Department of Physiology and Developmental Biology, Brigham Young University, Provo, Utah, USA.
J Appl Physiol (1985). 2011 Dec;111(6):1622-8. doi: 10.1152/japplphysiol.00160.2011. Epub 2011 Sep 8.
In liver, the AMP-activated protein kinase kinase (AMPKK) complex was identified as the association of liver kinase B1 (LKB1), mouse protein 25 (MO25α/β), and Ste-20-related adaptor protein (STRADα/β); however, this complex has yet to be characterized in skeletal muscle. We demonstrate the expression of the LKB1-MO25-STRAD complex in skeletal muscle, confirm the absence of mRNA splice variants, and report the relative mRNA expression levels of these proteins in control and muscle-specific LKB1 knockout (LKB1(-/-)) mouse muscle. LKB1 detection in untreated control and LKB1(-/-) muscle lysates revealed two protein bands (50 and 60 kDa), although only the heavier band was diminished in LKB1(-/-) samples [55 ± 2.5 and 13 ± 1.5 arbitrary units (AU) in control and LKB1(-/-), respectively, P < 0.01], suggesting that LKB1 is not represented at 50 kDa, as previously cited. The 60-kDa LKB1 band was further confirmed following purification using polyethylene glycol (43 ± 5 and 8.4 ± 4 AU in control and LKB1(-/-), respectively, P < 0.01) and ion-exchange fast protein liquid chromatography. Mass spectrometry confirmed LKB1 protein detection in the 60-kDa protein band, while none was detected in the 50-kDa band. Coimmunoprecipitation assays demonstrated LKB1-MO25-STRAD complex formation. Quantitative PCR revealed significantly reduced LKB1, MO25α, and STRADβ mRNA in LKB1(-/-) muscle. These findings demonstrate that the LKB1-MO25-STRAD complex is the principal AMPKK in skeletal muscle.
在肝脏中，AMP 激活的蛋白激酶激酶（AMPKK）复合物被鉴定为肝激酶 B1（LKB1）、小鼠蛋白 25（MO25α/β）和 STE20 相关适应蛋白（STRADα/β）的复合物；然而，该复合物在骨骼肌中尚未得到表征。我们证明了 LKB1-MO25-STRAD 复合物在骨骼肌中的表达，证实了其 mRNA 剪接变体的缺失，并报告了这些蛋白质在对照和肌肉特异性 LKB1 敲除（LKB1(-/-)）小鼠肌肉中的相对 mRNA 表达水平。在未经处理的对照和 LKB1(-/-)肌肉裂解物中检测到 LKB1 时，发现有两条蛋白带（50 和 60 kDa），尽管只有较重的带在 LKB1(-/-)样本中减少[分别为 55 ± 2.5 和 13 ± 1.5 任意单位（AU），P < 0.01]，表明 LKB1 不存在于之前引用的 50 kDa 处。使用聚乙二醇（43 ± 5 和 8.4 ± 4 AU 分别在对照和 LKB1(-/-)中，P < 0.01）和离子交换快速蛋白液相色谱进一步证实了 60 kDa LKB1 带的纯化。质谱分析证实了在 60 kDa 蛋白带中检测到 LKB1 蛋白，而在 50 kDa 带中未检测到。免疫共沉淀实验证明了 LKB1-MO25-STRAD 复合物的形成。定量 PCR 显示 LKB1(-/-)肌肉中的 LKB1、MO25α 和 STRADβ mRNA 显著减少。这些发现表明，LKB1-MO25-STRAD 复合物是骨骼肌中主要的 AMPKK。
