Mechanism For The Regulation Of TORC1 Signaling Pathway By Yeast Whi2 And Its Human Homolog KCTD Proteins

Aim: In this study, we would investigate the mechanism by which yeast Whi2 regulates the amino acid-sensing TORC1 signaling pathway, and the roles of its human homolog KCTD proteins in regulating mammalian TORC1(mTORC1).Methods: Use yeast growth assay to compare growth of different strains; Investigate yeast TORC1 activity by detecting the immunoblot of the phosphorylation of ribosomal protein S6; Detect oxygen consumption of different strains using oxygen electrode; Detect the expression of endogenous Whi2-TAP during amino acid deprivation; Detect how Whi2 regulates autophagy by using the prATG8-GFP-ATG8 plasmid; Detect the expression and localization of KCTD proteins by immunofluorescence; Screen out the KCTD proteins that can inhibit mTORC1 activity using amino acid deprivation and re-stimulation assay; Determine the function of human KCTD11 by over-expression and RNA interference technique.Results: Growth assays and oxygen consumption experiments indicated that yeast Whi2 might have tumor suppressor-like features. Through transfecting prATG8-GFP-ATG8 plasmid into different strains, we found that yeast Whi2 affected TORC1 activity differently in response to low concentrations of amino acids and glucose. Expression level of endogenous Whi2 also varied differently in response to low amino acids and glucose. The yeast growth assay and the TORC1 activity assay showed that the mammalian Phospho-S6(Ser235/236) antibody can be used to detect yeast TORC1 activity; Whi2 was a TORC1 negative regulator; Whi2 was at a parallel pathway of amino acid-sensing SEACIT-Gtr-TORC1 axis. Immunofluorescence showed that all the constructed KCTD plasmids can be expressed in cells normally, and their various distribution patterns indicated that they may function differently. Several human KCTD proteins have been suggested to be able to inhibit mTORC1 in the preliminary experiments. Over-expression and knockdown of human KCTD11 confirmed that human KCTD11 protein was a negative regulator of mTORC1.Conclusion: Yeast Whi2 is a negative regulator of TORC1 and is located in a parallel pathway of the SEACIT-Gtr-TORC1 axis in response to amino acid signaling. Several KCTD proteins including KCTD9, KCTD11, KCNRGβ have the potential to inhibit mTORC1. Human KCTD11 has been confirmed as a negative regulator of mTORC1, and may be a novel tumor suppressor.