Neumann Dietbert, Suter Marianne, Tuerk Roland, Riek Uwe, Wallimann Theo
ETH Zurich, Institute of Cell Biology, HPM D23, Schafmattstr 18, Zurich, Switzerland.
Mol Biotechnol. 2007 Jul;36(3):220-31. doi: 10.1007/s12033-007-0029-x.
The tumour suppressor LKB1 plays a critical role in cell proliferation, polarity and energy metabolism. LKB1 is a Ser/Thr protein kinase that is associated with STRAD and MO25 in vivo. Here, we describe the individual expression of the three components of the LKB1 complex using monocistronic vectors and their co-expression using tricistronic vectors that were constructed from monocistronic vectors using a fully modular cloning approach. The data show that among the three individually expressed components of the LKB1 complex, only MO25alpha can be expressed in soluble form, whereas the other two, LKB1 and STRADalpha are found almost exclusively in inclusion bodies. However, using the tricistronic vector system, functional LKB1-MO25alpha-STRADalpha complex was expressed and purified from soluble extracts by sequential immobilized-metal affinity and heparin chromatography, as shown by Western blotting using specific antibodies. In size exclusion chromatography, MO25alpha and STRADalpha exactly co-elute with LKB1 with an apparent molecular weight of the heterotrimeric complex of 160 kDa. The specific activity in the peak fraction of the size exclusion chromatography was 250 U/mg at approximately 25% purity. As shown by autoradiography, LKB1 and STRADalpha, both strongly autophosphorylate in vitro. Moreover, recombinant LKB1 complex activates AMPK by phosphorylation of the alpha-subunit at the Thr-172 site as shown (i) by Western blotting using phospho-specific antibodies after LKB1-dependent phosphorylation, (ii) by LKB1-dependent incorporation of radioactive phosphate into the alpha-subunit of kinase dead AMPK heterotrimer, and (iii) by activity determination of AMPK. Functional mammalian LKB1 complex is constitutively active, and when enriched from bacteria should prove to be a valuable tool for studying its molecular function and regulation.
肿瘤抑制因子LKB1在细胞增殖、极性和能量代谢中发挥着关键作用。LKB1是一种丝氨酸/苏氨酸蛋白激酶，在体内与STRAD和MO25相关联。在此，我们使用单顺反子载体描述了LKB1复合物三个组分的单独表达，并使用通过完全模块化克隆方法从单顺反子载体构建的三顺反子载体描述了它们的共表达。数据显示，在LKB1复合物单独表达的三个组分中，只有MO25α能够以可溶形式表达，而其他两个组分LKB1和STRADα几乎完全存在于包涵体中。然而，使用三顺反子载体系统，功能性LKB1-MO25α-STRADα复合物得以表达，并通过连续的固定金属亲和层析和肝素层析从可溶提取物中纯化出来，使用特异性抗体进行的蛋白质免疫印迹法证实了这一点。在尺寸排阻色谱中，MO25α和STRADα与LKB1精确共洗脱，异源三聚体复合物的表观分子量为160 kDa。尺寸排阻色谱峰馏分中的比活性在纯度约为25%时为250 U/mg。如放射自显影所示，LKB1和STRADα在体外均强烈自磷酸化。此外，重组LKB1复合物通过在Thr-172位点磷酸化α亚基来激活AMPK，这通过以下方式得以证明：(i) 在LKB1依赖性磷酸化后使用磷酸化特异性抗体进行蛋白质免疫印迹法；(ii) 通过LKB1依赖性将放射性磷酸盐掺入激酶失活的AMPK异源三聚体的α亚基中；以及(iii) 通过测定AMPK的活性。功能性哺乳动物LKB1复合物具有组成性活性，当从细菌中富集时，应该会被证明是研究其分子功能和调控的有价值工具。
