IntroductionThe hypoxia-inducible factor 1 (HIF-1) is an ubiquitously expressed transcriptional master regulator of many genes regulating mammalian oxygen homeostasis. 1 Among others, the corresponding gene products are involved in erythropoiesis, iron metabolism, angiogenesis, control of blood flow, glucose uptake and glycolysis, pH regulation, and cell-cycle control. 2 HIF-1 is a ␣ 1 ␤ 1 heterodimer specifically recognizing the HIF-binding site within cis-regulatory hypoxia response elements. 3 Under normoxic conditions, the von Hippel-Lindau tumor suppressor protein (pVHL) targets the HIF-1␣ subunit for rapid ubiquitination and proteasomal degradation. 4 Binding of the pVHL tumor suppressor protein requires the modification of HIF-1␣ by prolyl-4-hydroxylation at prolines 402 and 564 of human HIF-1␣. [5][6][7][8] A family of 3 oxygen-and iron-dependent prolyl-4-hydroxylases called PHD1, PHD2, PHD3, or HPH3, HPH2, HPH1, respectively, has been shown to hydroxylate HIF␣. 9,10 A fourth member, called PH-4, regulates HIF-1␣ in overexpression conditions only. 11 Thus, limited oxygen supply prevents HIF␣ hydroxylation and degradation. 12 This unusual mechanism of protein regulation provides the basis for the very rapid HIF-1␣ response to hypoxia. 13 In addition to protein stability, oxygen-dependent C-terminal asparagine hydroxylation of HIF-1␣ by factor inhibiting HIF (FIH) prevents transcriptional cofactor recruitment, thereby fine-tuning HIF-1 activity following a further decrease in oxygen availability. 14,15 Among the HIF-1 targets are the genes encoding transferrin, transferrin receptor, heme oxygenase-1, and ceruloplasmin, which coordinately regulate iron metabolism. [16][17][18][19][20] Increased iron uptake, release from the liver, plasma transport, and uptake in the bone marrow are essential to sustain the erythropoietic function of erythropoietin, the prototype HIF-1 target. Ceruloplasmin is a multicopper plasma protein containing ferroxidase activity necessary for Fe 3ϩ saturation of transferrin. 21 Hereditary aceruloplasminemia in humans as well as targeted deletion of the ceruloplasmin gene (Cp) in mice results in iron metabolism disorders characterized by anemia, hepatic iron overload, and neurodegeneration, demonstrating a tight connection between copper and iron metabolism. [22][23][24][25][26] Iron deficiency has been known for more than a decade to induce erythropoietin gene expression and HIF-1␣ protein stabilization. 27 Nowadays, these results are most likely explained by inactivation of the iron-dependent protein hydroxylases PHD1 to 3 and FIH. 12 Iron deficiency also results in mRNA induction of ceruloplasmin by HIF-1-dependent promoter activation and subsequent transcriptional up-regulation of the Cp gene. 20 Materials and methods Cell lines and cell cultureAll cell lines were cultured in Dulbecco modified Eagle medium (high glucose) as described previously. 29 Oxygen partial pressures in the hypoxic workstation (InVivO 2 -400; Ruskinn Technology, Leeds, United Kingdom) or in the incubator (M...

PASKIN links energy flux and protein synthesis in yeast, regulates glycogen synthesis in mammals, and has been implicated in glucose-stimulated insulin production in pancreatic beta-cells. Using newly generated monoclonal antibodies, PASKIN was localized in the nuclei of human testis germ cells and in the midpiece of human sperm tails. A speckle-like nuclear pattern was observed for endogenous PASKIN in HeLa cells in addition to its cytoplasmic localization. By yeast two-hybrid screening, we identified the multifunctional eukaryotic translation elongation factor eEF1A1 as a novel interaction partner of PASKIN. This interaction was mapped to the PAS A and kinase domains of PASKIN and to the C-terminus of eEF1A1 using mammalian two-hybrid and GST pull-down assays. Kinase assays, mass spectrometry and site-directed mutagenesis revealed PASKIN auto-phosphorylation as well as eEF1A1 target phosphorylation mainly but not exclusively at Thr432. Wild-type but not kinase-inactive PASKIN increased the in vitro translation of a reporter cRNA. Whereas eEF1A1 did not localize to the nucleus, it co-localizes with PASKIN to the cytoplasm of HeLa cells. The two proteins also showed a remarkably similar localization in the midpiece of the sperm tail. These data suggest regulation of eEF1A1 by PASKIN-dependent phosphorylation in somatic as well as in sperm cells. Key Words Energy homeostasis • Glycogen synthesis • Nitrogen fixation • Protein phosphorylation • Protein translation • TestisAbstract PASKIN links energy flux and protein synthesis in yeast, regulates glycogen synthesis in mammals, and has been implicated in glucose-stimulated insulin production in pancreatic β-cells. Using newly generated monoclonal antibodies, PASKIN was localized in the nuclei of human testis germ cells and in the midpiece of human sperm tails. A speckle-like nuclear pattern was observed for endogenous PASKIN in HeLa cells in addition to its cytoplasmic localization. By yeast twohybrid screening, we identified the multifunctional eukaryotic translation elongation factor eEF1A1 as a novel interaction partner of PASKIN. This interaction was mapped to the PAS A and kinase domains of PASKIN and to the C-terminus of eEF1A1 using mammalian two-hybrid and GST pull-down assays. Kinase assays, mass spectrometry and site-directed mutagenesis revealed PASKIN auto-phosphorylation as well as eEF1A1 target phosphorylation mainly but not exclusively at Thr432. Wild-type but not kinase-inactive PASKIN increased the in vitro translation of a reporter cRNA. Whereas eEF1A1 did not localize to the nucleus, it co-localizes with PASKIN to the cytoplasm of HeLa cells. The two proteins also showed a remarkably similar localization in the midpiece of the sperm tail. These data suggest regulation of eEF1A1 by PASKIN-dependent phosphorylation in somatic as well as in sperm cells.