, 2007). We found that NDR1-CA M166A used Benzyl-ATP-γ-S; however, the ATP analog usage was reduced (Figure S4B). To rescue NDR1 kinase activity we, mutated two residues known to be suppressor

mutations that can rescue kinase activity when the gatekeeper residue is mutated (Zhang et al., 2005) and obtained NDR1-as-CA with increased kinase activity (NDR1-CA with M166A, M152L, and S229A mutations; Figures 1D, 1E, and 5B). We used this kinase (NDR1-as-CA) in subsequent substrate identification experiments. To perform labeling reactions in which NDR1-as-CA would thiophosphorylate substrates with find more Benzyl-ATP-γ-S, we reacted 10 μg of purified kinase with 1 mg brain lysate protein. Labeled lysate was treated by covalent capture for substrate identification (Blethrow et al., 2008 and Hertz et al., 2010). Briefly, labeled protein lysate is digested by trypsin and then thiol-containing peptides (including thiophosphorylated substrates and cysteine-containing peptides) are captured by thiol reactive resin, whereas non-thiol-containing

peptides are washed away. In the third step, beads are treated with Oxone to oxidize sulfur and elute phosphopeptides by spontaneous hydrolysis of thiophosphate linkage, whereas cysteine-containing Selleck DAPT peptides remain attached to the beads by thioether bonds. Finally, the eluted peptides are analyzed by liquid chromatography/tandem mass spectrometry to identify not only the substrates but also the phosphorylation sites, which is a major advantage of the method (Figure 5D). In each experiment, we included two negative controls (lysate Calpain alone and lysate reacted with NDR1-KD) in parallel; with these controls we could disregard abundant proteins that are detected nonspecifically. We have carried out substrate labeling from brain lysates eight times, using

P3 (2X), P8 (5X), and P13 (1X) brains, to identify potential NDR1 targets. We identified five phospho-proteins that are specific to NDR1-as-CA and are detected in more than one experiment (Table 1). Strikingly, four of these contained the consensus sequence of HXRXXS/T, which is highly similar to the one reported for the NDR1 homolog Cbk1p (HXRRXS/T; Mazanka et al., 2008; Table 1). The remaining candidate was not included in the table, because the phosphorylation site was preceded by acidic amino acids, rendering it an unlikely NDR1 substrate. In addition, we cultured dissociated cortical neurons on transwell insert culture dishes in order to harvest neuronal processes but not cell bodies to simplify total protein content. We identified one additional candidate with the same consensus site: Rab11fip5 (Rab11 family interacting protein 5; Table 1). Proteins without the consensus sequence were not included in the table for this experiment.