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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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.
KeywordsNM23 Transgenic mice Liver Proteome Annexin IV EF-1Bα
Nucleoside diphosphate kinase
Two-dimensional gel electrophoresis
Colloidal Coomassie blue
Peptide mass fingerprinting
Liquid chromatography–tandem mass spectrometry
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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