Rassf Proteins as Modulators of Mst1 Kinase Activity

Aruna Bitra,Ruchi Anand,Harshada Malvi,Jessy Mariam,Srinivas Sistla

doi:10.1038/srep45020

Abstract

Rassf1A/5 tumor suppressors serve as adaptor proteins possessing a modular architecture with the C-terminal consisting of a coiled-coil SARAH (Salvador-Rassf-Hippo) domain and the central portion being composed of Ras associated (RA) domain. Here, we investigate the effect of Rassf effectors on Mst1 function by mapping the interaction of various domains of Rassf1A/5 and Mst1 kinase using surface plasmon resonance (SPR). The results revealed that apart from the C-terminal SARAH domain of Mst1 which interacts to form heterodimers with Rassf1A/5, the N-terminal kinase domain of Mst1 plays a crucial role in the stabilization of this complex. In addition, SPR experiments show that the RA domains play an important role in fine-tuning the Mst1-Rassf interaction, with Rassf5 being a preferred partner over a similar Rassf1A construct. It was also demonstrated that the activity profile of Mst1 in presence of Rassf adaptors completely switches. A Rassf-Mst1 complexed version of the kinase becomes apoptotic by positively regulating Mst1-H2B mediated serine 14 histone H2B phosphorylation, a hallmark of chromatin condensation. In contrast, the heterodimerization of Mst1 with Rassf1A/5 suppresses the phosphorylation of FoxO, thereby inhibiting the downstream Mst1-FoxO signalling pathway.

Highlights

Rassf1A/5 tumor suppressors serve as adaptor proteins possessing a modular architecture with the C-terminal consisting of a coiled-coil SARAH (Salvador-Rassf-Hippo) domain and the central portion being composed of Ras associated (RA) domain
The results revealed that apart from the C-terminal SARAH domain of Mst[1] which interacts to form heterodimers with Rassf1A/5, the N-terminal kinase domain of Mst[1] plays a crucial role in the stabilization of this complex
By employing surface plasmon resonance (SPR) as a primary tool we have interpreted the effect of different regions of both Mst[1] and Rassf1A/5A proteins towards their SARAH-SARAH domain association

Summary

OPEN Rassf Proteins as Modulators of

Aruna Bitra[1], Srinivas Sistla[2], Jessy Mariam[1], Harshada Malvi1 & Ruchi Anand[1] received: 26 September 2016 accepted: 17 February 2017 Published: 22 March 2017. Mst[1] is a pro-apoptotic kinase and its overexpression induces apoptosis in a variety of cellular backgrounds that involve activation of stress related pathways[11] It belongs to the serine/threonine family of sterile 20 (Ste20) proteins[12,13] and exists in two forms; a 54 kDa full length protein and a 36 kDa caspase cleaved version[14]. The full length cytosolic version of Mst[1] phosporylates FoxO at a conserved serine residue (Ser 207 in FoxO3 and FoxO1 at Ser 212)[20] within the winged helix DNA binding domain[21,22] In this region of FoxO, a variety of other posttranslational modifications occur that in consort mediate its transcriptional activity[23]. No binding 0.11–0.13 kd × 104 s 1.85–2.81 0.81–0.97 4.22–9.61 1.52–1.78 4.34–5.12 0.31–1.53 4.31–5.05 0.66–1.18 3.13–3.16

Results and Discussion

Methods

Additional Information