Identification of a nuclear export signal in the catalytic subunit of AMP-activated protein kinase.
Identification of a nuclear export signal in the catalytic subunit of AMP-activated protein kinase.
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DOI：
10.1091/mbc.e10-04-0347
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发表时间：
2010-10-01
影响因子：
3.3
通讯作者：
Brenman JE
中科院分区：
文献类型：
--
作者：
Kazgan N;Williams T;Forsberg LJ;Brenman JE
在这项研究中，我们利用遗传学和细胞生物学的方法来评估AMPKα c -末端的潜在功能。我们在体内发现了AMPKα羧基末端氨基酸的一个关键新功能，它影响AMPKα亚细胞定位、磷酸化和最终的生物体活力。代谢调节剂amp活化蛋白激酶（AMPK）通过调节参与能量产生和消耗途径的蛋白质来维持细胞稳态。尽管AMPK磷酸化的靶点包括细胞质和核蛋白，但调控AMPK定位的确切机制，以及其进入这些底物的途径尚不清楚。我们鉴定出高度保守的羧基端疏水氨基酸，它们在AMPK催化亚基（AMPKα）中作为leptomycin b敏感的、crm1依赖的核输出序列（NES）。当该序列被修饰时，AMPKα通过依赖于ran的进口途径显示出增加的核定位。细胞质定位可以通过将定义明确的snurportin-1或蛋白激酶A抑制剂（PKIA） crm1结合NESs取代到AMPKα中来恢复。我们在体内证明了AMPKα羧基末端NES的功能需求，因为表达AMPKα缺乏这种NES的转基因果蝇不能挽救AMPKα零突变体果蝇的致命性，并且在热休克应激下表现出激活环磷酸化降低。这个被截断的蛋白被隔离在细胞核中，在关键的Thr172激活残基上显示出高度降低的磷酸化，这表明AMPK激活主要发生在非胁迫条件下的细胞质中。因此，通过其c端NES调节crm1介导的AMPKα输出为细胞调节AMPK活性和定位提供了一种额外的机制。
In this study, we utilized genetic and cell biological approaches to evaluate potential functions for the AMPKα C-terminus. We identify a critical new function for the carboxy-terminal amino acids of AMPKα in vivo, which affects AMPKα subcellular localization, phosphorylation, and ultimately organismal viability. The metabolic regulator AMP-activated protein kinase (AMPK) maintains cellular homeostasis through regulation of proteins involved in energy-producing and -consuming pathways. Although AMPK phosphorylation targets include cytoplasmic and nuclear proteins, the precise mechanisms that regulate AMPK localization, and thus its access to these substrates, are unclear. We identify highly conserved carboxy-terminal hydrophobic amino acids that function as a leptomycin B–sensitive, CRM1-dependent nuclear export sequence (NES) in the AMPK catalytic subunit (AMPKα). When this sequence is modified AMPKα shows increased nuclear localization via a Ran-dependent import pathway. Cytoplasmic localization can be restored by substituting well-defined snurportin-1 or protein kinase A inhibitor (PKIA) CRM1-binding NESs into AMPKα. We demonstrate a functional requirement in vivo for the AMPKα carboxy-terminal NES, as transgenic Drosophila expressing AMPKα lacking this NES fail to rescue lethality of AMPKα null mutant flies and show decreased activation loop phosphorylation under heat-shock stress. Sequestered to the nucleus, this truncated protein shows highly reduced phosphorylation at the key Thr172 activation residue, suggesting that AMPK activation predominantly occurs in the cytoplasm under unstressed conditions. Thus, modulation of CRM1-mediated export of AMPKα via its C-terminal NES provides an additional mechanism for cells to use in the regulation of AMPK activity and localization.