The Hippo pathway is a conserved signaling circuit in multicellular organisms that regulates organ development by controlling cell proliferation through repression of programmed cell death, or apoptosis.1,2 Like most apoptosis-inhibiting pathways in higher organisms, dysregulation of this pathway is associated with several disease states, most notably cancer.2
The Hippo pathway works through a series of kinases that phosphorylate proteins, ultimately affecting transcription of several hundred genes. The Hippo kinase (MST1/2 in mammals) and Sav1 form a complex, phosphorylating the LATS1/2 kinase. The now-activated phospho-LATS1/2 kinase inhibits the activity of the transcription activator YAP/TAZ by phosphorylation. After dephosphorylation by various phosphatases (such as PP1/ASSP2), the YAP/TAZ complex translocates to the nucleus, where it forms a complex with TEAD1-4 and SMAD to upregulate genes responsible for cell proliferation and apoptosis inhibition. Despite extensive study in the past decade, scientists have not fully elucidated the Hippo pathway or the role that proteins may play in signaling.
In Couzens et al. (2013), the researchers conducted a systematic study of the protein–protein interaction network of the Hippo signaling pathway, in both the absence and presence of a chemical inhibitor of the phosphatases responsible for the dephosphorylation of the YAP/TAZ pathway.3 Using a combination of FLAG-tagged protein-based affinity purification (FLAG AP-MS) and BirA fusion protein-based biotinylation (BioID) coupled with mass spectrometry, they ultimately mapped 749 protein–protein contacts, including 599 previously unknown interactions.
Using human cervical cancer cell lines and human embryonic kidney cells expressing FLAG epitope-tagged versions of kinases such as MST1 or SAV1 and other proteins known in the Hippo pathway, 21 “bait” proteins isolated over 200 interacting partners. Interacting proteins were identified by mass spectrometry after digestion with trypsin; analysis took place on an LTQ ion trap mass spectrometer (Thermo Scientific). The research team searched peptide masses against the human protein database of the Global Proteome Machine, using MASCOT, and they repeated this procedure with the BioID protocol. Nineteen proteins fused to BirA were expressed in the two cell lines. Proteins that came in contact with one of the bait proteins were post-translationally biotinylated by the BirA fusions. Biotinylated proteins were isolated from cell free lysates. After isolation, the researchers once again analyzed proteins via mass spectrometry, resulting in an additional 487 protein identifications.3
Because it is previously established that the activity of both kinases and phosphatases alter protein–protein interactions in this pathway,1 Couzens et al. repeated the previously described experiment in the presence of the phosphatase inhibitor okadaic acid. As predicted, the overall number of proteins in the interactome of cells treated with the inhibitor were significantly perturbed. The lack of phosphatase activity induced interactions between MST1 (a kinase in the pathway) and MOB1A and MOB1B.3 Mutations resulting in alteration of basic residues in MOB1A abolish this interaction altogether, even in the presence of the inhibitor. This might indicate that MOB1A/MOB1B is fine-tuning the phosphorylation state of the pathway and could be a previously unidentified point of control for anti-apoptotic pathways.
Studies such as those performed by Couzens et al. provide valuable insight into the molecular mechanisms of cellular signaling. Due to the crucial role of cell signaling in carcinogenesis, comprehensive views of the total protein interactome may identify new potential biomarkers or drug targets in diseases that exploit the dysregulation of the Hippo pathway.
1. Zhao, B., Tumaneng, K., and Guan, K.L. (2011) “The Hippo pathway in organ size control, tissue regeneration and stem cell self-renewal,” Nature Cell Biology, 13(8) (pp. 877–83).
2. Pan, D. (2010) “The hippo signaling pathway in development and cancer,” Developmental Cell, 19(4) (pp. 491–505).
3. Couzens, A.L., Knight, J.D., Kean, M.J., Teo, G., Weiss, A., Dunham, W.H., Lin, Z.Y., Bagshaw, R.D., Sicheri, F., Pawson, T., Wrana, J.L., Choi, H., and Gingras, A.C. (2013, November) “Protein interaction network of the mammalian Hippo pathway reveals mechanisms of kinase-phosphatase interactions,” Science Signaling, 6(302) (p. rs15), doi: 10.1126/scisignal.2004712.
Post Author: Adam Humbard.