J. Cell Biol. 209, 895–912 (2015)

Rho-associated kinase (Rho kinase) regulates different cellular functions ranging from migration to adhesion through the phosphorylation of substrate proteins such as myosin light chain and tau. Although genetic and biochemical studies have identified a number of Rho kinase substrates, many potential substrates still remain uncharacterized. To ensure a more comprehensive identification of Rho kinase targets, Amano et al. developed a method called kinase-interacting substrate screening (KISS) that involved incubating brain lysates with affinity beads coated with the catalytic domain of Rho kinase in the presence or absence of ATP. These samples were run through a titanium oxide column to enrich for phosphorylated peptides, which were then analyzed by LC/MS/MS. Overall, the authors identified a total of 140 proteins containing 356 phosphorylation sites comprising known and novel Rho kinase substrates. In particular, they focused on the planar polarity protein Scribble (Scrib), which was phosphorylated at two sites in the C-terminal domain by Rho kinase. Additional proteomic studies found that a known Rho kinase interactor, Shroom2, interacted with the C-terminal domain of Scrib in a phosphorylation-dependent manner. Scrib, Rho kinase and Shroom2 formed a tertiary complex that was dependent on Scrib phosphorylation. Given that interactions between Shroom2 and Rho kinase were associated with actomyosin contractility, the authors wanted to confirm whether Scrib was also involved in this process. Scrib was co-localized at the cellular edge with Shroom2 and Rho kinase, where there was increased contractility; knockdown of Scrib blocked this activity. Finally, the authors were able to apply their KISS method to identify kinase substrates of nine different protein kinases, including PKA and MAPK1. The planned deposition of these findings into a public database will provide a great resource for researchers seeking to identify particular substrates that are required for a kinase-dependent activity.