RanBPM associates with CD39 and modulates ecto-nucleotidase activity

Harvard University, Cambridge, Massachusetts, United States
Biochemical Journal (Impact Factor: 4.4). 06/2006; 396(1):23-30. DOI: 10.1042/BJ20051568
Source: PubMed


CD39/ecto-NTPDase 1 (nucleoside triphosphate diphosphohydrolase 1) is an ecto-nucleotidase that influences P2 receptor activation to regulate vascular and immune cell adhesion and signalling events pivotal in inflammation. Whether CD39 interacts with other membrane or cytoplasmic proteins has not been established to date. Using the yeast two-hybrid system, we note that the N-terminus of CD39 binds to RanBPM (Ran binding protein M; also known as RanBP9), a multi-adaptor scaffolding membrane protein originally characterized as a binding protein for the small GTPase Ran. We confirm formation of complexes between CD39 and RanBPM in transfected mammalian cells by co-immunoprecipitation studies. Endogenous CD39 and RanBPM are also found to be co-expressed and abundant in cell membranes of B-lymphocytes. NTPDase activity of recombinant CD39, but not of N-terminus-deleted-CD39 mutant, is substantially diminished by RanBPM co-expression in COS-7 cells. The conserved SPRY [repeats in splA and RyR (ryanodine receptor)] moiety of RanBPM is insufficient alone for complete physical and functional interactions with CD39. We conclude that CD39 associations with RanBPM have the potential to regulate NTPDase catalytic activity. This intermolecular interaction may have important implications for the regulation of extracellular nucleotide-mediated signalling.

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Available from: Xiaofeng Sun
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    • "RanBP9 is also known to exist as a component of large protein complex which could be both cytoplasmic and nuclear [14], [15]. This type of differential subcellular localization is consistent with the postulated role of RanBP9 as a multifunctional scaffolding protein that interacts with cytoplasmic domains of a variety of membrane receptors such as integrin β subunit [11], c-Met [9], L1CAM [16], CD39 [17] and the calcium channel, Cav3.1 [18], mediating diverse transmembrane signaling. "
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    ABSTRACT: RanBP9 is known to act as a scaffolding protein bringing together a variety of cell surface receptors and intracellular targets thereby regulating functions as diverse as neurite and axonal outgrowth, cell morphology, cell proliferation, myelination, gonad development, myofibrillogenesis and migration of neuronal precursors. Though RanBP9 is ubiquitously expressed in all tissues, brain is one of the organs with the highest expression levels of RanBP9. In the neurons, RanBP9 is localized mostly in the cytoplasm but also in the neurites and dendritic processes. We recently demonstrated that RanBP9 plays pathogenic role in Alzheimer's disease. To understand the role of RanBP9 in the brain, here we generated RanBP9 null mice by gene-trap based strategy. Most of Ran-/- mice die neonatally due to defects in the brain growth and development. The major defects include smaller cortical plate (CP), robustly enlarged lateral ventricles (LV) and reduced volume of hippocampus (HI). The lethal phenotype is due to a suckling defect as evidenced by lack of milk in the stomachs even several hours after parturition. The complex somatosensory system which is required for a behavior such as suckling appears to be compromised in Ran-/- mice due to under developed CP. Most importantly, RanBP9 phenotype is similar to ERK1/2 double knockout and the neural cell adhesion receptor, L1CAM knockout mice. Both ERK1 and L1CAM interact with RanBP9. Thus, RanBP9 appears to control brain growth and development through signaling mechanisms involving ERK1 and L1CAM receptor.
    Full-text · Article · Jun 2013 · PLoS ONE
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    • "The lysates were sonicated briefly on ice and centrifuged at 14,000 rpm for 10 minutes at 4°C. The measurement of protein concentrations and detailed procedures of immunoblotting were described previously [29], [31]. "
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    ABSTRACT: Background Extracellular adenosine triphosphate (ATP) functions as a novel danger signal that boosts antitumor immunity and can also directly kill tumor cells. We have previously reported that chronic exposure of tumor cells to ATP provokes P2X7-mediated tumor cell death, by as yet incompletely defined molecular mechanisms. Methodology/Principal Findings Here, we show that acute exposure of tumor cells to ATP results in rapid cytotoxic effects impacting several aspects of cell growth/survival, leading to inhibition of tumor growth in vitro and in vivo. Using agonist and antagonist studies together with generation of P2X7 deficient tumor cell lines by lentiviral shRNA delivery system, we confirm P2X7 to be the central control node transmitting extracellular ATP signals. We identify that downstream intracellular signaling regulatory networks implicate two signaling pathways: the known P2X7-PI3K/AKT axis and remarkably a novel P2X7-AMPK-PRAS40-mTOR axis. When exposed to high levels of extracellular ATP, these two signaling axes perturb the balance between growth and autophagy, thereby promoting tumor cell death. Conclusions Our study defines novel molecular mechanisms underpinning the antitumor actions of P2X7 and provides a further rationale for purine-based drugs in targeted cancer therapy.
    Full-text · Article · Apr 2013 · PLoS ONE
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    • "These procedures were performed as previously [20] [21] [33] [37] "
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    ABSTRACT: Extracellular adenosine triphosphate (ATP) is known to boost immune responses in the tumor microenvironment but might also contribute directly to cancer cell death. CD39/ENTPD1 is the dominant ectonucleotidase expressed by endothelial cells and regulatory T cells and catalyzes the sequential hydrolysis of ATP to AMP that is further degraded to adenosine by CD73/ecto-5'-nucleotidase. We have previously shown that deletion of Cd39 results in decreased growth of transplanted tumors in mice, as a result of both defective angiogenesis and heightened innate immune responses (secondary to loss of adenosinergic immune suppression). Whether alterations in local extracellular ATP and adenosine levels as a result of CD39 bioactivity directly affect tumor growth and cytotoxicity has not been investigated to date. We show here that extracellular ATP exerts antitumor activity by directly inhibiting cell proliferation and promoting cancer cell death. ATP-induced antiproliferative effects and cell death are, in large part, mediated through P2X(7) receptor signaling. Tumors in Cd39 null mice exhibit increased necrosis in association with P2X(7) expression. We further demonstrate that exogenous soluble NTPDase, or CD39 expression by cocultured liver sinusoidal endothelial cells, stimulates tumor cell proliferation and limits cell death triggered by extracellular ATP. Collectively, our findings indicate that local expression of CD39 directly promotes tumor cell growth by scavenging extracellular ATP. Pharmacological or targeted inhibition of CD39 enzymatic activity may find utility as an adjunct therapy in cancer management.
    Full-text · Article · Mar 2011 · Neoplasia (New York, N.Y.)
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