| Extracellular regulated protein (ERK), which is a mitogen-actived protein kinase in mammalian cells, is one of the upstream regulators of the mammalian target of rapamycin (mTOR) signaling pathway. ERK/mTOR signal pathway can integrate signals from nutrient, growth factor, energy and environmental stress to regulate cell growth and metabolism. ERK/mTOR signal pathway has been investigated extensively in human cells and other model cells, whereas it is not yet studied in goat cells.Inner Mongolia Cashmere goat is a local species, which was isolated after long term of natural selection and breeding. The goats have some unique properties in utilization and metabolism of amino acids. Leucine (Leu) is the only ketone amino acids in animals, and is essential to protein metabolism among branched-chain amino acid. Leu could activate mTORC1and p70S6K, and consequently increase the synthesis of protein. This study aims to elucidate the molecular mechanism of ERK on regulation of goat fetal fibroblast (GFb cells) growth and its response to leucine signal, and try to obtain a preliminary understanding of ERK/mTOR signal pathway in regulation of the GFb cell growth.The whole study can be divided into two stages. First, we proved the important role of ERK on GFb cell growth through inhibition of ERK by U0126, which is a MEK/ERK specific inhibitor. Then we explored the putative mechanism of ERK/mTOR/p70S6K signaling pathway in sensing amino acid with the combination of U0126and Leu. In the former stage, MTT assay and Trypan blue exclusion assay were applied to make inhibition curve and cell growth curve respectively. And in the latter stage, ERK1/2expression and phosphorylation level were detected after Leu stimulation and the interaction between ERK and p70S6was investigated by Co-immunopricipitation (Co-IP) assay and yeast two hybrid.The results showed that, U0126inhibited GFb cell growth, and the IC50was1.52μM. Meanwhile, U0126could obviously inhibit the phosphorylation of p70S6K(T389), S6(T240/244) and4EBP1(T37/46) which are downstream effectors of the mTORC1. The phosphorylated level of ERK (T202/Y204) obviously increased after Leu stimulated for30min and Leu could also result in higher phosphorylation of ERK in the existence of U0126, compared with control. This result indicated a MEK-independent mechanism of Leu on the activation of ERK1/2phosphorylation.In order to explore the possible mechanism of how Leu activates ERK, we performed Co-IP assay and the results showed p70S6K could co-immunopricipitated with ERK1/2. This data indicated that there was a possible interaction between ERK1/2and p70S6K. Next, we performed yeast two hybrid assay, however, the results showed that there was no association between ERK2and p70S6K in our yeast two hybrid system, indicating that ERK2could not directly interact with p70S6K in vivo. However, whether there is any other potential indirect interaction between these two proteins need further research.In this study, we proved that ERK could regulate GFb cell growth and found that Leu promoted the phosphorylation of ERK1/2in GFb cells. These data indicate that the increased phosphorylation of ERK1/2is independent to its upstream regulator-MEK. The exacted mechanism need to be further investigated.Our data provide a scientific evidence to improve understanding on the important role of ERK/mTOR signaling pathway in mediating amino acid signals to regulate GFb cell growth. |
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