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

Am J Physiol Cell Physiol. 2014 May 1;306(9):C805-18. doi: 10.1152/ajpcell.00339.2013. Epub 2014 Feb 26.

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.

Keywords: Hippo; YAP/TAZ; cancer; interactomics; proteomics.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adaptor Proteins, Signal Transducing / genetics
  • Adaptor Proteins, Signal Transducing / metabolism*
  • Amino Acid Sequence
  • Animals
  • Cell Cycle Proteins
  • Cluster Analysis
  • Computational Biology
  • Gene Expression Regulation
  • Green Fluorescent Proteins / genetics
  • Green Fluorescent Proteins / metabolism
  • HEK293 Cells
  • Humans
  • Immunoprecipitation
  • Mice
  • Molecular Sequence Data
  • NIH 3T3 Cells
  • Phosphoproteins / genetics
  • Phosphoproteins / metabolism*
  • Protein Binding
  • Protein Interaction Mapping* / methods
  • Protein Interaction Maps*
  • Protein-Serine-Threonine Kinases / metabolism
  • Proteomics* / methods
  • Recombinant Fusion Proteins / metabolism
  • Signal Transduction
  • Tandem Mass Spectrometry
  • Transcription Factors / genetics
  • Transcription Factors / metabolism*
  • Transfection

Substances

  • Adaptor Proteins, Signal Transducing
  • Cell Cycle Proteins
  • Phosphoproteins
  • Recombinant Fusion Proteins
  • Taz protein, mouse
  • Transcription Factors
  • Yap1 protein, mouse
  • Green Fluorescent Proteins
  • Hippo protein, mouse
  • Protein-Serine-Threonine Kinases