Label-free quantitative proteomic analysis of the YAP/TAZ interactome

Abstract

The function of an individual protein is typically defined by protein-protein interactions orchestrating the formation of large complexes critical for a wide variety of biological processes. Over the last decade the analysis of purified protein complexes by mass spectrometry became a key technique to identify protein-protein interactions. We present a fast and straightforward approach for analyses of interacting proteins combining a Flp-in single-copy cellular integration system and single-step affinity purification with single-shot mass spectrometry analysis. We applied this protocol to the analysis of the YAP and TAZ interactome. YAP and TAZ are the downstream effectors of the mammalian Hippo tumor suppressor pathway. Our study provides comprehensive interactomes for both YAP and TAZ and does not only confirm the majority of previously described interactors but, strikingly, revealed uncharacterized interaction partners that affect YAP/TAZ TEAD-dependent transcription. Among these newly identified candidates are Rassf8, thymopoetin, and the transcription factors CCAAT/enhancer-binding protein (C/EBP)β/δ and core-binding factor subunit β (Cbfb). In addition, our data allowed insights into complex stoichiometry and uncovered discrepancies between the YAP and TAZ interactomes. Taken together, the stringent approach presented here could help to significantly sharpen the understanding of protein-protein networks.

Jade-1S phosphorylation induced by CK1α contributes to cell cycle progression

“…21 We used a single-copy integration Flp-In strategy 22 in NIH-3T3 cells to express C-terminal GFP-tagged wild-type Jade-1S (Jade-1S. GFP), CK1a phosphorylation motif mutant Jade-1S (Jade-1S S18/20A.GFP) or control GFP only (GFP.GFP) proteins in cycling cells.…”

“…60 Purified protein complexes were then eluted nonspecifically by direct in-column digestion with trypsin as previously described. 21 Digestion was stopped the next day by adding 1% trifluoracetic acid. Label-free pull-downs were performed as 4 biological replicates.…”

“…LFQ intensities for respective protein groups or site intensities were uploaded in Perseus 62 and analyzed as previously described. 21 Briefly, raw LFQ intensities were logarithmised and normalized by subtraction of the mean. At least 2 LFQ values per protein group needed to be present for the analysis.…”

Transformation by Polyomavirus Middle T Antigen Involves a Unique Bimodal Interaction with the Hippo Effector YAP

“…Interactions of PDZ domains of YAP and TAZ are also different (57). Not surprisingly, there are notable differences in the YAP and TAZ protein interactomes (92). The relevant targets will have to be identified in future experiments.…”

Cysteine S-Glutathionylation Promotes Stability and Activation of the Hippo Downstream Effector Transcriptional Co-activator with PDZ-binding Motif (TAZ)

“…On the protein level, TAZ and YAP display ϳ50% sequence similarity, including the conserved phosphorylation residues. They have many common but also few distinct protein-protein interaction partners that are essential for their stability and function (11). Interestingly, three conserved cysteine residues are only present in TAZ, highlighting its potential redox regulation.…”