Blundon Malachi A, Schlesinger Danielle R, Parthasarathy Amritha, Smith Samantha L, Kolev Hannah M, Vinson David A, Kunttas-Tatli Ezgi, McCartney Brooke M, Minden Jonathan S
Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, PA 15213, USA.
Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, PA 15213, USA
Development. 2016 Jul 15;143(14):2629-40. doi: 10.1242/dev.130567. Epub 2016 Jun 10.
Wnt signaling generates patterns in all embryos, from flies to humans, and controls cell fate, proliferation and metabolic homeostasis. Inappropriate Wnt pathway activation results in diseases, including colorectal cancer. The adenomatous polyposis coli (APC) tumor suppressor gene encodes a multifunctional protein that is an essential regulator of Wnt signaling and cytoskeletal organization. Although progress has been made in defining the role of APC in a normal cellular context, there are still significant gaps in our understanding of APC-dependent cellular function and dysfunction. We expanded the APC-associated protein network using a combination of genetics and a proteomic technique called two-dimensional difference gel electrophoresis (2D-DIGE). We show that loss of Drosophila Apc2 causes protein isoform changes reflecting misregulation of post-translational modifications (PTMs), which are not dependent on β-catenin transcriptional activity. Mass spectrometry revealed that proteins involved in metabolic and biosynthetic pathways, protein synthesis and degradation, and cell signaling are affected by Apc2 loss. We demonstrate that changes in phosphorylation partially account for the altered PTMs in APC mutants, suggesting that APC mutants affect other types of PTM. Finally, through this approach Aminopeptidase P was identified as a new regulator of β-catenin abundance in Drosophila embryos. This study provides new perspectives on the cellular effects of APC that might lead to a deeper understanding of its role in development.
Wnt信号通路在从果蝇到人类的所有胚胎中形成模式，并控制细胞命运、增殖和代谢稳态。Wnt信号通路的不适当激活会导致包括结直肠癌在内的疾病。腺瘤性息肉病 coli（APC）肿瘤抑制基因编码一种多功能蛋白，该蛋白是Wnt信号通路和细胞骨架组织的重要调节因子。尽管在确定APC在正常细胞环境中的作用方面已经取得了进展，但我们对APC依赖的细胞功能和功能障碍的理解仍存在重大差距。我们结合遗传学和一种称为二维差异凝胶电泳（2D-DIGE）的蛋白质组学技术扩展了APC相关蛋白网络。我们发现果蝇Apc2的缺失会导致蛋白质异构体变化，这反映了翻译后修饰（PTM）的失调，而这种失调不依赖于β-连环蛋白的转录活性。质谱分析表明，参与代谢和生物合成途径、蛋白质合成和降解以及细胞信号传导的蛋白质受到Apc2缺失的影响。我们证明磷酸化的变化部分解释了APC突变体中PTM的改变，这表明APC突变体影响其他类型的PTM。最后，通过这种方法，氨肽酶P被确定为果蝇胚胎中β-连环蛋白丰度的新调节因子。这项研究为APC的细胞效应提供了新的视角，可能会加深我们对其在发育中作用的理解。
