Mass spectrometry detection of histidine phosphorylation on NM23-H1

Phosphorylation is a ubiquitous protein post-translational modification that is intimately involved in most aspects of cellular regulation. Currently, most proteomic analyses are performed with phosphorylation searches for serine, threonine, and tyrosine modifications, as the phosphorylated residues of histidine and aspartic acid are acid labile and thus undetectable with most proteomic methodologies. Here, we present a novel buffer system to show histidine phosphorylation of NM23-H1, the product of the first identified putative human metastasis suppressor gene (NME1), which catalyzes the transfer of the γ-phosphate from nucleoside triphosphates to nucleoside diphosphates. On the basis of a pH titration of LC elution buffers and MS/MS identification, recombinant NM23-H1 subjected to autophosphorylation was shown to contain phosphorylated histidine at residue 118 at pH 5 and 6, with each level giving over 75% peptide coverage for identification. The solvent system presented permits the detection of all five possible phosphorylation moieties. Application of histidine and aspartic acid phosphorylation modifications to proteomic analyses will significantly advance the understanding of phosphorylation relay signaling in cellular regulation, including elucidation of the role of NM23-H1 in metastasis.     

Double mutant P96S/S120G of Nm23-H1 abrogates its NDPK activity and motility-suppressive ability

The Nm23-H1 gene is a metastasis suppressor gene. However, its biochemical mechanism of suppressing the metastatic potential of cancer cells is still unknown. The previous hypothesis that a histidine protein kinase activity may contributes to the motility-suppressive effect of Nm23-H1 could not explain why the H118F mutant, a kinase-deficient mutant, still had motility-suppressive ability. We conducted a study on the double mutant P96S/S120G of Nm23-H1 and succeeded in introducing the RP-HPLC method in NDPK assay. The results showed that the double mutant P96S/S120G, when expressed in the bacteria, was completely aggregated in inclusion bodies; this mutant abrogated not only its motility-suppressive ability, but also its NDPK activity. Based on previous work and this study, we prompted that the deficiency of motility-suppressive function of S120G, P96S, and P96S/S120G mutants was due to their altered structure, which might deprive Nm23-H1 of most activities including kinase activity or interactions with other proteins.     

表达NM23-H1/NDPK-A工程菌的遗传稳定性研究

目的:研究重组工程菌的遗传稳定性。方法:利用重组表达质粒pBVNM-H1转化宿主菌E.coli DH5α,筛选重组工程菌DH5α-pBVNM-H1。将新构建好的重组工程菌在无选择压力的条件下进行连续传代培养,比较菌落在LB(-)和LB(+)培养基上的生长状况,并对传代菌株目标蛋白的表达情况以及质粒数量和目的基因DNA进行电泳鉴定。结果:重组工程菌连续传代50次中,在LB(-)和LB(+)培养基上的生长状况相同,目标蛋白表达量无显著差异,质粒数量及目的基因DNA结构稳定。结论:重组工程菌DH5α-pBVNM-H1具有良好的遗传稳定性。     

Potential contributions of antimutator activity to the metastasis suppressor function of NM23-H1

nm23-h1 is a well-documented metastasis suppressor gene whose mechanism(s) of action have yet to be fully elucidated. The purpose of this report is to discuss recent advances in investigating the potential role of a novel 3′–5′ exonuclease activity identified recently in our laboratory, a biochemical function associated, in general, with DNA repair and replication. We have employed a site-directed mutagenesis approach to demonstrate that the 3′–5′ exonuclease activity of NM23-H1 is required for its metastasis suppressor function. Consistent with a role in DNA repair, we also observe that the single yeast NM23 homolog (YNK1) is required for the maintenance of genomic integrity and normal kinetics of DNA repair in response to exposure to ultraviolet radiation. These results and their implications for understanding the molecular mechanisms underlying NM23-H1 functions in cancer are discussed.     

Tumor suppressor NM23-H1 is a granzyme A-activated DNase during CTL-mediated apoptosis,and the nucleosome assembly protein SET is its inhibitor

Granzyme A (GzmA) induces a caspase-independent cell death pathway characterized by single-stranded DNA nicks and other features of apoptosis. A GzmA-activated DNase (GAAD) is in an ER associated complex containing pp32 and the GzmA substrates SET, HMG-2, and Ape1. We show that GAAD is NM23-H1, a nucleoside diphosphate kinase implicated in suppression of tumor metastasis, and its specific inhibitor (IGAAD) is SET. NM23-H1 binds to SET and is released from inhibition by GzmA cleavage of SET. After GzmA loading or CTL attack, SET and NM23-H1 translocate to the nucleus and SET is degraded, allowing NM23-H1 to nick chromosomal DNA. GzmA-treated cells with silenced NM23-H1 expression are resistant to GzmA-mediated DNA damage and cytolysis, while cells overexpressing NM23-H1 are more sensitive.     

