Understanding the role of Mammalian sterile-20 like kinases 1 (Mst1) in cells by identifying its interactors

The mammalian Ste20-like kinase 1 (Mst1) and 2 (Mst2) are serine/threonine protein kinases that are activated in response to various apoptotic or stress stimuli, including oxidative stress. Recently, Mst1/2 has become a subject of intense investigation due its role in tumor suppression. Despite this interest, very little is known about the regulation of Mst1/2 activity and its cellular targets. In this work, we sought to learn more about the regulation and function of Mst1 by identifying its interactors. We used tandem affinity chromatography to identify novel binding partners for Mst1 under basal and oxidative stress conditions. We identified two novel interactors of Mst1, Peroxiredoxin 1 (Prdx1) and Mst2. Prdx1 was identified only under oxidative stress condition while Mst2 was identified under both non-stress and under the conditions of oxidative stress.;Peroxiredoxin 1 (Prdx1) is an antioxidant enzyme that regulates the cellular redox state by reducing hydrogen peroxide to water and oxygen. We confirmed the association between Mst1 and Prdx1 by co-immunoprecipitation experiments and found that the interaction between Mst1 and Prdx1 is induced by oxidative stress. We also demonstrated that Prdx1 is a Mst1 substrate; Mst1 phosphorylates Prdx1 at three predominant sites Thr18, Thr90 and Thr183 in vitro. We further showed that phosphorylation of Prdx1 by Mst1 inactivates Prdx1, leading to accumulation of hydrogen peroxide in cells. Our results suggest that hydrogen peroxide stimulated Mst1 activates a positive feedback loop to sustain a pro-oxidative state in cells.;The Mst1 interaction screen revealed another Mst1 interactor, Mst2, suggesting that Mst1 and Mst2 form heterodimers in cells. Using co-immunoprecipitation experiments, we confirmed Mst1 association with Mst2 and also identified the domain required for Mst1/Mst2 complex formation. We evaluated dissociation constants (Kdis) of Mst1/2 homo- and heterodimers and found that the Kdis of Mst1/Mst2 heterodimer is much lower than Mst1/2 homodimers. We also compared the kinase activities of Mst1/2 homo- and heterodimers and found that heterodimers have much lower kinase activity than Mst1/2 homodimers. These findings suggest that Mst1 and Mst2 can associate with one another to form Mst1/Mst2 heterodimers, which are tighter and have lower kinase activity than corresponding homodimers.