Oxytocin induces dephosphorylation of eukaryotic elongation factor 2 in human myometrial cells.
The oxytocin (OT) receptor (OTR) mediates a wide spectrum of biological actions and is expressed in a large number of different tissues, including uterine, breast, and lung tumors. To define more completely the intracellular signaling mechanisms linked to OTR activation, we have used a phosphoproteomics approach and have characterized changes in the phosphorylation states of intracellular proteins in response to OTR activation in OTR-expressing cell lines. Using a specific antiphosphothreonine antibody, we observed several distinct changes in the threonine phosphorylation patterns. The most prominent change involved dephosphorylation of a 95-kDa moiety. Purification by ion exchange chromatography combined with one- and two-dimensional polyacrylamide gel electrophoresis followed by N-terminal micro-sequence analysis revealed that the 95-kDa moiety corresponded to eukaryotic elongation factor 2. This protein is a key regulator of cellular protein synthesis and mediates, upon dephosphorylation, the translocation step of peptide chain elongation. Dose-response curves in myometrial cells expressing the endogenous OTR indicated a significant effect of OT on eukaryotic elongation factor 2 dephosphorylation at 1 nM, a concentration close to the dissociation constant (K(d)) of OT. Time course analysis indicates that the effect is rapid with a significant effect occurring at 5 min. To determine directly the effect of OT on protein synthesis, the incorporation of [35S]Met into total protein was assessed. In myometrial cells, OTR activation led to significant 29% increase in total protein synthesis over a 2-h period. These findings establish a novel link between OTR activation and cellular protein synthesis and thus define a mechanism by which OT assumes a so far unrecognized, physiologically relevant trophic function.
Devost
Dominic
D
Department of Medicine, McGill University, Montreal, Quebec, Canada H3A 1A1.
Girotti
Milena
M
Carrier
Marie-Eve
ME
Russo
Caterina
C
Zingg
Hans H
HH
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000494:pharmacology; D016466:CHO Cells; D002478:Cells, Cultured; D006224:Cricetinae; D004305:Dose-Response Relationship, Drug; D015180:Electrophoresis, Gel, Two-Dimensional; D005260:Female; D015870:Gene Expression; D006801:Humans; D007526:Isoelectric Point; D009215:Myometrium / Q000187:drug effects* / Q000378:metabolism*; D010121:Oxytocin / Q000494:pharmacology*; D020652:Peptide Elongation Factor 2 / Q000737:chemistry / Q000378:metabolism*; D010446:Peptide Fragments / Q000737:chemistry; D010766:Phosphorylation; D010769:Phosphothreonine / Q000276:immunology; D051381:Rats; D018045:Receptors, Oxytocin / Q000235:genetics; D020539:Sequence Analysis, Protein; D013912:Threonine / Q000378:metabolism
D000906:Antibodies; D020652:Peptide Elongation Factor 2; D010446:Peptide Fragments; D018045:Receptors, Oxytocin; D010769:Phosphothreonine; D013912:Threonine; D010121:Oxytocin