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General Information
Name
Hippo_Pathway_Interactome
Species
D. melanogaster
Result type
FlyBase ID
FBlc0000668
Project
Data Provider
Title
204 high-confidence interactions involving components of the Hippo signaling pathway.
Status
Current
Accessions
    Biosample Source
    Overview
    Strain
    Stage
    Sex
    Tissue isolated
    Other tissues studied
    Cell component
    Key genes
    Sample preparation

    cDNAs for 12 components of the hippo signaling pathway were cloned into into pMK33-CTAP(SG) and stable S2R+ cell lines were generated. Cells were induced overnight with 140uM copper. Experiments were done in triplicate.

    Biosamples analyzed by this result (1)
    Biosample
    Type
    Title
    D. melanogaster, S2R+ cell line, stably transfected with bait protein, source for Hippo pathway affinity purifications.
    Data Analyzed
    Assay methods
    Key genes
    (bait_protein)
    Protocol

    Cells were lysed and soluble lysate was incubated with IgG sepharose in the first affinity purification step. Beads were collected, washed and bound proteins eluted by TEV protease cleavage. In a second purification step, TEV-eluted proteins were incubated with Streptavidin Plus resin. Purified proteins were then eluted with 2mM biotin.

    Mode of Assay

    Purified proteins were TCA precipitated, trypsin digested in solution, dried and resuspended. Peptides were analysed by LC-MS/MS. MS/MS spectra were matched to peptides using SEQUEST and a composite database of all translated D. melanogaster open reading frames in FlyBase (r5.23).

    Raw Data Analyzed (1)
    Assay / Reagent collection
    Type
    Title
    Coaffinity purification of interacting proteins and identification by mass spectrometry, S2R+ cells, Hippo pathway components.
    Processed Data Analyzed (0)
    Result
    Type
    Title
    Analysis
    Methods
    Reference Genome
    Reference Annotation
    Data analysis

    From twelve baits, 4560 unfiltered interactions among 1518 proteins were identified. SAINT analysis was used to assign confidence scores (PMID: 21131968). Six 'tag-only' purifications, and 8 purifications using 'JAK/STAT' pathway components as bait were used as negative controls. In order to test the performance of the SAINT analysis, a positive reference set of 21 interactions involving Hippo pathway proteins were curated from the literature. Additionally, a random reference set of 1000 interactions was randomly selected from DPiM-1 (FBlc0000184) interactions involving any one of 1348 'non-specific' interactors. Non-specific interactors were defined as proteins that pull down with approximately 1000 experiments in the unfiltered DPiM-1 dataset. A SAINT score threshold of ≥ 0.8 was set to define 204 high confidence interactions among 153 proteins.

    Comments

    23 of 26 interactions tested were validated by coimmunoprecipitation experiments.

    Associated Data
    Size
    Associated interactions
    204 Interactions
    Additional Information
    Synonyms and Secondary IDs (4)
    Reported As
    Symbol Synonym
    Hippo Signaling Pathway Interactome
    Hippo_Pathway_Interactome
    Name Synonyms
    204 high-confidence interactions involving components of the Hippo signaling pathway.
    Secondary FlyBase IDs
      References (3)