The SCFFbw7 Substrate Cycle: phosphodegron processing and nucleolar translocation

SCFFbw7 底物循环：磷酸化降解子加工和核仁转位

• 批准号: 8912922
• 负责人: Steven I Reed
• 金额: $ 39.14万
• 依托单位: SCRIPPS RESEARCH INSTITUTE, THE
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-03-01 至 2019-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8912922
• 关键词: Binding; Cell Line; Cell Nucleolus; Cells; Clustered Regularly Interspaced Short Palindromic Repeats; Complex; Consensus; Consensus Sequence; Coupled; Cyclin E; Environment; Event; Exclusion; Exhibits; Goals; Human; Individual; JUN gene; Knock-in Mouse; Ligase; Malignant Neoplasms; Mediating; Molecular; Mutate; Nuclear; Orthologous Gene; Pathway interactions; Peptide Hydrolases; Peptidylprolyl Isomerase; Phosphoserine; Phosphothreonine; Positioning Attribute; Process; Proline; Property; Protein Isoforms; Proteolysis; Regulation; Research; Specificity; Substrate Cycling; Technology; Testing; Ubiquitin; Ubiquitin-mediated Proteolysis Pathway; Variant; Yeasts; c-myc Genes; cis trans isomerization; design; genetic regulatory protein; human CCNE1 protein; mutant; prolyl-proline; public health relevance; research study; ubiquitin ligase

 DESCRIPTION (provided by applicant): SCFFbw7 is a multisubunit ubiquitin ligase that targets number important cellular regulatory proteins for ubiquitin mediated proteolysis. Targeting of these substrates is mediated via the specificity factor Fbw7, which recognizes a somewhat degenerate consensus sequence known as the CPD (for Cdc4 PhosphoDegron; Cdc4 is the yeast ortholog of Fbw7). Although there is significant variation between individual CPDs, the consensus is invariant in demanding a phosphothreonine (less frequently a phosphoserine) at position 0 and a proline at position +1. However, for most vertebrate substrates of SCFFbw7, there is a second proline at position +2, a sequence motif not observed in yeast substrates of SCFCdc4. Nevertheless, SCFCdc4 is competent to ubiquity late mammalian substrates containing the proline-proline sequence. This observation suggests that the proline-proline motif has evolved to carry out an additional function. We have shown for one substrate, human cyclin E1, that although the second proline is not important for ubiquitylation and proteolysis, per se, it does have a profound effect on certain aspects of cyclin E turnover. Specifically, this second proline is required for translocation of phosphorylated cyclin E into the nucleolus where ubiquitylation is carried out by SCF constituted with a nucleolus-specific Fbw7 isoform, Fbw7. Nucleolar translocation requires both the nuclear Fbw7 isoform, Fbw7a, and the prolyl peptidyl cis-trans isomerase Pin1, which collaborate to carry out a non-cannonical isomerization of the proline-proline bond rather than the phosphothreonine-proline bond, as is typically the case for Pin1. The general goal of the proposed research is to understand the mechanism whereby a proline-proline bond in conjunction with Fbw7a and Pin1 undergoes cis-trans isomerization and promotes cyclin E nucleolar translocation, and to determine why ubiquitin-mediated proteolysis of cyclin E has evolved to incorporate nucleolar translocation. Finally, we will determine whether other SCFFbw7 substrates that contain a proline-proline sequence at an equivalent position in their phosphodegrons also exhibit Pin1- dependent isomerization and nucleolar translocation coupled to their degradation.

 描述（由申请人提供）：SCFFbw7 是一种多亚基泛素连接酶，针对泛素介导的蛋白水解作用的许多重要细胞调节蛋白。这些底物的靶向是通过特异性因子 Fbw7 介导的，该因子识别称为 CPD 的稍微简并的共有序列（对于 Cdc4 PhosphoDegron；Cdc4 是 Fbw7 的酵母直系同源物）。尽管各个 CPD 之间存在显着差异，但共识是在位置 0 处要求磷酸苏氨酸（较少见的是磷酸丝氨酸）和在位置 +1 处要求脯氨酸。然而，对于大多数脊椎动物的 SCFFbw7 底物，在位置 +2 处有第二个脯氨酸，这是在 SCFCdc4 的酵母底物中未观察到的序列基序。然而，SCFCdc4 能够普遍存在含有脯氨酸-脯氨酸序列的晚期哺乳动物底物。这一观察结果表明脯氨酸-脯氨酸基序已经进化以执行额外的功能。我们已经证明，对于一种底物，人细胞周期蛋白 E1，虽然第二个脯氨酸对于泛素化和蛋白水解并不重要，但它本身确实对细胞周期蛋白 E 周转的某些方面具有深远的影响。具体来说，第二个脯氨酸是磷酸化细胞周期蛋白 E 易位至 核仁中的泛素化是由核仁特异性 Fbw7 同种型 Fbw7 构成的 SCF 进行的。核仁易位需要核 Fbw7 亚型 Fbw7a 和脯氨酰肽基顺反异构酶 Pin1，它们协同进行脯氨酸-脯氨酸键的非规范异构化，而不是像 Pin1 的典型情况那样进行磷酸苏氨酸-脯氨酸键的异构化。本研究的总体目标是了解脯氨酸-脯氨酸键与 Fbw7a 和 Pin1 结合发生顺反异构化并促进细胞周期蛋白 E 核仁转位的机制，并确定为什么泛素介导的细胞周期蛋白 E 蛋白水解已进化为合并核仁转位。最后，我们将确定在其磷酸降解子中的等效位置含有脯氨酸-脯氨酸序列的其他 SCFFbw7 底物是否也表现出与降解相关的 Pin1 依赖性异构化和核仁易位。