Negative Effects of the Amino Acids Lys, His, and Thr on S6K1 Phosphorylation in Mammary Epithelial Cells

The role of essential amino acids (AA) on protein synthesis via the mTOR pathway was studied in murine mammary epithelial cells cultured under lactogenic conditions. Len, Ile, and Val increased S6K1 phosphorylation compared to that measured in AA-deprived cells. Trp, Phe, and Met had no effect. Surprisingly, Lys, His, and Thr inhibited S6K1 phosphorylation in both murine and bovine mammary cells. Thr exhibited the most potent inhibition, being the only amino acid that competed with Leu's positive role. In non-deprived cells, there was no observable effect of Lys, His, or Thr on S6K1 phosphorylation at concentrations up to five times those in the medium. However, their addition as a mix revealed a synergistic negative effect. Supplementation of Lys, His, and Thr abrogated mTOR Ser 2448 phosphorylation, with no effect on Akt Ser473-an mTORC2 target. This confirms specific mTORC1 regulation of S6K1 phosphorylation. The individual supplementation of Lys, His, and Thr maintained a low level of IRS-1 phosphorylation, which was dose-dependently increased by their combined addition. Thus, in parallel to inhibiting S6K1 activity, these AA may act synergistically to activate an additional kinase, phosphorylating IRS-1 via an S6K1-independent pathway. In cultures supplemented by Lys, His, and Thr, cellular protein synthesis decreased by up to 65%. A more pronounced effect was observed on P-casein synthesis. These findings indicate that positive and negative signaling from AA to the mTOR pathway, combined with modulation of insulin sensitization, mediate the synthesis rates of total and specific milk proteins in mammary epithelial cells. J. Cell. Biochem. 105: 1038-1047, 2008. (C) 2008 Wiley-Liss, Inc.