Co-expression of LKB1, MO25α and STRADα in bacteria yield the functional and active heterotrimeric complex

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 M025 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 M025 alpha can be expressed in soluble form, whereas the other two, LKB1 and STRAD alpha are found almost exclusively in inclusion bodies. However, using the tricistronic vector system, functional LKB1-MO25 alpha-STRAD alpha 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, MO25 alpha and STRAD alpha 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 STRADi, both strongly autophosphorylate in vitro. Moreover, recombinant LKB1 complex activates AMPK by phosphorylation of the a-subunit at the Thr-172 site as shown (i) by Western blotting using phospho-specific antibodies after LKB1-dependent phosphorylation, (ii) by LKBIdependent incorporation of radioactive phosphate into the a-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.