AMP-activated protein kinase (AMPK) is a key regulator of cellular energy homeostasis, acting as a sensor of energy and nutrient status. As such, AMPK is considered a promising drug target for treatment of medical conditions particularly associated with metabolic dysfunctions. To better understand the downstream effectors and physiological consequences of AMPK activation, we have employed a chemical genetic screen in mouse primary hepatocytes in an attempt to identify novel AMPK targets. Treatment of hepatocytes with a potent and specific AMPK activator 991 resulted in identification of 65 proteins phosphorylated upon AMPK activation, which are involved in a variety of cellular processes such as lipid/glycogen metabolism, vesicle trafficking, and cytoskeleton organisation. Further characterisation and validation using mass spectrometry followed by immunoblotting analysis with phosphorylation site-specific antibodies identified AMPK-dependent phosphorylation of Gapex-5 (also known as GTPase-activating protein and VPS9 domain-containing protein 1 (GAPVD1)) on Ser902 in hepatocytes and starch-binding domain 1 (STBD1) on Ser175 in multiple cells/tissues. As new promising roles of AMPK as a key metabolic regulator continue to emerge, the substrates we identified could provide new mechanistic and therapeutic insights into AMPK-activating drugs in the liver.

中文翻译：

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化学遗传筛选将Gapex-5 / GAPVD1和STBD1鉴定为新型AMPK底物。

AMP激活的蛋白激酶（AMPK）是细胞能量稳态的关键调节剂，可作为能量和营养状况的传感器。因此，AMPK被认为是用于治疗特别是与代谢功能障碍有关的医学病症的有希望的药物靶标。为了更好地了解AMPK激活的下游效应子和生理后果，我们在小鼠原代肝细胞中进行了化学遗传筛选，以尝试鉴定新型AMPK靶标。用有效的和特定的AMPK激活剂991处理肝细胞导致鉴定到65种在AMPK激活后被磷酸化的蛋白，这些蛋白参与多种细胞过程，例如脂质/糖原代谢，囊泡运输和细胞骨架组织。使用质谱的进一步表征和验证，然后使用磷酸化位点特异性抗体进行免疫印迹分析，确定了肝细胞和肝癌细胞中Ser902上AMPK依赖的Gapex-5（也称为GTPase激活蛋白和含VPS9域的蛋白1（GAPVD1））的AMPK依赖性磷酸化。多个细胞/组织中Ser175上的淀粉结合结构域1（STBD1）。随着AMPK作为关键代谢调节剂的新有希望的作用不断出现，我们确定的底物可以为肝脏中AMPK活化药物提供新的机理和治疗见解。