Identifying Akt as a potential regulator of ACSS2 phosphorylation and understanding the consequence this has on ACSS2 activity

在哺乳动物细胞中，乙酰辅酶a是一种具有多种细胞功能的中心中间体，包括ATP的产生、脂肪酸合成的前体和赖氨酸乙酰化的乙酰基供体。赖氨酸n- e-乙酰化是一种可逆的蛋白质翻译后修饰，发生在组蛋白和非组蛋白上，可以影响转录、细胞增殖和蛋白质功能/定位。乙酰辅酶a是赖氨酸乙酰转移酶的底物，通过多种途径合成。核胞浆乙酰辅酶a由两种主要酶合成；atp -柠檬酸裂解酶（ACLY）催化柠檬酸转化为乙酰辅酶a，而乙酰辅酶a合成酶2 （ACSS2）则由乙酸合成乙酰辅酶a。已知ACSS2的磷酸化会影响其生物活性。例如，ampk介导的S659磷酸化暴露了一个核定位信号，导致ACSS2易位到核中，在核中合成乙酰辅酶a，乙酰辅酶a可以通过修饰染色质结构来增强基因转录。最近的证据表明，ACSS2有其他可磷酸化残基，这些残基对ACSS2的活性有功能影响。然而，这些位点的磷酸化作用以及上游激酶的作用仍未被探索。这个博士项目将致力于解决这些悬而未决的问题。PI3K-PKB/Akt通路是一个很好的信号通路，它调节一系列生物学功能1。PKB/Akt是一种丝氨酸/苏氨酸激酶，在质膜上通过PH结构域与pi3k生成的PIP3结合。一旦募集到质膜上，PKB/Akt分别被PDK1和mTORC2磷酸化Thr308和Ser473位点，导致完全激活。活性Akt可以磷酸化多种底物，包括转录因子、促凋亡蛋白和细胞周期调节因子。最近在该领域的工作，包括对磷酸化蛋白质组振荡谱的研究和饮食诱导的磷酸化蛋白质组变化，导致了ACSS2是Akt的潜在底物的假设，这在以前没有报道过。我们假设ACSS2、S12、S30和S267上有3个可能的候选位点可以被Akt靶向。最近，mTORC2被报道调控棕色前脂肪细胞中ACSS2的活性。然而，作者没有研究PKB/Akt的作用。此外，我们假设ACSS2活性与PI3K-PKB/Akt通路之间可能存在反馈回路。据报道，ACSS2、PKB/Akt和上游激酶PDK1、mTORC2均受乙酰化调节。因此，ACSS2生成的乙酰辅酶a水平的变化有可能影响负责ACSS2、PKB/Akt、PDK1和mTORC2乙酰化的赖氨酸乙酰转移酶的活性。因此，本项目的目的是确定S12、S30和S267位点ACSS2磷酸化的作用，并探索PKB/Akt是否与此激酶有关。这将通过产生缺乏候选磷酸化残基的ACSS2突变体来实现。负责磷酸化ACSS2的上游激酶将使用抑制剂和/或编辑技术来解决。此外，该项目将研究ACSS2活性是否影响PI3K-PKB/Akt通路。acss2基因操作的细胞将用于确定acss2产生的乙酰辅酶a在多个水平上对PI3K途径活性乙酰化的潜在影响。这包括通过选择性下拉和蛋白质组学技术测量PIP3水平、PKB/Akt磷酸化和mTORC1/2活性，以及I类pi3k、PDK1、PKB和mtorc复合物成员的乙酰化。

PhD project strategic theme: Bioscience for an integrated understanding of healthAcetyl-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.