Sci. Signal., 16 June 2009
Structural Biology Kinase Conformation, Not Activity, Required
Nancy R. Gough
Science Signaling, AAAS, Washington, DC 20005, USA
The kinase LKB1, which is a tumor suppressor and activates AMP-activated protein kinase (AMPK), is activated by interacting with two additional proteins, STRAD (Ste-related adaptor) and MO25. STRAD (one of two isoforms) is a pseudokinase, meaning that critical conserved residues required for catalytic activity are missing, and MO25 (one of two isoforms) is a scaffolding protein. Zeqiraj et al. characterized the crystal structure of STRAD (the pseudokinase domain, residues 59 to 431) and full-length MO25. In the structure, STRAD adopted a conformation similar to that of an active kinase and bound an ATP molecule. In contrast to catalytically active kinases, ATP binding did not require coordination with Mg2+; instead, STRAD appeared to position the ATP through interactions with specific amino acids (Arg215 and His200 for the β-phosphate and Lys197 for the -phosphate). In addition to the known interaction between the STRAD WEF motif and MO25, the structure revealed extensive interactions between MO25 on its concave surface and STRAD; the importance of these interactions for formation of a stable complex was confirmed by mutational analysis. Comparison of the structure of STRAD when bound to MO25 resembled that of CDK1 (cyclin-dependent kinase 1) bound to cyclin A and a complex between an active epidermal growth factor receptor (EGFR) and an EGFR in the inactive conformation, with the conformation characterized as "closed and active." Various biochemical assays demonstrated that MO25 increases the affinity of STRAD for ATP and that ATP increases the affinity of STRAD for MO25. Interactions with LKB1 permit MO25 and STRAD to form a heterotrimeric complex even when the interaction between MO25 and STRAD is disrupted by mutation. However, STRAD mutants that could not bind either MO25 or ATP failed to activate LKB1 even though they could form the heterotrimeric complex. Thus, ATP and MO25 appear to allosterically modulate STRAD, allowing it to adopt the closed and active conformation that is necessary for LKB1 activation. A mutation that produces a truncated STRAD is associated with polyhydramnios, megalencephaly, symptomatic epilepsy syndrome, and this would be expected to produce a functionally inactive form of STRAD due to loss of the C-terminal domain of the pseudokinase domain. Expression of this truncated form in cultured cells showed that the protein was less stable than full-length STRAD and failed to interact with or activate LKB1.
E. Zeqiraj, B. M. Filippi, S. Goldie, I. Navratilova, J. Boudeau, M. Deak, D. R. Alessi, D. M. F. van Aalten, ATP and MO25 regulate the conformational state of the STRAD pseudokinase and activation of the LKB1 tumour suppressor. PLoS Biol. 7, e1000126 (2009). [PubMed]
Citation: N. R. Gough, Kinase Conformation, Not Activity, Required. Sci. Signal. 2, ec199 (2009).
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