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Protein Interaction Network of the Mammalian Hippo Pathway Reveals Mechanisms of Kinase-Phosphatase Interactions
First Author: Couzens AL, Knight JDR, Kean MJ, Teo G, Weiss A, Dunham WH, Lin ZY, Bagshaw RD, Sicheri F, Pawson T, Wrana JL, Choi H, Gingras AC
  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. Here, 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) that prevented autophosphorylation disrupted its association with STRIPAK (Striatin-Interacting Phosphatase and Kinase complex). Deletion of the N-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 2, and with the trimeric protein phosphatase 6 complex (PP6). Mutation of three basic amino acids that are part of a phospho-serine and 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.

Manuscript available at Science Signaling. Also see accompanying Perspective article.

Interaction data was deposited in IntAct and BioGRID

The raw mass spectrometry data was contributed to ProteomeXchange through the MassIVE repository dataset 1, dataset 2, dataset 3.