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.

T he Per-ARNT-Sim (PAS) domain proteins often serve as environment sensors, regulating the cellular metabolism and behavior of microorganisms in response to, among others, oxygen or light. In nitrogen-fixing Rhizobium species, for example, the oxygen sensor protein FixL contains a heme group within its PAS domain. Oxygen bound to heme inhibits the histidine kinase domain. Under oxygen-free conditions, kinase activity is de-repressed and activates FixJ, the master transcriptional inducer of genes involved in nitrogen fixation.We and others previously identified a novel mammalian PAS protein, termed PASKIN (1), or PAS kinase (2). The domain architecture of PASKIN resembles that of FixL. PASKIN contains two PAS domains (PAS A and PAS B) and a serine/threonine kinase domain related to AMP kinases that might be regulated in cis by the binding of thus far unknown (possibly metabolic) ligands to the PAS domain (3). After de-repression, autophosphorylation in trans results in the "switch-on" of the kinase domain of PASKIN (2). The budding yeast PASKIN homologs PSK1 and PSK2 phosphorylate three translation factors and two enzymes involved in the regulation of glycogen and trehalose synthesis, thereby coordinately controlling translation and sugar flux (4). Under stress conditions (nutrient restriction combined with high temperature), PASKIN kinase activity results in downregulation of protein synthesis and carbohydrate storage in yeast. In mammalian cells, PASKIN-dependent phosphorylation inhibits the activity of mammalian glycogen synthase (5).A recent report suggested that PASKIN kinase activity followed by mRNA and protein expression is increased in MIN6 cells and in isolated pancreatic ␤-cells after exposure to high glucose concentrations (6). Increased PASKIN activity appeared to be required for glucose-dependent transcriptional induction of the pancreatic duodenum homeobox 1 (PDX-1) transcription factor, leading to transcriptional induction of preproinsulin but not glucokinase or uncoupling protein 2 gene expression. The authors concluded that decreases in PASKIN activity in ␤-cells might contribute to some forms of type 2 diabetes (6). However, no in vivo data were provided in the report.We previously generated PASKIN null mice by targeted replacement of the kinase domain of the mouse Paskin gene by a lacZ-neo fusion construct in embryonic stem cells (7,8). Surprisingly, PASKIN expression is strongly upregulated in postmeiotic germ cells during spermatogenesis, as revealed by both ␤-galactosidase staining as well as mRNA blotting. In fact, PASKIN mRNA levels in testis are several magnitudes higher than in all other organs tested. No other "sensory" organs, including pancreas, carotid bodies, or photoreceptor cells, stained positive for ␤-galactosidase. At least under laboratory conditions, fertility as well as sperm production and sperm motility were not affected in PASKIN knockout mice. To examine the role of PASKIN in glucose-stimulated insulin production, we used pancreatic ␤-cells derived from wildtype and kno...