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Endothelial FAT1 inhibits angiogenesis by controlling YAP/TAZ protein degradation via E3 ligase MIB2

ABSTRACT: Activation of endothelial YAP/TAZ signaling is crucial for physiological and pathological angiogenesis. The mechanisms of endothelial YAP/TAZ regulation are, however, incompletely understood. Here we report that the protocadherin FAT1 acts as a critical upstream regulator of endothelial YAP/TAZ which limits the activity of these transcriptional cofactors during developmental and tumor angiogenesis by promoting their degradation. We show that loss of endothelial FAT1 results in increased endothelial cell proliferation in vitro and in various angiogenesis models in vivo. This effect is due to perturbed YAP/TAZ protein degradation, leading to increased YAP/TAZ protein levels and expression of canonical YAP/TAZ target genes. We identified the E3 ubiquitin ligase Mind Bomb-2 (MIB2) as a novel FAT1-interacting protein mediating FAT1-induced YAP/TAZ ubiquitination and degradation. Loss of MIB2 expression in endothelial cells in vitro and in vivo recapitulated the effects of FAT1 depletion and caused decreased YAP/TAZ degradation and increased YAP/TAZ signaling. Our data identify a pivotal mechanism of YAP/TAZ regulation involving FAT1 and its associated E3 ligase MIB2, which is essential for YAP/TAZ-dependent angiogenesis. Samples for proteomics were obtained from HUVECs transduced with the FAT1 intracellular part fused with the extracellular part and transmembrane domain of the IL-2 receptor and carrying a Flag tag on the C terminus (see above) for 36 h followed by lysis in IP buffer. Lysates were incubated with magnetic anti-FLAG beads (M8823, Sigma/Aldrich) overnight at 4Deg. Affinity purified samples were subjected to in-gel (4-12% NuPAGE Bis-Tris gels, ThermoFisher Scientific) digestion as described (DOI: 10.1038/nprot.2006.468). In brief, gel lanes were cut into nine blocks/fractions and finely diced. Embedded proteins were reduced (10 mM dithiothreitol) and alkylated (55 mM iodoacetamide), followed by overnight digestion using trypsin (Serva). Peptides were extracted by increasing concentrations of acetonitrile and lyophilized. Final desalting, concentration and storage utilized "stop and go extraction" (STAGE) tips (DOI: 10.1021/ac026117i). Eluted peptides where subjected to electrospray ionization (ESI)-mediated liquid chromatography/tandem mass spectrometry (LC/MS2) using in house-packed column emitters (15 cm length, 70 um ID, 1.9 um ReprsoSil-Pur 120 C18-AQ, Dr. Maisch) and a buffer system comprising solvent A (0.1% formic acid) and solvent B (80% acetonitrile, 0.1% formic acid). Instrumentation details and parameters were extracted and summarized using MARMoSET (DOI: 10.1074/mcp.TIR119.001505) and are repository deposited. Peptide/spectrum matching and label free quantitation were performed using the MaxQuant suite of algorithms (DOIs: 10.1038/nbt.1511, 10.1021/pr101065j, 10.1074/mcp.M113.031591) against the human UniProt database (canonical & isoforms; downloaded on 2019/01/23). Parameters are included here. Downstream statistical analysis used the limma-based (DOI: 10.1093/nar/gkv007) in-house package autonomics (https://doi.org/doi:10.18129/B9.bioc.autonomics).

INSTRUMENT(S): Q Exactive

ORGANISM(S): Homo Sapiens (ncbitaxon:9606)

SUBMITTER: Stefan Offermanns

PROVIDER: MSV000091279 | MassIVE | Tue Feb 14 01:05:00 GMT 2023

SECONDARY ACCESSION(S): PXD040114

REPOSITORIES: MassIVE

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Endothelial FAT1 inhibits angiogenesis by controlling YAP/TAZ protein degradation via E3 ligase MIB2.

Li Rui R Shao Jingchen J Jin Young-June YJ Kawase Haruya H Ong Yu Ting YT Troidl Kerstin K Quan Qi Q Wang Lei L Bonnavion Remy R Wietelmann Astrid A Helmbacher Francoise F Potente Michael M Graumann Johannes J Wettschureck Nina N Offermanns Stefan S

Nature communications 20230408 1

Activation of endothelial YAP/TAZ signaling is crucial for physiological and pathological angiogenesis. The mechanisms of endothelial YAP/TAZ regulation are, however, incompletely understood. Here we report that the protocadherin FAT1 acts as a critical upstream regulator of endothelial YAP/TAZ which limits the activity of these transcriptional cofactors during developmental and tumor angiogenesis by promoting their degradation. We show that loss of endothelial FAT1 results in increased endothel ...[more]

PMID: 37031213

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