phase, in which it invades and migrates through mucosal epithelial cells (9). It is likely that secreted products are involved in survival and development of parasites in both environments. Consistent with this assumption, infective larvae possess a large organelle termed the stichosome, which is the major source of secreted proteins which can be recovered from in vitro culture of parasites (9).We have previously demonstrated that T. spiralis infective larvae secrete serine/threonine protein kinases (3). During the course of these studies, it became apparent that phosphorylation of a protein with an estimated mass of 17 kDa was regulated independently of both exogenous substrates and the major endogenous parasite substrate for protein kinase activity, a doublet of 50 and 55 kDa. We hypothesized that this may result from the activity of an additional enzyme and demonstrate here that T. spiralis se...

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“…4). The identities of these proteins are unknown, but NDPKs from other sources have also been shown to copurify with a number of other proteins (10,28).…”

Section: Discussionmentioning

confidence: 99%

Secreted Variant of Nucleoside Diphosphate Kinase from the Intracellular Parasitic Nematode Trichinella spiralis

et al. 2001

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“…Samples containing equal amounts of protein were solubilized in SDS-PAGE sample buffer with 20 mM DTT, alkylated with 60 mM iodoacetamide and resolved in 15% or 4-20% minigels. Proteins were transferred to nitrocellulose and immunoblotted with antibodies to Rac and NM23 as described previously (Otero, 1997). Immunoblotting with the plasma membrane marker Na + ,K + -ATPase was used to verify equal loading of control and serum-treated samples.…”

Section: Subcellular Fractionationmentioning

confidence: 99%

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

Gallagher

Parrott

Szabó

et al. 2003

Journal of Cell Science

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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.

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“…The biochemical mechanism of metastasis suppression is thought to involve attenuation of signaling for tumor cell motility, invasion, and colonization. At least four classes of Nm23 biochemical activities may contribute to altered signaling, including proteinprotein interactions (4,12,16,28,44,47,51,55,57,58,66), regulation of GTP-binding protein function (15,48,50,72,79,82), DNA-associated activities (14,53,54,63), and histidinedependent protein phosphotransferase activity (11,73,74). A role for KSR1 in Nm23-H1 attenuation of Erk activation was investigated.…”

mentioning

confidence: 99%

Nm23-H1 Metastasis Suppressor Expression Level Influences the Binding Properties, Stability, and Function of the Kinase Suppressor of Ras1 (KSR1) Erk Scaffold in Breast Carcinoma Cells

Salerno

Palmieri

Bouadis

et al. 2005

Molecular and Cellular Biology

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Metastatic disease is a significant contributor to cancer patient mortality. We previously reported that the Kinase Suppressor of Ras1 (KSR1) scaffold protein for the Erk mitogen-activated protein kinase pathway coimmunoprecipitated the metastasis suppressor protein Nm23-H1. We now hypothesize that altered expression levels of Nm23-H1 influence the binding properties, stability, and function of the KSR1 scaffold. Increased coimmunoprecipitation of Hsp90 with KSR1 was observed in either stable or transient transfectants of nm23-H1 in MDA-MB-435 human breast carcinoma cells. Similar trends were also observed in the cytoplasmic and nuclear fractions of cells. Cells expressing high levels of Nm23-H1 exhibited increased KSR1 degradation in the presence of either cycloheximide or an Hsp90-directed drug currently in clinical trial, 17-allylamino-17-demethoxygeldanamycin (17-AAG). In agreement with KSR1 degradation data, high-Nm23-H1-expression cells were preferentially inhibited in anchorage-independent colonization assays by 17-AAG. KSR1 scaffold binding patterns are dynamic in both the cytoplasmic and nuclear compartments, modulated by metastasis suppressor expression. Metastasis suppressor expression levels can impact traditional signaling pathways, such as the Erk pathway, resulting in altered tumor cell sensitivity to cancer therapeutics.

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Copurification of Vimentin, Energy Metabolism Enzymes, and a MER5 Homolog with Nucleoside Diphosphate Kinase

Cited by 49 publications

References 33 publications

Secreted Variant of Nucleoside Diphosphate Kinase from the Intracellular Parasitic Nematode Trichinella spiralis

Secreted Variant of Nucleoside Diphosphate Kinase from the Intracellular Parasitic Nematode Trichinella spiralis

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

Nm23-H1 Metastasis Suppressor Expression Level Influences the Binding Properties, Stability, and Function of the Kinase Suppressor of Ras1 (KSR1) Erk Scaffold in Breast Carcinoma Cells

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