mEAK-7 Functions Downstream of Rag Proteins to Regulate mTORC1 Signaling in Human Cancer

Objectives: To determine the cellular location and role of mEAK-7 relative to the Rag Proteins in mTORC1 signaling in human cancer cells.
Methods: Human cancer cell lines (H1975, MDA-MB-231, and H1299) and human embryonic kidney cell line HEK-293T were treated with 100 nM siRNA control, mEAK-7, RagA, RagB, RagC, or RagD for 48 hours in 10% serum-containing medium. Cells were overexpressed with GST-RagA/C and simultaneously treated with the previously mentioned siRNAs to assess interaction with mTORC1 components. Confocal microscopy imaging was used to ascertain the cellular location of the Rag Proteins.
Results: Mammalian EAK-7 (mEAK-7) is a crucial gene for the regulation of mTORC1 signaling in human cancer cells, as mEAK-7 knockdown results in the elimination of mTORC1 signaling. To determine this, we relied on immunoblot analysis of p-S6 (Ser^240/244), a readout of mTORC1 pathway activation. Furthermore, it is believed that RagA and RagC, two of the four Rag Proteins in the mTORC1 pathway, form a necessary heterodimer to recruit mTORC1 to the lysosome in response to amino acids. However, we found that mTORC1 signaling in response to amino acids may not occur in of the presence of the RagA/C heterodimer. We also determined that mEAK-7 protein stability and function is dependent on the presence of the RagA/C heterodimer. Therefore, we provide evidence that mEAK-7 is downstream of the four Rag Proteins (RagA, RagB, RagC, and RagD), in order to regulate mTORC1 signaling by directly interacting with the RagA/C heterodimer.
Conclusions: The Rag A/C heterodimer is necessary for mEAK-7 protein stability and function, while being dispensable to the mTORC1 signaling pathway; the heterodimer affects mTORC1 by affecting mEAK-7.