Co-expression of LKB1, MO25alpha and STRADalpha 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 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.
Neumann
Dietbert
D
ETH Zurich, Institute of Cell Biology, HPM D23, Schafmattstr 18, Zurich, Switzerland. dietbert.neumann@cell.biol.ethz.ch
Suter
Marianne
M
Tuerk
Roland
R
Riek
Uwe
U
Wallimann
Theo
T
D000091162:AMP-Activated Protein Kinase Kinases; D055372:AMP-Activated Protein Kinases; D048868:Adaptor Proteins, Signal Transducing / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D033942:Adaptor Proteins, Vesicular Transport / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D000818:Animals; D002135:Calcium-Binding Proteins; D002850:Chromatography, Gel; D004926:Escherichia coli / Q000235:genetics*; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D002479:Inclusion Bodies / Q000737:chemistry; D018832:Molecular Chaperones / Q000096:biosynthesis / Q000235:genetics; D009097:Multienzyme Complexes / Q000737:chemistry / Q000378:metabolism; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D012995:Solubility
D048868:Adaptor Proteins, Signal Transducing; D033942:Adaptor Proteins, Vesicular Transport; C000629659:Cab39 protein, mouse; D002135:Calcium-Binding Proteins; D018832:Molecular Chaperones; D009097:Multienzyme Complexes; D011994:Recombinant Proteins; C476400:STRADA protein, human; D017346:Protein Serine-Threonine Kinases; C109790:STK11 protein, human; D000091162:AMP-Activated Protein Kinase Kinases; D055372:AMP-Activated Protein Kinases