Identifying Akt as a potential regulator of ACSS2 phosphorylation and understanding the consequence this has on ACSS2 activity
Lead Research Organisation:
University of Cambridge
Department Name: School of Biological Sciences
Abstract
PhD project strategic theme: Bioscience for an integrated understanding of health
Acetyl-CoA is a central intermediate with multiple cellular functions, including ATP production, precursor for fatty acid synthesis and the acetyl donor for lysine acetylation in mammalian cells. Lysine N-e-acetylation is a reversible protein post-translational modification that occurs on histones and non-histones, and can impact transcription, cell proliferation and protein function/localisation. Acetyl-CoA, the substrate of lysine acetyltransferases, is synthesised through various pathways. Nuclear-cytosolic acetyl-CoA is synthesised by two major enzymes; ATP-citrate lyase (ACLY) catalyses the conversion of citrate to acetyl-CoA whereas acetyl CoA synthetase2 (ACSS2) synthesises acetyl-CoA from acetate. Phosphorylation of ACSS2 is known to influence its biological activity. For example, AMPK-mediated phosphorylation of S659 exposes a nuclear-localisation signal, causing ACSS2 translocation to the nucleus where it synthesises acetyl-CoA which can enhance gene transcription by modifying chromatin structure. Recent evidence suggests ACSS2 has other phosphorylatable residues which have a functional consequence on ACSS2 activity. However, the role of phosphorylation at these sites, and the upstream kinase responsible, remains unexplored. This PhD project will aim to address these unanswered questions.
The PI3K-PKB/Akt pathway is a well characterised signalling pathway, which regulates a range of biological functions1. PKB/Akt, a serine/threonine kinase, binds PI3K-generated PIP3 at the plasma membrane via its PH domain. Once recruited to the plasma membrane, PKB/Akt is phosphorylated at Thr308 and Ser473 by PDK1 and mTORC2, respectively, resulting in full activation. Active Akt can phosphorylate multiple substrates, including transcription factors, pro-apoptotic proteins and cell cycle regulators.
Recent work in the field, including investigations into the oscillatory profiles of the phosphoproteome and diet-induced changes to the phosphoproteome lead to the hypothesis that ACSS2 is a potential substrate of Akt, something which has not been previously reported. We hypothesise that there are 3 possible candidate sites on ACSS2, S12, S30 and S267, which could be targeted by Akt. Recently, mTORC2 has been reported to regulate ACSS2 activity in brown preadipocytes2. However, the authours did not investigate a role for PKB/Akt. Additionally we hypothesise that a feedback loop between ACSS2 activity and PI3K-PKB/Akt pathway might exist. ACSS2, PKB/Akt, and the upstream kinases PDK1, and mTORC2, are all reported to be regulated by acetylation. Consequently, changes in the levels of ACSS2-generated acetyl-CoA has the potential to impact the activities of the lysine acetyltransferases responsible for acetylating ACSS2, PKB/Akt, PDK1 and mTORC2.
Therefore, the objectives of this project are to determine the role of ACSS2 phosphorylation at S12, S30 and S267, and to explore if PKB/Akt is the kinase responsible. This will be achieved by producing ACSS2 mutants that lack candidate phosphorylated residues. The upstream kinase responsible for phosphorylating ACSS2 will be addressed using inhibitor and/or editing techniques. Additionally, the project will investigate if ACSS2 activity impacts the PI3K-PKB/Akt pathway. ACSS2-genetically manipulated cells will be used to determine the potential impact of ACSS2-generated acetyl-CoA on acetylation of PI3K pathway activity at a number of levels. This includes measurement of PIP3 levels, PKB/Akt phosphorylation and mTORC1/2 activity, and the acetylation of class I PI3Ks, PDK1, PKB and mTORC-complex members by selective pulldown and proteomic techniques.
Acetyl-CoA is a central intermediate with multiple cellular functions, including ATP production, precursor for fatty acid synthesis and the acetyl donor for lysine acetylation in mammalian cells. Lysine N-e-acetylation is a reversible protein post-translational modification that occurs on histones and non-histones, and can impact transcription, cell proliferation and protein function/localisation. Acetyl-CoA, the substrate of lysine acetyltransferases, is synthesised through various pathways. Nuclear-cytosolic acetyl-CoA is synthesised by two major enzymes; ATP-citrate lyase (ACLY) catalyses the conversion of citrate to acetyl-CoA whereas acetyl CoA synthetase2 (ACSS2) synthesises acetyl-CoA from acetate. Phosphorylation of ACSS2 is known to influence its biological activity. For example, AMPK-mediated phosphorylation of S659 exposes a nuclear-localisation signal, causing ACSS2 translocation to the nucleus where it synthesises acetyl-CoA which can enhance gene transcription by modifying chromatin structure. Recent evidence suggests ACSS2 has other phosphorylatable residues which have a functional consequence on ACSS2 activity. However, the role of phosphorylation at these sites, and the upstream kinase responsible, remains unexplored. This PhD project will aim to address these unanswered questions.
The PI3K-PKB/Akt pathway is a well characterised signalling pathway, which regulates a range of biological functions1. PKB/Akt, a serine/threonine kinase, binds PI3K-generated PIP3 at the plasma membrane via its PH domain. Once recruited to the plasma membrane, PKB/Akt is phosphorylated at Thr308 and Ser473 by PDK1 and mTORC2, respectively, resulting in full activation. Active Akt can phosphorylate multiple substrates, including transcription factors, pro-apoptotic proteins and cell cycle regulators.
Recent work in the field, including investigations into the oscillatory profiles of the phosphoproteome and diet-induced changes to the phosphoproteome lead to the hypothesis that ACSS2 is a potential substrate of Akt, something which has not been previously reported. We hypothesise that there are 3 possible candidate sites on ACSS2, S12, S30 and S267, which could be targeted by Akt. Recently, mTORC2 has been reported to regulate ACSS2 activity in brown preadipocytes2. However, the authours did not investigate a role for PKB/Akt. Additionally we hypothesise that a feedback loop between ACSS2 activity and PI3K-PKB/Akt pathway might exist. ACSS2, PKB/Akt, and the upstream kinases PDK1, and mTORC2, are all reported to be regulated by acetylation. Consequently, changes in the levels of ACSS2-generated acetyl-CoA has the potential to impact the activities of the lysine acetyltransferases responsible for acetylating ACSS2, PKB/Akt, PDK1 and mTORC2.
Therefore, the objectives of this project are to determine the role of ACSS2 phosphorylation at S12, S30 and S267, and to explore if PKB/Akt is the kinase responsible. This will be achieved by producing ACSS2 mutants that lack candidate phosphorylated residues. The upstream kinase responsible for phosphorylating ACSS2 will be addressed using inhibitor and/or editing techniques. Additionally, the project will investigate if ACSS2 activity impacts the PI3K-PKB/Akt pathway. ACSS2-genetically manipulated cells will be used to determine the potential impact of ACSS2-generated acetyl-CoA on acetylation of PI3K pathway activity at a number of levels. This includes measurement of PIP3 levels, PKB/Akt phosphorylation and mTORC1/2 activity, and the acetylation of class I PI3Ks, PDK1, PKB and mTORC-complex members by selective pulldown and proteomic techniques.