Expression of metastasis associated proteins,CD44v6 and NM23-H1, in pediatric acute lymphoblastic leukemia

Two metastasis associated proteins, CD44v6 and NM23-H1, are expressed by normal lymphoid cells, the former serving as activation marker and the later as a constitutive protein. CD44v6 is considered as a marker of poor prognosis of various hematological cancers but its expression was not demonstrated in childhood acute lymphoblastic leukemia (ALL). On the other hand, NM23-H1 is considered as a differentiation inhibitory factor in various hematological cancers and as a marker of poor prognosis. Therefore we have analyzed the expression of CD44v6 and NM23-H1 in bone marrow of sixteen pediatric ALL patients using immunocytochemistry. For the first time, we have demonstrated the expression of CD44v6 protein epitopes on leukemic cells in a proportion of ALL cases (6/16), primarily in the medium/high risk group (except one case), suggesting a possible association to an unfavorable outcome. On the other hand, NM23-H1 protein expression was maintained in leukemic cells in 50% of both low and medium/high risk ALL cases. The majority of the pediatric ALL cases expressed only one of the metastasis associated proteins (10/16). This feature is highly similar to the observations made in several adult solid cancers. The potential of CD44v6 expression in leukemic cells as prognosticator in pediatric ALL has to be evaluated in a larger clinical trial.     

NM23-H2, an estrogen receptor beta-associated protein, shows diminished expression with progression of atherosclerosis

While estrogen receptor (ER) profile plays an important role in response to estrogens, receptor coregulators act as critical determinants of signaling. Although the clinical effects of ovarian hormones on various normal and pathological processes are an active area of research, the exact signaling effects on, for example, the vessel wall, are incompletely understood. Hence, we sought to discover proteins that associate with ERbeta, the isoform that shows upregulated mRNA expression after arterial injury. Using a yeast two-hybrid screen we identified NM23-H2, a multifaceted metastasis suppressor candidate protein, as an ERbeta-associated protein. Although NM23-H2 was immunodetected in arteries from young subjects (27 +/- 6 yr, 14 men and 6 women) with benign intimal hyperplasia, expression was diminished in fatty streaks/atheromas and altogether absent in advanced atherosclerotic lesions. Both nm23-H2 mRNA and protein were expressed by vascular cells in vitro. Treatment with 17beta-estradiol and an ERbeta-selective agonist, diarylpropionitrile, increased protein expression of NM23-H2; an effect that was not seen with an ERalpha-selective agonist, propylpyrazole-triol. Estrogen also prompted nuclear localization of NM23-H2 protein in human coronary smooth muscle cells (SMCs). An in vitro mimic of inflammation decreased the expression of NM23-H2 in SMCs, which was restored on addition of estrogen and dependent on the estrogen receptor. In summary, we report the novel association of NM23-H2 with ERbeta and show for the first time its expression in vascular cells and demonstrate regulation of its expression and localization by estrogen. In that the abundance of NM23-H2 diminishes with both the advancement of atherosclerosis and inflammation, this ERbeta-associated protein may play an important role in mediating the vasculoprotective effects of estrogens.     

Protein phosphorylation corrects the folding defect of the neuroblastoma (S120G) mutant of human nucleoside diphosphate kinase A/Nm23-H1

Human nucleoside diphosphate (NDP) kinase A is a 'house-keeping' enzyme essential for the synthesis of nonadenine nucleoside (and deoxynucleoside) 5'-triphosphate. It is involved in complex cellular regulatory functions including the control of metastatic tumour dissemination. The mutation S120G has been identified in high-grade neuroblastomas. We have shown previously that this mutant has a folding defect: the urea-denatured protein could not refold in vitro. A molten globule folding intermediate accumulated, whereas the wild-type protein folded and associated into active hexamers. In the present study, we report that autophosphorylation of the protein corrected the folding defect. The phosphorylated S120G mutant NDP kinase, either autophosphorylated with ATP as donor, or chemically prosphorylated by phosphoramidate, refolded and associated quickly with high yield. Nucleotide binding had only a small effect. ADP and the non-hydrolysable ATP analogue 5'-adenyly-limido-diphosphate did not promote refolding. ATP-promoted refolding was strongly inhibited by ADP, indicating protein dephosphorylation. Our findings explain why the mutant enzyme is produced in mammalian cells and in Escherichia coli in a soluble form and is active, despite the folding defect of the S120G mutant observed in vitro. We generated an inactive mutant kinase by replacing the essential active-site histidine residue at position 118 with an asparagine residue, which abrogates the autophosphorylation. The double mutant H118N/S120G was expressed in inclusion bodies in E. coli. Its renaturation stops at a folding intermediate and cannot be reactivated by ATP in vitro. The transfection of cells with this double mutant might be a good model to study the cellular effects of folding intermediates.     

