Leucine‐Induced Signaling through mTOR is Inhibited by 1‐Butanol: A Possible Role for Phospholipase D in Leucine Signaling

It has been previously demonstrated that administration of leucine to animals in vivo or cells in culture, enhances signaling through the mammalian target of rapamycin (mTOR) pathway, leading to phosphorylation of downstream targets, such as ribosomal protein S6 (S6K)1 and eukaryotic initiation factor (eIF4E) binding protein (4E‐BP)1. Similarly, activation of phospholipase D in cells in culture increases mTOR‐dependent phosphorylation of S6K1 and 4E‐BP1. To determine whether or not leucine enhances signaling through mTOR in a phospholipase D‐dependent manner, Rat2 cells were deprived of serum and leucine for 2 h and then leucine was returned to the culture medium in the presence or absence of 1‐butanol, an inhibitor of phospholipase D function. As previously reported, leucine enhanced signaling through mTOR was assessed by phosphorylation of S6K1 and 4E‐BP1. Similarly, lysophosphatidic acid (LPA) promoted phosphorylation of S6K1 and 4E‐BP1 in serum‐ and leucine‐deprived cells. Administration of 1‐butanol significantly attenuated both leucine‐ and LPA‐dependent mTOR activation, suggesting leucine acts to enhance signaling through mTOR in a phospholipase D‐dependent manner. To establish the role of phospholipase D1 versus phospholipase D2 in the effect, siRNA is currently being used to repress the expression of the enzymes alone, and in combination. Overall, the results suggest a novel role for phospholipase D in the regulation of mTOR by leucine. (Supported by NIH Grants DK‐13499 and DK‐15658)