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. 2009 Dec 18;326(5960):1707-11.
doi: 10.1126/science.1178377. Epub 2009 Nov 5.

Structure of the LKB1-STRAD-MO25 complex reveals an allosteric mechanism of kinase activation

Elton Zeqiraj  1 , Beatrice Maria Filippi, Maria Deak, Dario R Alessi, Daan M F van Aalten
Affiliations

Structure of the LKB1-STRAD-MO25 complex reveals an allosteric mechanism of kinase activation

Elton Zeqiraj et al. Science. .

Abstract

The LKB1 tumor suppressor is a protein kinase that controls the activity of adenosine monophosphate-activated protein kinase (AMPK). LKB1 activity is regulated by the pseudokinase STRADalpha and the scaffolding protein MO25alpha through an unknown, phosphorylation-independent, mechanism. We describe the structure of the core heterotrimeric LKB1-STRADalpha-MO25alpha complex, revealing an unusual allosteric mechanism of LKB1 activation. STRADalpha adopts a closed conformation typical of active protein kinases and binds LKB1 as a pseudosubstrate. STRADalpha and MO25alpha promote the active conformation of LKB1, which is stabilized by MO25alpha interacting with the LKB1 activation loop. This previously undescribed mechanism of kinase activation may be relevant to understanding the evolution of other pseudokinases. The structure also reveals how mutations found in Peutz-Jeghers syndrome and in various sporadic cancers impair LKB1 function.

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Figures

FIGURE 1
FIGURE 1. Overall structure and LKB1-STRADα-MO25α complex interactions
A) Cartoon representation of the heterotrimeric complex and two bound AMP-PNP molecules are shown in sticks representations (LKB1, yellow carbons; STRADα, magenta carbons). The γ-P for AMP-PNP bound to LKB1 was not visible due to disorder. The WEF motif at the C-terminus of STRADα, for which connectivity could not be unambiguously identified due to disorder of the linkers, is shown in cyan. B) Detailed view of LKB1-STRADα interaction. STRADα p+1 and αEF-αF loops are coloured green and orange respectively. C) Interaction of the LKB1 CFTL with STRADα and LKB1 N- and C-lobes. The proline-rich CFTL is coloured red. D) Detailed view of LKB1-MO25α interaction. LKB1 activation loop is coloured magenta. E) Detailed view of anchor point LKB1 A-loop interactions. Backbone atoms of residues 208-210 and 230-234 are shown, whereas residues Asp208, Thr230 and Ser232 mutated in PJS are labelled and their side chains displayed. A salt bridge between Glu199 and Lys175 depicted as dashed lines, represent the interaction of LKB1 activation segment with its catalytic loop (C-loop). The corresponding interaction found in PKA (PDBID 1ATP) between the phospho-threonine 197 (pThr) and Arg165 is also shown, with PKA residues represented as transparent sticks (carbon atoms coloured cyan). The typical “activatory” threonine (Thr202) present in LKB1 A-loop is labelled. Secondary structure elements are labelled according to the structure of PKA [15].
FIGURE 2
FIGURE 2. Characterisation of the LKB1-STRADα-MO25α interactions and LKB1 activation
A & C) The indicated constructs of GST-LKB1 and Flag-STRADα were expressed in 293 cells in the absence of MO25α. Cells at 36 h post-transfection were lysed and GST-LKB1 affinity purified on glutathione-Sepharose. The purified GST-LKB1 preparation (upper panel) as well as the cell extracts (lower panel) were immunoblotted with the indicated antibodies. Similar results were obtained in three separate experiments. B, D & F) 293 cells were co-transfected with the indicated constructs of GST-LKB1, Flag-STRADα and Myc-MO25α. Cells at 36 h post-transfection were lysed and GST-LKB1 affinity purified and assayed for the ability to activate heterotrimeric AMPK complex expressed in E. coli as described in the Materials and Methods. Kinase activities are representative of three independent assays carried out in triplicate (error bars represent the SD for a single triplicate experiment). Affinity purified GST-LKB1 preparation (upper panel) as well as cell extracts (lower panel) were immunoblotted with the indicated antibodies.
FIGURE 3
FIGURE 3. Map of oncogenic mutations on the LKB1 kinase domain and the CFTL
A) Location of LKB1 residues that are mutated in PJS and other types of cancer. The CFT region is coloured red and dashed lines represent areas that were not well-defined by electron density. B) Surface exposed residues that are mutated in PJS and other types of cancer.
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