title: Male germ cell expression of the PAS domain kinase PASKIN and its novel target eukaryotic translation elongation factor eEF1A1.
abstract: 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.
authors.affiliation: Institute of Physiology and Zürich Center for Integrative Human Physiology, University of Zürich, Zürich, Switzerland.
authors.forename: Katrin
authors.initials: K
authors.lastname: Eckhardt
authors.forename: Juliane
authors.initials: J
authors.lastname: Troger
authors.forename: Jana
authors.initials: J
authors.lastname: Reissmann
authors.forename: Dörthe M
authors.initials: DM
authors.lastname: Katschinski
authors.forename: Klaus F
authors.initials: KF
authors.lastname: Wagner
authors.forename: Petra
authors.initials: P
authors.lastname: Stengel
authors.forename: Uwe
authors.initials: U
authors.lastname: Paasch
authors.forename: Peter
authors.initials: P
authors.lastname: Hunziker
authors.forename: Emanuela
authors.initials: E
authors.lastname: Borter
authors.forename: Sandra
authors.initials: S
authors.lastname: Barth
authors.forename: Philipp
authors.initials: P
authors.lastname: Schlafli
authors.forename: Patrick
authors.initials: P
authors.lastname: Spielmann
authors.forename: Daniel P
authors.initials: DP
authors.lastname: Stiehl
authors.forename: Gieri
authors.initials: G
authors.lastname: Camenisch
authors.forename: Roland H
authors.initials: RH
authors.lastname: Wenger
mesh_terms: D000911:Antibodies, Monoclonal; D001483:Base Sequence; D002467:Cell Nucleus / Q000378:metabolism; D002474:Cell-Free System; D003593:Cytoplasm / Q000378:metabolism; D017931:DNA Primers / Q000235:genetics; D015870:Gene Expression; D006367:HeLa Cells; D006801:Humans; D066298:In Vitro Techniques; D008297:Male; D020648:Peptide Elongation Factor 1 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D014176:Protein Biosynthesis; D025941:Protein Interaction Mapping; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D013082:Sperm Tail / Q000378:metabolism; D013094:Spermatozoa / Q000378:metabolism*; D014162:Transfection; D020798:Two-Hybrid System Techniques
chemical_list: D000911:Antibodies, Monoclonal; D017931:DNA Primers; C094814:EEF1A1 protein, human; D020648:Peptide Elongation Factor 1; D011994:Recombinant Proteins; C116037:PAS domain kinases; D017346:Protein Serine-Threonine Kinases