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

博士项目战略主题：乙酰辅酶A是具有多种细胞功能的中心中间体，包括ATP生产，脂肪酸合成的前体和哺乳动物细胞中赖氨酸乙酰化的乙酰基供体。赖氨酸N-e-乙酰化是发生在组蛋白和非组蛋白上的可逆蛋白质翻译后修饰，并且可以影响转录、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（ACSS 2）从乙酸合成乙酰辅酶A。已知ACSS 2的磷酸化影响其生物活性。例如，AMPK介导的S659磷酸化暴露了核定位信号，导致ACSS 2易位到核中，在那里它合成乙酰辅酶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，ACSS 2有其他磷酸化的残基，对ACSS 2的活性有功能性的后果。然而，在这些网站的磷酸化的作用，上游激酶负责，仍然没有探索。PI 3 K-PKB/Akt信号通路是一个非常有特色的信号通路，它调节着一系列的生物学功能1。PKB/Akt是一种丝氨酸/苏氨酸激酶，通过其PH结构域在质膜上结合PI 3 K产生的PIP 3。一旦募集到质膜，PKB/Akt分别被PDK 1和mTORC 2在Thr 308和Ser 473处磷酸化，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。该领域的最新工作，包括对磷酸化蛋白质组的振荡曲线和饮食诱导的磷酸化蛋白质组变化的研究，导致了ACSS 2是Akt的潜在底物的假设，这是以前没有报道过的。我们假设ACSS 2上有3个可能的候选位点，S12，S30和S267，它们可以被Akt靶向。最近，mTORC 2被报道调节棕色前脂肪细胞中的ACSS 2活性2。然而，作者没有调查PKB/Akt的作用。此外，我们推测ACSS 2活性和PI 3 K-PKB/Akt通路之间可能存在反馈环。ACSS 2、PKB/Akt和上游激酶PDK 1和mTORC 2均被报道受乙酰化调节。因此，ACSS 2产生的乙酰辅酶A水平的变化可能会影响负责乙酰化ACSS 2的赖氨酸乙酰转移酶、PKB/Akt、PDK 1和mTORC 2的活性。因此，本项目的目的是确定ACSS 2在S12、S30和S267磷酸化的作用，并探讨PKB/Akt是否是负责乙酰化ACSS 2的激酶。这将通过产生缺乏候选磷酸化残基的ACSS 2突变体来实现。负责磷酸化ACSS 2的上游激酶将使用抑制剂和/或编辑技术来解决。此外，该项目将调查ACSS 2活性是否影响PI 3 K-PKB/Akt途径。将使用ACSS 2基因操作细胞确定ACSS 2生成的乙酰辅酶A在多个水平上对PI 3 K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PIP 3水平、PKB/Akt磷酸化和mTORC 1/2活性，以及I类PI 3 Ks、PDK 1、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.