Protein Interaction Network of the Mammalian Hippo Pathway Reveals Mechanisms of Kinase-Phosphatase Interactions

Abstract

The Hippo pathway regulates organ size and tissue homeostasis in response to multiple stimuli, including cell density and mechanotransduction. Pharmacological inhibition of phosphatases can also stimulate Hippo signaling in cell culture. We defined the Hippo protein-protein interaction network with and without inhibition of serine and threonine phosphatases by okadaic acid. We identified 749 protein interactions, including 599 previously unrecognized interactions, and demonstrated that several interactions with serine and threonine phosphatases were phosphorylation-dependent. Mutation of the T-loop of MST2 (mammalian STE20-like protein kinase 2), which prevented autophosphorylation, disrupted its association with STRIPAK (striatin-interacting phosphatase and kinase complex). Deletion of the amino-terminal forkhead-associated domain of SLMAP (sarcolemmal membrane-associated protein), a component of the STRIPAK complex, prevented its association with MST1 and MST2. Phosphatase inhibition produced temporally distinct changes in proteins that interacted with MOB1A and MOB1B (Mps one binder kinase activator-like 1A and 1B) and promoted interactions with upstream Hippo pathway proteins, such as MST1 and MST2, and with the trimeric protein phosphatase 6 complex (PP6). Mutation of three basic amino acids that are part of a phospho-serine- and phospho-threonine-binding domain in human MOB1B prevented its interaction with MST1 and PP6 in cells treated with okadaic acid. Collectively, our results indicated that changes in phosphorylation orchestrate interactions between kinases and phosphatases in Hippo signaling, providing a putative mechanism for pathway regulation.

PP6 Controls T Cell Development and Homeostasis by Negatively Regulating Distal TCR Signaling

“…PP6 also suppresses IL-1-stimulated TAK1 activation by dephosphorylating TAK1 in 293 cells (15). Recent evidence suggests that PP6 may also regulate Hippo signaling via interaction with MOB1B (16). However, the physiological functions of PP6, especially in T cell biology, are not known.…”

Probing nuclear pore complex architecture with proximity-dependent biotinylation

“…BioID has been used successfully to screen for constituents of the relatively insoluble mammalian nuclear lamina (1), the trypanosome bilobe (2), cell junction complexes (3)(4)(5), and centrosomes (6,7). The method also has been used to screen for proteins involved in the Hippo signaling pathway (8).…”

Discovering the Hippo pathway protein-protein interactome

“…They then tested the function of these proteins by RNAi knock-down and found that 102 of them affected the transcriptional activity of Yorkie. The two other studies by Wang et al [6] and Couzens et al, [7] from the groups of Junjie Chen and Anne-Claude Gingras, characterized the protein-protein interaction network of the human Hippo pathway and identified high-confidence networks of 343 and 445 proteins, respectively. All three studies rediscovered many of the known interactions between pathway components.…”

“…Couzens et al [7] addressed how phosphorylation affects the Hippo pathway interaction network. They found that phosphorylation triggered remodeling of the interactomes of MST, LATS, and the MOB cofactors for LATS.…”

“…Three recent reports now describe proteomic studies uncovering extensive protein-protein interaction networks that connect the Hippo pathway to proteins involved in other signaling pathways and processes [5][6][7]. All three papers used many of the known Hippo pathway components as baits to identify interacting proteins by affinity purification and mass spectrometry (AP-MS) in cultured cells.…”