Background:Oncogenic mutations in theKRASgene are detected in >90% of pancreatic cancers (PC). In genetically engineered mouse models of PC, oncogenicKRASdrives the formation of precursor lesions and their progression to invasive PC. The Yes-associated Protein (YAP) is a transcriptional coactivator required for transformation by the RAS oncogenes and the development of PC. In Ras-driven tumors, YAP can also substitute for oncogenic KRAS to drive tumor survival after the repression of the oncogene. Ras oncoproteins exert their transforming properties through their downstream effectors, including the PI3K kinase, Rac1 GTPase, and MAPK pathways.Methods:To identify Ras effectors that regulate YAP, YAP levels were measured in PC cells exposed to inhibitors of oncogenic K-Ras and its effectors.Results:In PC cells, the inhibition of Rac1 leads to a time-dependent decline in YAP protein, which could be blocked by proteosome inhibitor MG132. This YAP degradation after Rac1 inhibition was observed in a range of cell lines using different Rac1 inhibitors, Rac1 siRNA, or expression of dominant negative Rac1T17Nmutant. Several E3 ubiquitin ligases, including SCFβTrCP, regulate YAP protein stability. To be recognized by this ligase, the βTrCP degron of YAP (amino acid 383–388) requires its phosphorylation by casein kinase 1 at Ser384 and Ser387, but these events must first be primed by the phosphorylation of Ser381 by LATS1/2. Using Flag-tagged mutants of YAP, we show that YAP degradation after Rac1 inhibition requires the integrity of this degron and is blocked by the silencing of βTrCP1/2 and by the inhibition of casein kinase 1. Unexpectedly, YAP degradation after Rac1 inhibition was still observed after the silencing of LATS1/2 or in cells carrying a LATS1/2 double knockout.Conclusions:These results reveal Rac1 as an oncogenic KRAS effector that contributes to YAP stabilization in PC cells. They also show that this regulation of YAP by Rac1 requires the SCFβTrCPligase but occurs independently of the LATS1/2 kinases.
背景：超过90%的胰腺癌（PC）中可检测到KRAS基因的致癌突变。在胰腺癌的基因工程小鼠模型中，致癌KRAS驱动前体病变的形成及其向侵袭性胰腺癌的进展。Yes相关蛋白（YAP）是RAS癌基因转化和胰腺癌发展所必需的转录共激活因子。在Ras驱动的肿瘤中，YAP也可以在癌基因被抑制后替代致癌KRAS来驱动肿瘤存活。Ras癌蛋白通过其下游效应因子（包括PI3K激酶、Rac1 GTP酶和MAPK通路）发挥其转化特性。 方法：为鉴定调控YAP的Ras效应因子，测量了暴露于致癌K-Ras及其效应因子抑制剂的PC细胞中的YAP水平。 结果：在PC细胞中，抑制Rac1会导致YAP蛋白呈时间依赖性下降，这一过程可被蛋白酶体抑制剂MG132阻断。使用不同的Rac1抑制剂、Rac1 siRNA或表达显性负性Rac1T17N突变体，在一系列细胞系中均观察到Rac1抑制后的YAP降解。包括SCFβTrCP在内的几种E3泛素连接酶调控YAP蛋白稳定性。YAP的βTrCP降解基序（氨基酸383-388）需被酪蛋白激酶1在Ser384和Ser387位点磷酸化才能被该连接酶识别，但这些事件首先需要LATS1/2对Ser381进行磷酸化启动。通过使用Flag标记的YAP突变体，我们发现Rac1抑制后的YAP降解需要该降解基序的完整性，并且可被βTrCP1/2沉默和酪蛋白激酶1抑制所阻断。出乎意料的是，在沉默LATS1/2后或在携带LATS1/2双敲除的细胞中，仍能观察到Rac1抑制后的YAP降解。 结论：这些结果表明Rac1作为致癌KRAS效应因子，有助于PC细胞中YAP的稳定。研究还显示，Rac1对YAP的这种调控需要SCFβTrCP连接酶，但独立于LATS1/2激酶发生。
Rac1 GTPase Regulates the βTrCP-Mediated Proteolysis of YAP Independently of the LATS1/2 Kinases
肿瘤(癌症)患者之家