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Proteomic analysis of NME1/NDPK A null mouse liver: evidence for a post-translational regulation of annexin IV and EF-1Bα

  • Arnaud Bruneel
  • Dominique Wendum
  • Valérie Labas
  • Odile Mulner-Lorillon
  • Joelle Vinh
  • Nelly Bosselut
  • Eric Ballot
  • Bruno Baudin
  • Chantal Housset
  • Sandrine Dabernat
  • Marie-Lise Lacombe
  • Mathieu Boissan
ORIGINAL ARTICLE

Abstract

NME/NDPK family proteins are involved in the control of intracellular nucleotide homeostasis as well as in both physiological and pathological cellular processes, such as proliferation, differentiation, development, apoptosis, and metastasis dissemination, through mechanisms still largely unknown. One family member, NME1/NDPK-A, is a metastasis suppressor, yet the primary physiological functions of this protein are still missing. The purpose of this study was to identify new NME1/NDPK-A-dependent biological functions and pathways regulated by this gene in the liver. We analyzed the proteomes of wild-type and transgenic NME1-null mouse livers by combining two-dimensional gel electrophoresis and mass spectrometry (matrix-assisted laser desorption/ionization time of flight and liquid chromatography–tandem mass spectrometry). We found that the levels of three proteins, namely, phenylalanine hydroxylase, annexin IV, and elongation factor 1 Bα (EF-1Bα), were strongly reduced in the cytosolic fraction of NME1−/− mouse livers when compared to the wild type. This was confirmed by immunoblotting analysis. No concomitant reduction in the corresponding messenger RNAs or of total protein level was observed, however, suggesting that NME1 controls annexin IV and EF-1Bα amounts by post-translational mechanisms. NME1 deletion induced a change in the subcellular location of annexin IV in hepatocytes resulting in enrichment of this protein at the plasma membrane. We also observed a redistribution of EF-1Bα in NME1−/− hepatocytes to an intracytoplasmic compartment that colocalized with a marker of the reticulum endoplasmic. Finally, we found reduced expression of annexin IV coincident with decreased NME1 expression in a panel of different carcinoma cell lines. Taken together, our data suggest for the first time that NME1 might regulate the subcellular trafficking of annexin IV and EF-1Bα. The potential role of these proteins in metastatic dissemination is discussed.

Keywords

NM23 Transgenic mice Liver Proteome Annexin IV EF-1Bα 

Abbreviations

NDPK

Nucleoside diphosphate kinase

2-DE

Two-dimensional gel electrophoresis

CCB

Colloidal Coomassie blue

PMF

Peptide mass fingerprinting

LC-MS/MS

Liquid chromatography–tandem mass spectrometry

ER

Endoplasmic reticulum

Notes

Acknowledgments

We are very grateful to Drs. J. Dijk and M. Kaetzel for the gift of anti-EF-1Bα and anti-annexin IV antibodies, respectively, to Dr. V. Barbu for advice on real-time PCR, and to Dr. N. Chignard for helpful comments. This work was supported by the Institut National de la Santé et de la Recherche Médicale (INSERM), the Université Pierre et Marie Curie (UPMC), and grants (to MLL) from the Groupement des Entreprises Françaises contre le Cancer (GEFLUC) and from the Association pour la Recherche contre le Cancer (ARC).

Supplementary material

210_2011_639_Fig8_ESM.jpg (84 kb)
Supplementary Figure 1

Representative 2-DE gels of NME1+/+ (left) and NME1−/− (right) mouse liver cytosolic fractions obtained in the broad range pH 3.0–10.0 (JPEG 84 kb)

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High resolution image (TIFF 2042 kb)
210_2011_639_Fig9_ESM.jpg (65 kb)
Supplementary Figure 2a

Details (triplicate enlarged areas) of the six differentially modulated protein spots in 2D gels shown in Fig. 1 from NME1+/+ and NME1−/− mouse liver cytosolic fractions. a Spots 1 and 2 (JPEG 64 kb)

210_2011_639_MOESM2_ESM.tif (2 mb)
High resolution image (TIFF 2042 kb)
210_2011_639_Fig10_ESM.jpg (69 kb)
Supplementary Figure 2b

