Molecular Pharmacology and Pathology Program, Department of Pathology and Bosch Institute, Medical Foundation Building (K25), University of Sydney, Sydney, New South Wales 2006, Australia.
Molecular Pharmacology and Pathology Program, Department of Pathology and Bosch Institute, Medical Foundation Building (K25), University of Sydney, Sydney, New South Wales 2006, Australia.
Biochim Biophys Acta Mol Basis Dis. 2019 Jun 1;1865(6):1361-1378. doi: 10.1016/j.bbadis.2019.02.008. Epub 2019 Feb 11.
N-myc downstream regulated gene 1 (NDRG1) is an intriguing metastasis suppressor protein, which plays an important role in suppressing multiple oncogenic signaling pathways. Interestingly, multiple isoforms of NDRG1 have been identified, although the molecular mechanisms involved in their generation remains elusive. Herein, we demonstrate the role of two mechanisms involving autophagic and proteasomal machinery as part of an intricate system to generate different NDRG1 isoforms. Examining multiple pancreatic cancer cell-types using immunoblotting demonstrated three major isoforms of NDRG1 at approximately 41-, 46- and 47-kDa. The top NDRG1 band at 47-kDa was shown to be processed by the proteasome, followed by autophagic metabolism of the middle NDRG1 band at 46-kDa. The role of the proteasomal and autophagic pathways in NDRG1 processing was further confirmed by co-localization analysis of confocal images using PSMD9 and LC3 as classical markers of these respective pathways. All NDRG1 isoforms were demonstrated to be, at least in part, phosphorylated forms of the protein. Inhibition of two well-characterized upstream kinases of NDRG1, namely GSK3β and SGK1, resulted in decreased levels of the top NDRG1 band. Studies demonstrated that inhibition of GSK3β decreased levels of the top 47-kDa NDRG1 band, independent of its kinase activity, and this effect was not mediated via the proteasomal pathway. In contrast, the decrease in the top NDRG1 band at 47-kDa after SGK1 inhibition, was due to suppression of its kinase activity. Overall, these studies elucidated the complex and intricate regulatory pathways involving both proteasomal and autophagic processing of the metastasis suppressor protein, NDRG1.
N- MYC 下游调节基因 1（NDRG1）是一种有趣的转移抑制蛋白，在抑制多种致癌信号通路中发挥重要作用。有趣的是，已经鉴定出多个 NDRG1 同工型，尽管其产生的分子机制仍不清楚。在此，我们证明了两种机制的作用，包括自噬和蛋白酶体机制，作为产生不同 NDRG1 同工型的复杂系统的一部分。使用免疫印迹法检查多种胰腺癌细胞系，证明 NDRG1 有三个主要同工型，大约为 41、46 和 47 kDa。47 kDa 的 NDRG1 主要条带被证明被蛋白酶体处理，然后是 46 kDa 的中间 NDRG1 条带的自噬代谢。通过使用 PSMD9 和 LC3 作为这些各自途径的经典标志物对共焦图像进行共定位分析，进一步证实了蛋白酶体和自噬途径在 NDRG1 加工中的作用。所有 NDRG1 同工型均被证明至少部分是该蛋白的磷酸化形式。两种 NDRG1 的特征性上游激酶 GSK3β 和 SGK1 的抑制导致顶部 NDRG1 条带的水平降低。研究表明，抑制 GSK3β 降低了顶部 47 kDa NDRG1 条带的水平，而与激酶活性无关，并且这种作用不是通过蛋白酶体途径介导的。相反，SGK1 抑制后顶部 NDRG1 条带在 47 kDa 处的减少是由于其激酶活性的抑制。总体而言，这些研究阐明了涉及转移抑制蛋白 NDRG1 的蛋白酶体和自噬加工的复杂而复杂的调节途径。
