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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

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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.

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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

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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).

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Author notes

  1. Mathieu Boissan

    Present address: Dynamique de la Membrane et du Cytosquelette, Institut Curie, CNRS UMR 144, Paris, France

Authors and Affiliations

  1. Service de Biochimie A, Hôpital Saint-Antoine, AP-HP, 75012, Paris, France

    Arnaud Bruneel, Nelly Bosselut & Bruno Baudin

  2. UPRES EA 4530, Université Paris-Sud 11, Châtenay-Malabry, France

    Arnaud Bruneel & Bruno Baudin

  3. UPMC Université Paris 06, 75005, Paris, France

    Dominique Wendum, Odile Mulner-Lorillon, Chantal Housset, Marie-Lise Lacombe & Mathieu Boissan

  4. INSERM UMR_S938, Centre de Recherches Saint-Antoine, 75571, Paris Cedex 12, France

    Dominique Wendum, Chantal Housset, Marie-Lise Lacombe & Mathieu Boissan

  5. Laboratoire d’Anatomie Pathologique, Hôpital Saint-Antoine, 75012, Paris, France

    Dominique Wendum

  6. CNRS USR 3149, Spectrométrie de Masse Biologique et Protéomique, ESPCI-ParisTech, Paris, France

    Valérie Labas & Joelle Vinh

  7. UMR 7150 CNRS, Station Biologique de Roscoff, 29682, Roscoff Cedex, France

    Odile Mulner-Lorillon

  8. Service d’Immunologie et Hématologie Biologique, Hôpital Saint-Antoine, 75012, Paris, France

    Eric Ballot

  9. EA DRED 3674, Laboratoire de Biologie de la Différenciation et du Développement, Université de Bordeaux 2, Bordeaux, France

    Sandrine Dabernat

  10. Service de Biochimie et Hormonologie, AP-HP, Hôpital Tenon, 75020, Paris, France

    Mathieu Boissan

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  1. Arnaud Bruneel
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Correspondence to Mathieu Boissan.

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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)

High resolution image (TIFF 2042 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)

High resolution image (TIFF 2042 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)

High resolution image (TIFF 2042 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)

High resolution image (TIFF 741 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)

High resolution image (TIFF 454 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)

High resolution image (TIFF 1455 kb)

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Bruneel, A., Wendum, D., Labas, V. et al. Proteomic analysis of NME1/NDPK A null mouse liver: evidence for a post-translational regulation of annexin IV and EF-1Bα. Naunyn-Schmiedeberg's Arch Pharmacol 384, 407–419 (2011). https://doi.org/10.1007/s00210-011-0639-5

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