Details (triplicate enlarged areas) of the six differentially modulated protein spots in 2D gels shown in Fig. 1 from NME1+/+ and NME1−/− mouse liver cytosolic fractions. b Spots 3–6 (JPEG 69 kb)

210_2011_639_MOESM3_ESM.tif (2 mb)
High resolution image (TIFF 2042 kb)
210_2011_639_Fig11_ESM.jpg (140 kb)
Supplementary Figure 3

MS/MS spectrum and corresponding sequence interpretation obtained after the fragmentation of a doubly-charged tryptic peptide (m/Z = 674.37; sequence SIQADGLVWGSSK) derived from spot 2 (EF-1Bα). Surrounded values correspond to matching fragmentation peptide m/Z values. “b1 to b13”, N-terminal peptide fragments; “y1 to y13, C-terminal peptide fragments (JPEG 140 kb)

210_2011_639_MOESM4_ESM.tif (741 kb)
High resolution image (TIFF 741 kb)
210_2011_639_Fig12_ESM.jpg (17 kb)
Supplementary Figure 4

Plasma concentrations of phenylalanine (μM) were measured after intraperitoneal injection of 1 mg of L-phenylalanine per gram of body weight in NME1+/+ (empty circle) and NME1−/− (filled circle) mice. Plasma concentrations are the mean±SEM from seven mice per group (JPEG 17 kb)

210_2011_639_MOESM5_ESM.tif (455 kb)
High resolution image (TIFF 454 kb)
210_2011_639_Fig13_ESM.jpg (34 kb)
Supplementary Figure 5

NME1 expression correlates positively with annexin IV expression in a panel of independent human carcinoma cell lines. a Immunoblot analysis of lysates from C-100 and H1-177 cell lines probed for NME1, annexin IV, and actin proteins with appropriate antibodies. b Western blotting of extracts of human colon (HCT8/S11) and liver (HepG2, PLC/PRF/5, Mahlavu) cancer cell lines with antibodies specific for NME1, annexin IV, and actin. c Western blotting of breast carcinoma cell lines with antibodies specific for NME1, annexin IV and actin (JPEG 34 kb)

210_2011_639_MOESM6_ESM.tif (1.4 mb)
High resolution image (TIFF 1455 kb)

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Copyright information

© Springer-Verlag 2011

Authors and Affiliations

  • Arnaud Bruneel
    • 1
    • 2
  • Dominique Wendum
    • 3
    • 4
    • 5
  • Valérie Labas
    • 6
  • Odile Mulner-Lorillon
    • 3
    • 7
  • Joelle Vinh
    • 6
  • Nelly Bosselut
    • 1
  • Eric Ballot
    • 8
  • Bruno Baudin
    • 1
    • 2
  • Chantal Housset
    • 3
    • 4
  • Sandrine Dabernat
    • 9
  • Marie-Lise Lacombe
    • 3
    • 4
  • Mathieu Boissan
    • 3
    • 4
    • 10
    • 11
  1. 1.Service de Biochimie AHôpital Saint-Antoine, AP-HPParisFrance
  2. 2.UPRES EA 4530Université Paris-Sud 11Châtenay-MalabryFrance
  3. 3.UPMC Université Paris 06ParisFrance
  4. 4.INSERM UMR_S938Centre de Recherches Saint-AntoineParis Cedex 12France
  5. 5.Laboratoire d’Anatomie PathologiqueHôpital Saint-AntoineParisFrance
  6. 6.CNRS USR 3149, Spectrométrie de Masse Biologique et ProtéomiqueESPCI-ParisTechParisFrance
  7. 7.UMR 7150 CNRSStation Biologique de RoscoffRoscoff CedexFrance
  8. 8.Service d’Immunologie et Hématologie BiologiqueHôpital Saint-AntoineParisFrance
  9. 9.EA DRED 3674, Laboratoire de Biologie de la Différenciation et du DéveloppementUniversité de Bordeaux 2BordeauxFrance
  10. 10.Service de Biochimie et HormonologieAP-HP, Hôpital TenonParisFrance
  11. 11.Dynamique de la Membrane et du CytosqueletteInstitut Curie, CNRS UMR 144ParisFrance

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