Fig 2: Impact of T-Loop and CPE Cys-to-Ala mutations on BRSK redox sensitivity in a cellular EGFP-Tau HEK-293T co-expression system. Representative immunoblot of EGFP-Tau co-expressed with WT and Cys-to-Ala mutants of (a) BRSK1 and (b) BRSK2 (left panels). Transiently transfected HEK-293T cells were treated with or without 10 mM H2O2 for 10 mins before the addition of 20 mM GSH. Whole cell lysates were harvested after a further 15 mins. Signal density for phospho Tau S262 and total Tau (GFP) was obtained using ImageStudio software (Licor) and results from at least 3 biological replicates were anaylzed with Graphpad Prism software using one way anova to determine significance (right panels). Data shown is mean and SE. All values are normalized to Tau pS262 signals from control (buffer only treatment) WT BRSK and Tau co-transfections. Data shown is mean and SD. * = P < 0.05, **= P < 0.01, ***= P < 0.001.

Fig 3: (a) Model of BRSK1/2 regulation. Schematic diagram demonstrating ways in which residues within BRSK kinases permit fine-tuning of catalytic activity through a variety of oxidative modifications, potentially including inter and intramolecular disulfide bonds. Cartoon representation of kinase domain with N-lobe colored dark blue/purple and the C-lobe colored light blue/purple. (b) ARK family member BRSK2, MELK, and MARK2/3 crystal structures demonstrate the ability to form asymetric dimers bringing T + 2 cys into proximity. Crystal structures for MARK2, and MELK both contain intermolecular disulfide bonds between T + 2 cys (Marx et al. 2010; Marx et al. 2006; Murphy et al. 2007; Cao et al. 2013).

Fig 4: Intramolecular disulfide bonds form in the kinase domains of BRSK1 and 2. (a) Full length BRSK1 and 2 were affinity-purified from HEK-293T cells and subjected to LC-MS/MS analysis. LC-MS/MS spectrum mapping revealed disulfide bridges formation between C147BRSK1 - C153BRSK1, C191BRSK1 - C198BRSK1, C132BRSK2 - C138BRSK2, and C176BRSK2 - C183BRSK2. Peptide coverage was 74 and 78 % for BRSK1 and BRSK2 respectively. (b) Alphafold structures demonstrating the location of disulfide bonds within the kinase domains of BRSK1 and BRSK2. (c) Real time phosphorylation of fluorescent AMARA peptide by the kinase domains of BRSK1 and 2 (100 ng) purified from E.coli. BRSK1 (29–358) and BRSK2 (14–341) were activated by incubation with LKB1 and assayed in the presence or absence of 1 mM DTT.

Fig 5: BRSK1/2 are redox sensitive. (a) Schematic representation of BRSK domain architecture, including Kinase domain, Ubiquitin Associated (UBA) domain, Proline-Rich Region (PRR), Kinase Associated Domain(KA1), and Autoinhibitory Sequence (AIS). (b) Immunoblotting showing BRSK dependent phosphorylation of Tau at Ser262 (pS262), from lysates of HEK-293T cells overexpressing full-length FLAG-BRSK1 or 2 (wild type [WT] or kinase dead [KD]) and EGFP-Tau. (c) Real time phosphorylation of fluorescent AMARA peptide by full length BRSK1 and 2 (200 ng) purified from Sf21 cells. BRSK proteins were incubated with buffer or 1 mM H2O2 for 10 mins, reactions were then initiated with the addition of ATP and peptide substrate in the presence (where indicated) of 10 mM DTT. Dose response curves for (d) DTT and (e) H2O2 with 200 ng full-length BRSK1 and BRSK2. All kinases assays are shown as mean and SD of three experiments. (f) Immunoblott (left) of pS262 in transiently co-transfected HEK-293T cells incubated with the indicated concentration of H2O2 for 10 mins. Signal density for phospho Tau S262 and total Tau (GFP) was obtained using ImageStudio software (Licor) and results from at least 3 biological replicates were anaylzed with Graphpad Prism software using one way anova to determine significance. Data shown is mean and SE. (g) Representative immunoblot (left) of transiently co-transfected HEK-293T cells treated with 10 mM H2O2 for 10 mins before the addition of 20 mM GSH. Whole cell lysates were harvested after a further 15 mins. Normalized densitometry of Tau pS262 signal (right) was calculated from 3 independent experiments. Data shown is mean and SD. * = P < 0.05, **= P < 0.01, ***= P < 0.001.