Full-length RAG-2, and not full-length RAG-1, specifically suppresses RAG-mediated transposition but not hybrid joint formation or disintegration

RAG-1 and RAG-2 initiate V(D)J recombination by introducing DNA breaks at recombination signal sequences flanking a pair of antigen receptor gene segments. Occasionally, the RAG proteins mediate two other alternative DNA rearrangements in vivo: the rejoining of signal and coding ends and the transposition of signal ends into unrelated DNA. In contrast, truncated, catalytically active "core" RAG proteins readily catalyze these reactions in vitro, suggesting that full-length RAG proteins directly or indirectly suppress these undesired reactions in vivo. To discriminate between direct and indirect suppression models, full-length RAG proteins were purified and characterized in vitro. From mammalian cells, full-length RAG-1 is readily purified with core RAG-2 but not full-length RAG-2 and vice versa. Despite differences in DNA binding activity, recombinase containing either core or full-length RAG-1 or RAG-2 possess comparable cleavage, rejoining, and end-processing activity, as well as similar usage preferences for canonical versus cryptic recombination signals. However, recombinase containing full-length RAG-2, but not full-length RAG-1, exhibits dramatically reduced transposition activity in vitro. These data suggest RAG-mediated transposition and rejoining are differentially regulated by the full-length RAG proteins in vivo (the former directly by RAG-2 and the latter indirectly through other factors) and argue that noncore portions of the RAG proteins have little or no direct influence over V(D)J recombinase site specificity.     

Intravenous CCK-8, but not GLP-1, suppresses ghrelin and stimulates PYY release in healthy men

We have investigated the effects of exogenous CCK-8 and GLP-1, alone and in combination, on ghrelin and PYY secretion. Nine healthy males were studied on four occasions. Plasma ghrelin and PYY concentrations were measured during 150 min intravenous infusions of: (i) isotonic saline, (ii) CCK-8 at 1.8 pmol/kg/min, (iii) GLP-1 at 0.9 pmol/kg/min or (iv) CCK-8 and GLP-1 combined. CCK-8 markedly suppressed ghrelin and stimulated PYY when compared with control between t=0-120 min (P<0.001 for both). GLP-1 had no effect on ghrelin, but decreased PYY slightly at 120 min (P<0.05). During infusion of CCK-8+GLP-1, there was comparable suppression of ghrelin (P<0.001), but the stimulation of PYY was less (P<0.001), than that induced by CCK-8, between t=20-120 min. In conclusion, in healthy subjects, in the doses evaluated, exogenous CCK-8 suppresses ghrelin and stimulates PYY, and exogenous GLP-1 has no effect on ghrelin and attenuates the effect of CCK-8 on PYY.     

Autocrine activation of adenosine A1 receptors blocks D1A but not D1B dopamine receptor desensitization

Adenosine is known to modulate dopamine responses in several brain areas. Here, we show that tonic activation of adenosine receptors is able to impede desensitization of D1 dopamine receptors. As measured by cAMP accumulation in transfected COS-7 cells, long-term exposure to dopamine agonists promoted desensitization of D1B receptor but not that of D1A receptor. The inability of D1A receptor to desensitize was a result of the adenosine present in culture medium acting through activation of adenosine A1 receptors. Cell incubation with either adenosine deaminase, CGS-15943, a generic adenosine receptor antagonist, or the A1 antagonist DPCPX restored the long-term desensitization time-course of D1A receptors. In Ltk cells stably expressing A1 adenosine receptors and D1A dopamine receptors, pre-treatment of cells with R(-)-PIA, a full A1 receptor agonist, did not significantly inhibit the acute increase in cAMP levels induced by D1 receptor agonists, but blocked desensitization of D1A receptors. However, simultaneous activation of A1 and D1A receptors promoted a delayed D1A receptor desensitization. This suggests that functional interaction between A1 and D1A receptors may depend on the activation kinetics of components regulating D1 receptor responses, acting differentially on D1A and D1B receptors.     

