TAZ (WWTR1) Interactome Analysis using nanotrap based affinity purification-mass spectrometry.

Abstract

Characterization of protein interactions is a fundamental goal in the post-genomic era. Here, we document a generally applicable approach to identify cellular protein interactomes using a combination of nanobody based affinity purification (A-P) coupled with tandem mass spectrometry (MS/MS). The Hippo signalling regulator TAZ functions as a transcriptional co-repressor/activator depending on its protein:protein interaction (PPI) network, we therefore undertook an unbiased proteomic screen to identify TAZ PPI's in striated muscle cells. A GFP-Nanotrap based A-P approach coupled with LC-MS/MS protein identification was used to document a comprehensive list of known and novel TAZ interactome components. Informatic analysis of the interactome documented known components of Hippo signalling and multiple epigenetic regulators such as the NuRD, FACT, SWI/SNF complexes and the pro-myogenic CARM1 methyltransferase. Hippo pathway reporter gene (HOP/HIP) analysis indicated that CARM1 represses TAZ transcriptional co-activator function, promoting TAZ Ser89 phosporylation and TAZ cytoplasmic sequestration. M-S analysis revealed that CARM1 dimethylates TAZ at Arg77 in a PGPR*LAGG consensus peptide resulting in enhanced TAZ Ser89 phoshorylation. These studies underline the utility of a nanobody based A-P approach for interactome analysis.