Kinetic Regulation of the Mammalian Sterile 20-like Kinase 2 (MST2)

Canonically, MST1/2 functions as a core kinase of the Hippo pathway and noncanonically during both apoptotic signaling and with RASSFs in T-cells. Faithful signal transduction by MST1/2 relies on both appropriate activation and regulated substrate phosphorylation by the activated kinase. Considerable progress has been made in understanding the molecular mechanisms regulating the activation of MST1/2 and identifying downstream signaling events. Here, we investigated the ability of MST2 to phosphorylate a peptide substrate and how that activity is regulated. Using a steady-state kinetic system, we parse the contribution of different factors to substrate phosphorylation, including the domains of MST2, phosphorylation, caspase cleavage, and complex formation. We found that in the unphosphorylated state, the SARAH domain stabilizes interactions with a peptide substrate and promotes turnover. Phosphorylation drives the activity of MST2, and once activated, MST2 is not further regulated by complex formation with other Hippo pathway components (SAV1, MOB1A, and RASSFS). We also show that the phosphorylated, caspase-cleaved MST2 is as active as the full-length one, suggesting that caspase-stimulated activity arises through noncatalytic mechanisms. The kinetic analysis presented here establishes a framework for interpreting how signaling events and posttranslational modifications contribute to the signaling of MST2 in vivo.

Automated summary

In this study, the ability of MST2 to phosphorylate a peptide substrate and its regulation were investigated using a steady-state kinetic system. It was found that the SARAH domain of unphosphorylated MST2 stabilizes interactions with a peptide substrate and promotes its phosphorylation. Phosphorylation drives the activity of MST2, and the phosphorylated, caspase-cleaved MST2 is as active as the full-length one.