Regulation of transcriptional coactivator p300 by posttranslational modification

转录辅活化子p300具有内在的组蛋白乙酰转移酶(HAT)活性，在许多细胞过程中起重要作用。因此，严格控制p300对于确保基因位点特异性的组蛋白乙酰化和基因调控至关重要。我们以前已经证实，p300受翻译后修饰和不同的细胞分布的动态调节。我们首次证明p300受细胞质蛋白酶体系统的调节。此外，我们已经确定Akt/蛋白激酶B是p300的正调节因子，而蛋白磷酸酶2A的B563调节亚基(PP2A)是负调节因子。我们的工作为细胞运输和空间重新分布如何控制p300的蛋白质稳定性和转录活性提供了新的见解。最近，我们发现在肌源性分化过程中，p300以一种谱系特异性的方式进行调控。因此，我们打算利用我们的发现来确定与成肌细胞分化相关的谱系特异性p300调控的分子机制。 我们假设可逆的磷酸化在控制p300活性中起关键作用，p300在成肌细胞分化过程中反过来调节p300功能，并建议定义B563调节亚基Akt和附加的激酶与p300在成肌细胞分化环境中的功能相互作用。我们将研究p300的磷酸化谱，并通过获得和功能丧失的方法描述p300的可逆磷酸化的调节。我们将产生模拟候选位点磷酸化的突变体，并产生针对位点特异性磷酸化的抗体，以研究可逆磷酸化对p300稳定性和转录活性的影响。我们的目标是确定翻译后修饰介导的p300调控的分子基础以及它们对p300作为HAT、支架或染色质上与基因转录相关的桥梁的功能的影响。我们将确定与细胞分化相关的不同信号通路和p300调控的分子机制。我们的长期目标是确定这些调控机制如何控制依赖于p300的基因表达，从细胞内运输、蛋白质周转到转录激活，并以网络生物学的方式确定p300功能的分子基础。 这个研究计划是基于我们最近的发现，并以p300为基础，p300是体内不同细胞过程和骨骼肌发生所必需的。许多HQP将通过建议的研究接受培训，正如我们经过验证的记录所表明的那样，因此，他们将完全准备好成功地进入自己的职业道路，并为我们的研究领域和不同的加拿大组织做出重大贡献。

The transcriptional coactivator p300 possess an intrinsic histone acetyltransferase (HAT) activity and is important for many cellular processes. A tight control of p300 is therefore critical to ensure locus-specific histone acetylation and gene regulation. We have established previously that p300 is dynamically regulated by posttranslational modification and distinct cellular distribution. We are the first to show that p300 is modulated by the cytoplasmic proteasome system. In addition, we have established that Akt/protein kinase B is a positive regulator of p300, while the B563 regulatory subunit of protein phosphatase 2A (PP2A) is a negative regulator. Our works have provided novel insights into how cellular trafficking and spatial redistribution control the protein stability and transcriptional activity of p300. Recently, we found that p300 is regulated in a lineage specific manner during myogenic differentiation. As such, we intend to leverage our findings to define the molecular mechanisms underlying lineage-specific p300 regulation associated with myoblast differentiation. We hypothesize that reversible phosphorylaton plays critical roles in the control of p300 activity, which in turn modulates p300 function during myoblast differentiation, and propose to define the functional interactions of the B563 regulatory subunit, Akt, and additional kinases with p300, in milieu of myoblast differentiation. We will survey p300 phosphorylation profiles and delineate the regulation of p300 by reversible phosphorylation with gain- and loss-of-function approaches. We will generate mutants that mimic candidate site phosphorylation and produce antibodies against site-specific phosphorylation to study the impact of reversible phosphorylation on p300 stability and transcriptional activity. Our goals are to determine the molecular basis for posttranslational modification-mediated p300 regulation and their consequences on the function of p300 as a HAT, a scaffold or a bridge on chromatin with respect to gene transcription. We will define the molecular mechanisms underlying different signaling pathways and p300 regulation pertinent to cellular differentiation. Our long term goals are to determine how these regulatory mechanisms control p300-dependent gene expression from intracellular trafficking, protein turnover to transcriptional activation, and to determine the molecular basis for p300 function in a network biology fashion. This research program is founded upon our recent findings and anchored on the p300 which is essential for different cellular processes and skeletal myogenesis in vivo. Many HQP will be trained through the proposed research as shown by our proven track record, and as a result, they will be well equipped to navigate into their own career path successfully and to contribute to our research field and different Canadian organizations significantly.