Phosphorylation of DEPDC5 by the Pim-1 protein kinase, a cancer driver, stimulates mTORC1 activity by regulating the DEPDC5-Rag GTPase interaction.

The serine/threonine protein kinase Pim-1 has been implicated as a driver of both triple-negative breast cancer and advanced prostate cancer. Regulation of tumor growth by Pim has been closely tied with the ability of this kinase to stimulate protein synthesis by activating mTORC1 signaling. The addition of small-molecule inhibitors of Pim to the prostate cancer PC3-LN4 or breast cancer cells MDA- MB231 decreases their growth and mTORC1 activity. mTORC1 activity is controlled by amino acid levels within the cell that regulate the interactions of multiple protein complexes, e.g., GATOR 1, 2 and KICSTOR, which modulate the ability of the mTORC1 regulator Rag GTPases. We have investigated whether these complexes could be the target for Pim-1 kinase, since a bioinformatics analysis using the Pim phosphorylation consensus RXR[H/R]Xp[S/T] revealed that DEPDC5 (DEP domain containing protein 5), and SZT2 (Seizure threshold 2), components of GATOR1 and KICSTOR complexes respectively, are potential Pim kinase phosphorylation substrates. Our results demonstrate that the knock-out of DEPDC5 and SZT2 blocks the ability of Pim and AKT inhibitors to suppress tumor growth and mTORC1 activity in breast and prostate cancer cells. The overexpression of a constitutively active Rag B mutant stimulates mTORC1 activity but blocks the ability of a Pim inhibitor to regulate mTORC1. Importantly, during amino acid starvation the overexpression of Pim-1 overcame the inhibitor protein cascade and activated mTORC1. Together these results suggest that Pim-1 is regulating the identical protein cascade that is being modulated by varying levels of amino acids. Phosphorylation of DEPDC5 and SZT2 could be detected with a commercial antibody that recognizes phospho-Akt substrates. Examining breast and prostate cancer cells using phospho-specific antibodies and transfecting phospho-inactive mutants of the DEPDC5 (S1002A and S1530A) confirmed its phosphorylation by Pim kinase. Further, CRISPR knock-in mutation of DEPDC5 S1530A suppresses mTORC1 activity, suggesting that the phospho-inactive mutant mimics Pim inhibitor conditions. In a colony formation growth assay, PC3LN4 cells harboring a knock-in DEPDC5 S1530E mutation acquired resistance to Pim and AKT inhibitors. Further, co-immunoprecipitation experiments using 293T cells expressing DEPDC5 wild-type suggested that phosphorylation of this molecule controls the physical interaction of DEPDC5 with Rag A/C. Thus, the Pim-1 mediated phosphorylation partially disables DEPDC5 activity, removing its ability to inhibit the activity of Rag protein complex to promote cancer cell growth. Our results reveal a novel phosphorylation-dependent regulatory mechanism targeting DEPDC5 through which Pim-1 acts as an upstream effector of mTORC1 to facilitate proliferation and survival of breast and prostate cancer cells. This abstract is also being presented as Poster A14. Citation Format: Sathish K.R. Padi, Neha Singh, Ghassan Mouneimne, Andrew S. Kraft, Koichi Okumura. Phosphorylation of DEPDC5 by the Pim-1 protein kinase, a cancer driver, stimulates mTORC1 activity by regulating the DEPDC5- Rag GTPase interaction [abstract]. In: Proceedings of the AACR Special Conference on Targeting PI3K/mTOR Signaling; 2018 Nov 30-Dec 8; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(10_Suppl):Abstract nr PR01.