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First published online 23 November 2004
doi: 10.1242/jcs.01571

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Analysis of the LKB1-STRAD-MO25 complex

¹ MRC Protein Phosphorylation Unit, MSI/WTB complex, University of Dundee, Dow Street, Dundee, DD1 5EH, Scotland
² Division of Molecular Physiology, MSI/WTB complex, University of Dundee, Dow Street, Dundee, DD1 5EH, Scotland
³ Division of Biological Chemistry and Molecular Microbiology, MSI/WTB complex, University of Dundee, Dow Street, Dundee, DD1 5EH, Scotland
⁴ Division of Cell Biology and Immunology, MSI/WTB complex, University of Dundee, Dow Street, Dundee, DD1 5EH, Scotland
⁵ Sez, Genetica Medica DIMIMP, Università di Bari, Piazza G. Cesare 11, 70124 Bari, Italy

View larger version (56K): [in a new window]   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.

View larger version (39K): [in a new window]   Fig. 2. Characterization of the MO25 WEF-binding site. (A) Structure of the WEF-binding pocket of MO25 (ribbon + surface) in which the residues interacting with the WEF motif (sticks with green carbons) of STRAD are labelled. (B,C) HEK293 cells were transfected with the indicated constructs and analysis performed as described in the legend to Fig. 1A. Results shown are the mean±s.d. of two independent assays carried out in triplicate.

View larger version (53K): [in a new window]   Fig. 3. Localisation of WEF-binding pocket MO25 mutants in cells. HeLa cells were transfected with the construct encoding wild-type or indicated mutants of Myc-MO25 in the absence or presence of GFP-LKB1 and Flag-STRAD. Twenty-four hours post-transfection, the cells were fixed in 4% (v/v) paraformaldehyde and immunostained with the anti-MO25 antibody to detect MO25 (TR anti-sheep secondary antibody, red channel) and anti-Flag antibody to detect STRAD (Cy5 anti-mouse secondary antibody, blue channel). GFP-LKB1 localization was visualized directly through the GFP fluorescence (green channel). The cells were imaged using a Zeiss LSM 510 META confocal microscope. The cells shown are representative images obtained in three separate experiments.

View larger version (54K): [in a new window]   Fig. 4. Characterisation of the Arg240 binding site on MO25. (A) Structure showing the concave surface of MO25, in which the repeated Arg-Arg/His residues are labelled. (B-D) HEK293 cells were transfected with the indicated constructs and analysis performed as described in the legend to Fig. 1A. Results shown are the mean±s.d. of two independent assays carried out in triplicate.

View larger version (36K): [in a new window]   Fig. 5. Activation of LKB1 does not require T-loop phosphorylation. (A) Amino acid sequence alignment of the T-loop of LKB1 and protein kinases of the AMPK subfamily (Manning et al., 2002). The identical residues are boxed in black and the conserved residues are shaded in grey. The T-loop Thr is indicated with an asterisk. The conserved Leu residue found on AMPK subfamily kinases is marked with an arrow. (B) HEK293 cells were transfected with the indicated constructs and analysis performed as described in the legend to Fig. 1A. Results shown are the mean±s.d. of two independent assays carried out in triplicate.

View larger version (38K): [in a new window]   Fig. 6. The STRAD pseudokinase is capable of binding ATP. (A) The wild-type and mutant GST-STRAD proteins were incubated with increasing concentrations of [-32P]ATP, in the presence or absence of 5 mM Mg2+, and binding of ATP to the proteins was measured as described in the Materials and Methods. Results shown are the means of three separate experiments carried out in duplicate. Data were fitted to a single-site binding model: bound=[ATP]/(Kd+[ATP]). (B,C) Displacement of ATP from wild-type STRAD by ADP (B) or AMP (C). A fixed concentration of [-32P]ATP (200 µM) was incubated with the GST-STRAD protein in the presence of increasing concentrations of either ADP or ATP, and in the presence or absence of 5 mM Mg2+; binding of ATP to the proteins was measured as described in the Materials and Methods. Data were fitted to the binding models: bound=[ATP]/([ATP]+ Kd ATP(1+[ADP]/Kd ADP)) or bound=[ATP]/([ATP]+Kd ATP(1+[AMP]/Kd AMP)). (D) HEK293 cells were transfected with the indicated constructs and analysis performed as described in the legend to Fig. 1A. Results shown are the mean±s.d. of two independent assays carried out in triplicate. (E) HeLa cells were transfected with the construct encoding wild-type or indicated mutant of Flag-STRAD in the presence of GFP-LKB1 and Myc-MO25, and analysed as described in the legend to Fig. 3.