The SCFFbw7 Substrate Cycle: phosphodegron processing and nucleolar translocation

 描述(由申请人提供)：SCFFbw7是一种多亚基泛素连接酶，针对一些重要的细胞调控蛋白进行泛素介导的蛋白分解。这些底物的靶向是通过特异性因子Fbw7介导的，它识别被称为CPD的某种简并的共识序列(对于CDC4磷酸脱氧核糖核酸；CDC4是Fbw7的酵母同源基因)。尽管不同的CPD之间存在显著的差异，但共识是一致的，要求0位的磷酸苏氨酸(较少见的是磷酸丝氨酸)和+1位的脯氨酸。然而，对于大多数脊椎动物底物SCFFbw7，在+2位存在第二个Pro，这是SCFCDC4的酵母底物中没有观察到的序列基序。然而，SCFCDC4能适应哺乳动物晚期底物中普遍存在的含有Pro-Pro序列的底物。这一观察表明，Pro-Pro基序已经进化为执行另一种功能。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泛素化和蛋白分解并不重要，但它本身对细胞周期蛋白E的某些方面的周转有深远的影响。具体地说，这第二个脯氨酸是将磷酸化的细胞周期蛋白E转位到 核仁，其中泛素化是由由核仁特异的Fbw7亚型Fbw7组成的SCF完成的。核仁易位需要核Fbw7异构体Fbw7a和Pro基肽顺式-反式异构酶Pin1，这两种异构酶协同进行Pro-Pro键的非常规异构化，而不是像Pin1典型的情况那样进行磷酸苏氨酸-Pro键的异构化。这项拟议研究的总体目标是了解与Fbw7a和Pin1结合的Pro-Pro键经历顺反异构化并促进Cyclin E核仁易位的机制，并确定为什么泛素介导的Cyclin E蛋白分解进化为包括核仁易位。最后，我们将确定是否其他SCFFbw7底物在其磷酸降解物中的相同位置包含Pro-Pro序列也表现出依赖Pin1的异构化和与其降解相耦合的核仁移位。

 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.