Figure 4:

Cysteine residues within the kinase domain fine-tune BRSK activity. In vitro kinase assays (right panels) showing normalized rates of peptide phosphorylation by WT and Cys-to-Ala variants of (a) BRSK1 and (b) BRSK2. 100 ng of LKB1 activated BRSK kinase domain was assayed in the presence or absence of 1 mM DTT. The positions of mutated Cys residues are modelled on the kinase domain as coloured spheres (left panel). Real time in vitro assays using (c) 50 ng BRSK1 and (d) 20 ng BRSK2. LKB1-activated BRSK proteins were incubated on ice in the presence or absence of 250 μM DTT for 30 mins. Assays were initiated by the addition of ATP and fluorescent peptide substrate in the presence or absence of 1 mM H₂O₂. All data is mean and SD of 3 experiments and activities are normalized to LKB1-phosphorylated BRSK signal (Supp Fig 4 b).