Figure 1

L123A SAD-1 is functional and can be inactivated with a PP1 analog in vitro . (A) Alignment of C. elegans SAD-1 and murine SAD kinases with other kinases successfully engineered using the chemical-genetic strategy. The gate-keeper residue is indicated in red. (B) In vitro kinase assay for SAD-1 activity (described in Materials and methods). In the absence of LKB1, both wild-type (WT) and analog-sensitive (as) versions of SAD-1 were inactive. Following treatment with LKB1, both versions became active towards tau [S262], but the kinase activity of the L123A SAD-1 was reduced 70%. Inclusion of 1NA-PP1 at the concentrations indicated caused significant inhibition of SAD-1as but not of wild-type SAD-1. ***p < 0.001, paired t-test, between DMSO- and 1NA-PP1-treated L123A SAD-1 samples. The y-axis indicates signal from ELISA assay for phospho-tau [S262]. Error bars indicate the standard error of the mean.