Devost Dominic, Wrzal Paulina, Zingg Hans H
Department of Pharmacology, McGill University, Montreal, Quebec, Canada.
Prog Brain Res. 2008;170:167-76. doi: 10.1016/S0079-6123(08)00415-9.
The great diversity of the expression sites and proposed function of the oxytocin (OXT) receptor (OXTR) is paralleled by a diversity of its signalling pathways, many of which have still remained unexplored. We have used different approaches to discover novel pathways. By means of a phosphoproteomics approach, we have detected several distinct OXT-induced changes in tyrosine as well as threonine phosphorylation states of intracellular protein in myometrial cells. The most prominent change involved dephosphorylation of a 95-kDa phosphothreonine moiety. By N-terminal amino acid microsequence analysis, this moiety was shown to correspond to eukaryotic translation factor eEF2. This protein is a key regulator of protein synthesis and mediates, upon dephosphorylation, the translocation step of peptide chain elongation. These findings define a novel mechanism by which OXT assumes a so far unrecognized trophic function. We next elucidated the intracellular pathway(s) involved. We found that this effect is not mediated by any of the known pathways known to induce eEF2 dephosphorylation (mTOR, ERK1/2 or p38) but by protein kinase C. Consistent with this idea, we also found that direct stimulation of protein kinase C with a phorbol ester induced eEF2 dephosphorylation in myometrial cells. Using phosphoERK antibodies, we discovered by Western blotting that OXT induced phosphorylation of a higher molecular weight ERK-related protein. We were able to show that this band corresponded to "big MAP kinase1" or ERK5. ERK5 is part of a distinct MAPK cascade and promotes expression of the myosin light chain gene and plays an obligatory role in muscle cell development and differentiation. The role of ERK5 in myometrium has remained unexplored, but it is likely to represent an important novel pathway mediating OXT's effects on smooth muscle function. Further elucidation of these novel signalling pathways will have significant relevance for the development of novel pathway-specific OXTR agonists and antagonists.
催产素（OXT）受体（OXTR）表达位点和推测功能的巨大多样性与其信号通路的多样性相平行，其中许多信号通路仍未被探索。我们采用了不同方法来发现新的信号通路。通过磷酸化蛋白质组学方法，我们检测到子宫肌层细胞内蛋白质酪氨酸和苏氨酸磷酸化状态中几种不同的OXT诱导变化。最显著的变化涉及一个95 kDa磷酸苏氨酸部分的去磷酸化。通过N端氨基酸微序列分析，该部分显示对应于真核翻译因子eEF2。这种蛋白质是蛋白质合成的关键调节因子，去磷酸化后介导肽链延伸的转位步骤。这些发现定义了一种新机制，通过该机制OXT发挥了迄今未被认识的营养功能。接下来我们阐明了所涉及的细胞内信号通路。我们发现这种效应不是由任何已知的诱导eEF2去磷酸化的信号通路（mTOR、ERK1/2或p38）介导的，而是由蛋白激酶C介导的。与此观点一致，我们还发现用佛波酯直接刺激蛋白激酶C可诱导子宫肌层细胞中eEF2去磷酸化。使用磷酸化ERK抗体，我们通过蛋白质印迹法发现OXT诱导了一种分子量更高的ERK相关蛋白的磷酸化。我们能够证明这条带对应于“大MAP激酶1”或ERK5。ERK5是一个独特的MAPK级联反应的一部分，促进肌球蛋白轻链基因的表达，并且在肌肉细胞发育和分化中起重要作用。ERK5在子宫肌层中的作用尚未被探索，但它可能代表了一条介导OXT对平滑肌功能影响的重要新信号通路。进一步阐明这些新的信号通路对于开发新的通路特异性OXTR激动剂和拮抗剂具有重要意义。