Null mutation of the dam or seqA gene suppresses temperature-sensitive lethality but not hypersensitivity to novobiocin of muk null mutants

Escherichia coli mukF, mukE, and mukB null mutants have common phenotypes such as temperature-dependent colony formation, anucleate cell production, chromosome cutting by septum closure, and abnormal localization of SeqA-DNA clusters. We show here that the associated muk null mutations cause hypersensitivity to novobiocin. Null mutation of either dam or seqA suppressed partially the temperature-sensitive lethality but failed to suppress the anucleate cell production and the hypersensitivity to novobiocin caused by muk null mutations.     

p70 S6K1 nuclear localization depends on its mTOR-mediated phosphorylation at T389, but not on its kinase activity towards S6

The protein kinase p70 S6K1 is regulated in response to cytokines, nutrients and growth factors, and plays an important role in the development of a variety of human diseases. Mammalian target of rapamycin (mTOR) is known to phosphorylate and thereby activate p70 S6K1. p70 S6K1 phosphorylates different cytoplasmic and nuclear substrates involved in the regulation of protein synthesis, cell cycle, cell growth and survival. Recently, we have shown that mTOR-mediated phosphorylation of p70 S6K1 at T389 also regulates its nucleocytoplasmic localization. Since this phosphorylation is associated with its kinase activity the question whether p70 S6K1 phosphorylation or kinase activity is essential for its proper localization remained elusive. Recently, the chemical compound PF-4708671 has been demonstrated to block p70 S6K1 kinase activity while inducing its phosphorylation at T389. This potential of PF-4708671 to separate p70 S6K1 activity from its T389 phosphorylation allowed us to demonstrate that the proper nucleocytoplasmic localization of this kinase depends on its mTOR-mediated phosphorylation but not on its kinase activity. These findings provide important insights into the regulation of p70 S6K1 and allow a more detailed understanding of subcellular enzyme localization processes.     

Chronic potassium supplementation of newborn dogs increases cortical Na,K-ATPase but not urinary potassium excretion

The distal nephron of the newborn dog cannot secrete an acute potassium load as efficiently as can that of the adult dog. Distal nephron potassium secretion is dependent upon basolateral Na,K-ATPase activity. Because Na,K-ATPase activity is lower in the immature than the mature distal nephron, it was hypothesized that lower Na,K-ATPase activity may be responsible for the lower potassium secretory capacity of the immature nephron. In the adult, chronic high dietary potassium intake increases renal tubular potassium secretory capacity by increasing Na/K pump abundance in distal nephron segments responsible for potassium secretion. Therefore, in order to test the above hypothesis, renal cortical and outer medullary Na,K-ATPase activity under Vmax conditions (a measure of pump abundance) and urinary potassium excretion during acute potassium loading were determined in 7 age-matched, litter mate pairs (chronically potassium supplemented versus control) newborn dogs. The potassium supplemented member of each pair received 6 mmol.day-1.kg-1 of KCl as a 150 mM solution for 7-21 days after birth and the control member received an equal volume of water for the same period of time. This protocol resulted in a doubling of renal cortical Vmax Na,K-ATPase activity in the potassium supplemented animals (from 369 +/- 186 to 718 +/- 286 nmol Pi liberated.h-1.micrograms DNA-1, P = 0.025). There was no significant change in outer medullary enzyme activity. Contrary to the above hypothesis, this increase in cortical enzyme activity was not associated with increased potassium excretion at baseline or during acute potassium loading.(ABSTRACT TRUNCATED AT 250 WORDS)     

Unliganded T3R, but not its oncogenic variant, v-erbA, suppresses RAR-dependent transactivation by titrating out RXR.

V-erbA is thought to be an antagonist of thyroid hormone receptor (T3R) function. Here we show that unliganded T3R, but not v-erbA, suppresses retinoic acid (RA)-dependent induction of the RAR-beta 2 promoter by competing for the common dimerization partner, the retinoid X receptor (RXR). Firstly, T3R suppression can be alleviated by co-transfection of RXR. Secondly, T3R, but not v-erbA, competes with RAR for RXR and causes the dissociation of a preformed RAR/RXR-RARE ternary complex in vitro. A single point mutation located in the dimerization interface of v-erbA (Pro349 to Ser) abolishes the transdominant phenotype when introduced at the respective position in T3R. The hypertransforming v-erbA variant r12, in which this mutation is reversed (Ser349 to Pro) suppresses RA-induced differentiation in chicken erythroid progenitors, while v-erbA does not. Our data thus suggest that unliganded T3R and v-erbA act as dominant suppressors through mechanistically distinct pathways.