The SCFFbw7 Substrate Cycle: phosphodegron processing and nucleolar translocation

 描述（由申请人提供）：SCFFbw 7是一种多亚基泛素连接酶，靶向许多重要的细胞调节蛋白，用于泛素介导的蛋白水解。这些底物的靶向通过特异性因子Fbw 7介导，其识别称为CPD的略微退化的共有序列（对于Cdc 4磷酸降解决定子; Cdc 4是Fbw 7的酵母直系同源物）。虽然个体CPD之间存在显著变化，但共识在要求位置0处的磷酸苏氨酸（不太常见的是磷酸丝氨酸）和位置+1处的脯氨酸方面是不变的。然而，对于SCFFbw 7的大多数脊椎动物底物，在位置+2处存在第二脯氨酸，这是在SCFCdc 4的酵母底物中未观察到的序列基序。然而，SCFCdc 4是主管遍在晚期哺乳动物基板含有脯氨酸-脯氨酸序列。这一观察结果表明，脯氨酸-脯氨酸基序已经进化到执行额外的功能。我们已经表明，一个基板，人类细胞周期蛋白E1，虽然第二脯氨酸是不重要的泛素化和蛋白水解，本身，它确实有一个深刻的影响，细胞周期蛋白E营业额的某些方面。具体地说，这第二个脯氨酸是磷酸化细胞周期蛋白E转位到细胞周期蛋白E中所必需的。 核仁，其中通过由核仁特异性Fbw 7同种型Fbw 7构成的SCF进行泛素化。核仁易位需要核Fbw 7同种型Fbw 7a和脯氨酰肽基顺反异构酶Pin 1，它们合作进行脯氨酸-脯氨酸键的非经典异构化，而不是磷酸苏氨酸-脯氨酸键，这是Pin 1的典型情况。拟议研究的总体目标是了解脯氨酸-脯氨酸键与Fbw 7a和Pin 1一起进行顺反异构化并促进细胞周期蛋白E核仁易位的机制，并确定为什么泛素介导的细胞周期蛋白E蛋白水解已演变为纳入核仁易位。最后，我们将确定是否其他SCFFbw 7基板，含有脯氨酸-脯氨酸序列在其磷酸降解决定子的同等位置也表现出Pin 1依赖的异构化和核仁易位耦合到他们的降解。

 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.