Increasing kinase domain proximity promotes MST2 autophosphorylation during Hippo signaling

Abstract

The Hippo pathway plays an important role in developmental biology, mediating organ size by controlling cell proliferation through the activity of a core kinase cassette. Multiple upstream events activate the pathway, but how each controls this core kinase cassette is not fully understood. Activation of the core kinase cassette begins with phosphorylation of the kinase MST1/2 (also known as STK3/4). Here, using a combination of in vitro biochemistry, and cell-based assays including chemically induced dimerization and single-molecule pull down we revealed that increasing the proximity of adjacent kinase domains, rather than formation of a specific protein assembly, is sufficient to trigger autophosphorylation. We validate this mechanism in cells and demonstrate that multiple events associated with the active pathway, including SARAH-domain mediated homodimerization, membrane recruitment, and complex formation with the effector protein SAV1, each increase the kinase domain proximity and autophosphorylation of MST2. Together, our results reveal that multiple and distinct upstream signals each utilize the same common molecular mechanism to stimulate MST2 autophosphorylation. This mechanism is likely conserved among MST2 homologs. Our works also highlights potential differences in Hippo signal propagation between each activating event owing to differences in the dynamics and regulation of each protein ensemble that triggers MST2 autophosphorylation and possible redundancy in activation.

Activated MST2 kinase is free of kinetic regulation

“…Attempts to purify either full-length SAV1 or the SARAH-domain of SAV1 were unsuccessful, instead we used the SARAH domain from the Drosophila homolog (dSAV-SARAH). Drosophila Salvador shares both sequence, structural, and functional similarity with SAV1, and previous studies of mixed systems (Drosophila Salvador and human MST2) have displayed similar behavior as equivalent mammalian systems 8,18,30,32,35,50 . Then, we confirmed that each purified variant did indeed form a complex with pMST2-FL using an in vitro pull-down assay (Figure 5).…”

“…All three binding partners examined mediate larger assemblies which may influence the cellular localization of MST2 or interactions with additional regulatory proteins that influence downstream signaling without altering the kinase activity of MST1/2. For example, SAV1 increases the level of active MST2 by promoting activation and preventing dephosphorylation of active MST1/2, thus promoting downstream signal transduction 8,18,33,34 ; hMOB1A may stabilize a ternary complex with LATS1/2 which would increase the effective concentration of a substrate (LATS1/2) for MST1/2 20,21 ; and the RASSFs link MST1/2 to other signaling pathways. All of these mechanisms would entail alterations to Hippo pathway activity without a catalytic change in the activity of activated MST2, and any additional analysis of the contribution of these complexes to downstream studies should focus on these non-catalytic contributions.…”

“…Activation of MST1/2 relies on phosphorylation of the T-loop on residue T180/183 by transautophosphorylation [3][4][5] or, less frequently, phosphorylation by Tao-1 6,7 . For Hippo signaling, events that increase proximity of MST1/2 to itself trigger autophosphorylation and, thus, activation 8 . Following activation MST1/2 is primed to phosphorylate downstream substrates, and the identity of these subsequent steps have been established.…”

“…The SARAH domain of MST1/2 either forms homodimers, which stimulate autophosphorylation, or heterodimers with the SARAH-domains of either SAV1 or RASSF family members, and each of these interactions tune pathway activity 5,17,30,31 . SAV1 promotes Hippo pathway activity in part by contributing to MST1/2 activation by multiple routes --increasing the effective concentration of MST1/2, translocating it to the membrane, and inhibiting the activity of the STRIPAK phosphatase 8,18,[32][33][34][35] . Additionally, SAV1 scaffolds MST1/2 to its substrate LATS1/2 36 but whether this complex contributes to downstream signaling remains to be determined.…”

“…MST1/2 fragments, isolated from cells, lacking the linker region displayed both higher levels of activity and different substrate selectivity than the full-length enzyme [10][11][12][13][14][15][16] . The SARAH domain promotes autophosphorylation 5,8,11,[17][18][19] but has not yet been implicated in directly regulating substrate phosphorylation.…”