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Fig. 1. Characterisation of LKB1 mutants found in human cancers. (A) HEK293 cells were transfected with 3 µg of plasmids encoding wild-type or the indicated mutants of GST-LKB1 in the presence or absence of 3 µg of plasmids encoding Flag-STRAD
and Myc-MO25. Thirty-six hours post-transfection, the GST-tagged proteins were affinity purified from the cell lysates using glutathione-Sepharose as described in Materials and Methods. Similar amounts of the purified GST fusion proteins were subjected to SDS-PAGE and immunoblotted with the anti-Flag and anti-Myc antibodies to detect co-purified Flag-STRAD and Myc-MO25, respectively, and with the anti-GST antibody to ensure that comparable amounts of the GST-tagged proteins were present in each lane (upper panels). 10 µg of total cell lysates prior to affinity purification were also immunoblotted with the anti-Flag and anti-Myc antibodies to ensure that Flag-STRAD and Myc-MO25 were expressed at similar levels in each condition (lower panels). The purified LKB1 proteins were tested for their ability to phosphorylate the LKBtide peptide substrate as described in Materials and Methods. The results are expressed as the peptide kinase activity generated per mg of affinity purified protein added to the assay. Results shown are the mean±s.d. of two independent assays carried out in triplicate. Bars marked with an asterisk indicate LKB1 mutants that fail to bind STRAD and MO25; bars marked with an inverted triangle indicate LKB1 mutants that are catalytically inactive but still bind STRAD and MO25. (B) Model of the LKB1 catalytic domain in which residues found to abolish binding of LKB1 to STRAD are indicated. A sequence alignment of LKB1 with the structurally most related Aurora-related kinase-1 [30%, 1MUO (Cheetham et al., 2002)] was generated. The surface exposed residues that correspond to impaired LKB1 function/complex formation are shown in green patches on the grey surface representation of the kinase fold, and are mapped onto the structure of Aurora-related kinase-1, which is shown as a ribbon. (C) HEK293 cells were transfected with the indicated constructs and analysis performed as described in A. Results shown are the mean±s.d. of two independent assays carried out in triplicate.
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