Male germ cell expression of the PAS domain kinase PASKIN and its novel target eukaryotic translation elongation factor eEF1A1.
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.
Eckhardt
Katrin
K
Institute of Physiology and Zürich Center for Integrative Human Physiology, University of Zürich, Zürich, Switzerland.
Troger
Juliane
J
Reissmann
Jana
J
Katschinski
Dörthe M
DM
Wagner
Klaus F
KF
Stengel
Petra
P
Paasch
Uwe
U
Hunziker
Peter
P
Borter
Emanuela
E
Barth
Sandra
S
Schlafli
Philipp
P
Spielmann
Patrick
P
Stiehl
Daniel P
DP
Camenisch
Gieri
G
Wenger
Roland H
RH
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
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