Development of an Mst2 construct for future studies of conformational regulation

Winona, Minnesota

The serine/threonine protein kinases Mst2 and Mst1 are key members of the Hippo pathway, a signaling pathway that regulates apoptosis by inhibiting the transcriptional cofactor yes-associated protein (YAP).1 Mst2 is activated by phosphorylation at threonine-180, allowing it to phosphorylate large tumor suppressor kinases 1/2 (LATS1/2) which can then inhibit YAP.1,2 The conformation of Mst2, particularly at the activation loop, is predicted to change when it is activated by phosphorylation. We believe that the stability and relatively small size of Mst2 may allow us to farther study its conformation using fluorescence-based techniques. Proteins can be labeled for these techniques using cysteine-maleimide crosslinking, where a "Cys-lite" background is prepared, and cysteine residues are inserted at specific positions that can be labeled under typical lab conditions. Using a Quikchange Lightning site-directed mutagenesis kit, we have made constructs of the His-tagged Mst2 kinase domain with a cysteine labeling position in a Cys-lite background to direct a fluorescent donor or acceptor tag. We have validated a high-yield purification protocol and checked the folding of our construct using circular dichroism (CD), and the nucleotide binding of our construct using fluorescence-based thermal denaturation. We have also prepared this construct with and without co-expression with lambda phosphatase.