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Identification of Notch binding protiens and localization of Notch to focal adhesions
Author(s) -
Havel Shelby Lee,
Gazdik Tana Ranae,
Wood Travis Lee,
LaFoya Bryce,
Albig Allan
Publication year - 2018
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2018.32.1_supplement.652.28
Subject(s) - notch signaling pathway , notch proteins , microbiology and biotechnology , focal adhesion , ankyrin repeat , hes3 signaling axis , immunoprecipitation , lim domain , proximity ligation assay , chemistry , biotinylation , signal transduction , biology , transcription factor , receptor , biochemistry , zinc finger , gene
The Notch signaling pathway begins at the cell surface when Notch receptors interact with Notch ligands on adjacent cells triggering an intramolecular cleavage that releases the NICD (or Notch Intracellular Domain fragment) that then enters the nucleus to drive transcription. In addition to this cannonical Notch mechanism, our lab previously demonstrated that Notch signaling is modified by integrin ligation, via a previously unknown non‐cannonical mechanism. In unpublished research performed by LaFoya et al, SRC kinase has now been determined to bind to, and phosphorylate a small peptide near the ankyrin domain of Notch 1, resulting in modification of Notch signaling. The goal of our project is to expand upon these results by discovering other proteins that may interact with this region of Notch. To accomplish this goal, we constructed a fusion protein of the Notch peptide and the BirA protein, and performed a proximity biotin ligation screen in 293T cells for interacting proteins. Biotinylated proteins were isolated by streptavidin magnetic beads, and identified by MS/MS. Amongst the many hits, we identified members of the SRC family of kinases and several proteins commonly associated with focal adhesions and/or tight junctions. Western blotting confirmed the presence of focal adhesion proteins α‐actinin 1 and α‐actinin 4, and tight junction protiens cingulin, and angiomotin, in the biotin precipitated fractions. Subsequent co‐immunoprecipitation experiments in HMEC endothelial cells positively determined an interaction between the NICD domain and α‐actinin1, but failed to confirm interactions with the other proteins. The functional significance of this Notch‐actinin interaction is currently unknown, although given that SRC and actinin both localize to focal adhesions, we speculate that Notch may localize to focal adhesions where it is phosphorylated by SRC kinases. Support or Funding Information NIH Grant 2R15GM102852‐02 This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .