Chen X, Sahasrabuddhe A A, Szankasi P, Chung F, Basrur V, Rangnekar V M, Pagano M, Lim M S, Elenitoba-Johnson K S J
Department of Pathology, University of Michigan Medical School, 2037 BSRB 109 Zina Pitcher Place, Ann Arbor, MI 48109, USA.
ARUP Laboratories, 500 Chipeta Way, Salt Lake City, UT 84108, USA.
Cell Death Differ. 2014 Oct;21(10):1535-45. doi: 10.1038/cdd.2014.92. Epub 2014 Jul 4.
Prostate apoptosis response protein 4 (Par-4) also known as PRKC apoptosis WT1 regulator is a tumor suppressor that selectively induces apoptosis in cancer cells. However, its post-translational regulation by ubiquitin-mediated proteolysis and the cellular machinery that is responsible for its proteasomal degradation are unknown. Using immunopurification and an unbiased mass spectrometry-based approach, we show that Par-4 interacts with the SPRY-domain containing E3 ubiquitin ligase Fbxo45 through a short consensus sequence motif. Fbxo45 interacts with Par-4 in the cytoplasm and mediates its ubiquitylation and proteasomal degradation. Fbxo45 silencing results in stabilization of Par-4 with increased apoptosis. Importantly, a Par-4 mutant that is unable to bind Fbxo45 is stabilized and further enhances staurosporine-induced apoptosis. Co-expression of Fbxo45 with Par-4 protects cancer cells against Par-4-induced apoptosis. Our studies reveal that Fbxo45 is the substrate-receptor subunit of a functional E3 ligase for Par-4 that has a critical role in cancer cell survival.
前列腺凋亡反应蛋白4(Par-4),也被称为PRKC凋亡WT1调节因子,是一种肿瘤抑制因子,可选择性地诱导癌细胞凋亡。然而,其通过泛素介导的蛋白水解作用进行的翻译后调控以及负责其蛋白酶体降解的细胞机制尚不清楚。我们利用免疫纯化和基于无偏差质谱的方法,发现Par-4通过一个短共有序列基序与含SPRY结构域的E3泛素连接酶Fbxo45相互作用。Fbxo45在细胞质中与Par-4相互作用,并介导其泛素化和蛋白酶体降解。Fbxo45沉默导致Par-4稳定,同时凋亡增加。重要的是,一个无法与Fbxo45结合的Par-4突变体被稳定下来,并进一步增强了星形孢菌素诱导的凋亡。Fbxo45与Par-4共表达可保护癌细胞免受Par-4诱导的凋亡。我们的研究表明,Fbxo45是Par-4功能性E3连接酶的底物受体亚基,在癌细胞存活中起关键作用。