Understanding the underlying mechanism behind of mTOR control of glutaminase C activity and identification and characterization of glutaminase C post-translational modifications

Author(s)

Carolline Fernanda Rodrigues Ascenção

• Document type: Doctoral Thesis
• Press: Campinas, SP.
• Institution: Universidade Estadual de Campinas (UNICAMP). Instituto de Biologia
• Defense date: 2018-02-20

Examining board members

Sandra Martha Gomes Dias; Jörg Kobarg; Talita Miguel Marin; Ana Karina de Oliveira; Juliana Helena Costa Smetana

• Advisor: Sandra Martha Gomes Dias

Abstract

Glutaminases (GLS) are enzymes involved in tumor growth and progression due to their role in maintaining redox homeostasis, energy production and increased cellular biomass. In this sense, GLS have been characterized as important for migration and invasion processes. The PI3K / mTOR pathway is closely related to increased biomass and tumor growth and is frequently dysregulated in tumors. mTOR regulates glutaminases in RNA stability in a mechanism involving c-MYC translational control, which in its turn, inhibits miRNA expression, impacting on increased GLS protein levels. In this work, the mTOR pathway was disturbed using a chemical inhibitor, rapamycin, through the knockdown of TSC2 and the expression of wild-type RagB and the constitutively active mutant. In all cases, we observed a positive regulation of mTOR on GLS activity without changes in glutaminase protein levels. Intrigued by the mechanism by which mTOR was regulating glutaminases, we investigated, using mass spectrometry, whether glutaminase C (one of the GLS isoforms) underwent post-translational modifications or binding to interaction partners, dependent on the action of mTOR. As a possible candidate, we have identified Sec13 (GATOR2 component), HNRNP H1 (Heterogeneous nuclear ribonucleoprotein H1), EEFD (Ekaryotic elongation factor delta) and ASS1 (Argininosuccinate synthase) proteins. On the other hand, we also identified a phosphorylation in serine 95 (S95) and acetylation in lysine 197 (K197), which, however, is not under mTOR control. We have found that expression of phosphomimetic S95D led to a decrease in glutamine activity. Accordingly, an expression of GAC.S95D led to a slight decrease in proliferation and cell migration phenotypes. Interestingly, the mutant S95A, which had little impact on glutaminase activity, led to a prominent increase in proliferation, migration, and levels of epithelial-mesenchymal transition markers (EMT). Taken together, our results show that GAC has the potential to be regulated by mTOR through protein interaction. In addition, we show that the N-terminal of GAC undergoes phosphorylation in residue S95 and, through the use of mutants, it was identified that this modification has the potential to affect an endogenous glutaminase enzyme function and cellular parameters such as proliferation and migration (AU)

• FAPESP's process: 13/23510-4 - Understanding how PI3K/AKt/mTOR and AMPK signaling pathways affect glutaminase activity.
• Grantee: Carolline Fernanda Rodrigues Ascenção
• Support Opportunities: Scholarships in Brazil - Doctorate