Hypoxia-inducible factor1 (HIF-1) is an essential transcription factor for cellular adaptation to decreased oxygen availability. In normoxia the oxygen-sensitive α-subunit of HIF-1 is hydroxylated on Pro564 and Pro402 and thus targeted for proteasomal degradation. Three human oxygen-dependent HIF-1α prolyl hydroxylases (PHD1, PHD2, and PHD3) function as oxygen sensors in vivo. Furthermore, the asparagine hydroxylase FIH-1 (factor inhibiting HIF) has been found to hydroxylate Asp803 of the HIF-1 C-terminal transactivation domain, which results in the decreased ability of HIF-1 to bind to the transcriptional coactivator p300/CBP. We have fused these enzymes to the N-terminus of fluorescent proteins and transiently transfected the fusion proteins into human osteosarcoma cells (U2OS). Three-dimensional 2-photon confocal fluorescence microscopy showed that PHD1 was exclusively present in the nucleus, PHD2 and FIH-1 were mainly located in the cytoplasm and PHD3 was homogeneously distributed in cytoplasm and nucleus. Hypoxia did not influence the localisation of any enzyme under investigation. In contrast to FIH-1, each PHD inhibited nuclear HIF-1α accumulation in hypoxia. All hydroxylases suppressed activation of a cotransfected hypoxia-responsive luciferase reporter gene. Endogenous PHD2mRNA and PHD3mRNA were hypoxia-inducible, whereas expression of PHD1mRNA and FIH-1mRNA was oxygen independent. We propose that PHDs and FIH-1 form an oxygen sensor cascade of distinct subcellular localisation.

An important regulator involved in oxygen-dependent gene expression is the transcription factor HIF (hypoxia-inducible factor), which is composed of an oxygen-sensitive alpha-subunit (HIF-1alpha or HIF-2alpha) and a constitutively expressed beta-subunit. In normoxia, HIF-1alpha is destabilized by post-translational hydroxylation of Pro-564 and Pro-402 by a family of oxygen-sensitive dioxygenases. The three HIF-modifying human enzymes have been termed prolyl hydroxylase domain containing proteins (PHD1, PHD2 and PHD3). Prolyl hydroxylation leads to pVHL (von-Hippel-Lindau protein)-dependent ubiquitination and rapid proteasomal degradation of HIF-1alpha. In the present study, we report that human PHD2 and PHD3 are induced by hypoxia in primary and transformed cell lines. In the human osteosarcoma cell line, U2OS, selective suppression of HIF-1alpha expression by RNA interference resulted in a complete loss of hypoxic induction of PHD2 and PHD3. Induction of PHD2 by hypoxia was lost in pVHL-deficient RCC4 cells. These results suggest that hypoxic induction of PHD2 and PHD3 is critically dependent on HIF-alpha. Using a VHL capture assay, we demonstrate that HIF-alpha prolyl-4-hydroxylase capacity of cytoplasmic and nuclear protein extracts was enhanced by prolonged exposure to hypoxia. Degradation of HIF-1alpha after reoxygenation was accelerated, which demonstrates functional relevance of the present results. We propose a direct, negative regulatory mechanism, which limits accumulation of HIF-1alpha in hypoxia and leads to accelerated degradation on reoxygenation after long-term hypoxia.

Mammalian PASKIN, a PAS-serine/threonine kinase related to bacterial oxygen sensors Footnotes:The following mouse Paskin genomic nucleotide sequence has been deposited in the EMBL/GenBank/DDBJ databases (accession number AJ318757).1 The first two authors contributed equally to this work KEY WORDSGene expression; hypoxia-inducible factor; iron; LOV-domain; nitrogen fixation; oxygen sensor; PAS-domain; protein phosphatase.Hofer et al.3

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.

Raoultella terrigena ATCC 33257, recently reclassified from the genus Klebsiella, is a drinking water isolate and belongs to a large group of non-typeable Klebsiella and Raoultella strains. Using an O-antiserum against a capsule-deficient mutant of this strain, we could show a high prevalence (10.5%) of the R. terrigena O-serotype among non-typeable, clinical Klebsiella and Raoultella isolates. We observed a strong serological cross-reaction with the K. pneumoniae O12 reference strain, indicating that a large percentage of these non-typeable strains may belong to the O12 serotype, although these are currently not detectable by the K. pneumoniae O12 reference antiserum in use. Therefore, we analyzed the O-polysaccharide (O-PS) structure and genetic organization of the wb gene cluster of R. terrigena ATCC 33257, and both confirmed a close relation of R. terrigena and K. pneumoniae O12. The two strains possess an identical O-PS, lipopolysaccharide core structure, and genetic organization of the wb gene cluster. Heterologous expression of the R. terrigena wb gene cluster in Escherichia coli K-12 resulted in the WecA-dependent synthesis of an O-PS reactive with the K. pneumoniae O12 antiserum. The serological data presented here suggest a higher prevalence of the O12-serotype among Klebsiella and Raoultella isolates than generally assumed.