title: Evidence of non-canonical NOTCH signaling: Delta-like 1 homolog (DLK1) directly interacts with the NOTCH1 receptor in mammals.
abstract: Canonical NOTCH signaling, known to be essential for tissue development, requires the Delta-Serrate-LAG2 (DSL) domain for NOTCH to interact with its ligand. However, despite lacking DSL, Delta-like 1 homolog (DLK1), a protein that plays a significant role in mammalian development, has been suggested to interact with NOTCH1 and act as an antagonist. This non-canonical interaction is, however controversial, and evidence for a direct interaction, still lacking in mammals. In this study, we elucidated the putative DLK1-NOTCH1 interaction in a mammalian context. Taking a global approach and using Dlk1(+/+) and Dlk1(-/-) mouse tissues at E16.5, we demonstrated that several NOTCH signaling pathways indeed are affected by DLK1 during tissue development, and this was supported by a lower activation of NOTCH1 protein in Dlk1(+/+) embryos. Likewise, but using a distinct Dlk1-manipulated (siRNA) setup in a mammalian cell line, NOTCH signaling was substantially inhibited by DLK1. Using a mammalian two-hybrid system, we firmly established that the effect of DLK1 on NOTCH signaling was due to a direct interaction between DLK1 and NOTCH1. By careful dissection of this mechanism, we found this interaction to occur between EGF domains 5 and 6 of DLK1 and EGF domains 10-15 of NOTCH1. Thus, our data provide the first evidence for a direct interaction between DLK1 and NOTCH1 in mammals, and substantiate that non-canonical NOTCH ligands exist, adding to the complexity of NOTCH signaling.
authors.affiliation: Laboratory of Molecular and Cellular Cardiology, Dep. of Clinical Biochemistry and Pharmacology, Odense University Hospital (OUH), Winsloewparken 21 3rd, 5000 Odense C, Denmark; Cardiovascular and Renal Research (University of Southern Denmark), OUH, Denmark.
authors.forename: Gunnhildur Ásta
authors.initials: GÁ
authors.lastname: Traustadóttir
authors.affiliation: Laboratory of Molecular and Cellular Cardiology, Dep. of Clinical Biochemistry and Pharmacology, Odense University Hospital (OUH), Winsloewparken 21 3rd, 5000 Odense C, Denmark; The Danish Centre for Regenerative Medicine (danishcrm@com), OUH, Denmark.
authors.forename: Charlotte H
authors.initials: CH
authors.lastname: Jensen
authors.affiliation: Dep. of Clinical Genetics (OUH), OUH, Denmark; Clinical Institute (University of Southern Denmark), OUH, Denmark.
authors.forename: Mads
authors.initials: M
authors.lastname: Thomassen
authors.affiliation: Clinical Institute (University of Southern Denmark), OUH, Denmark; Dep. of Clinical Biochemistry and Pharmacology (OUH), OUH, Denmark.
authors.forename: Hans Christian
authors.initials: HC
authors.lastname: Beck
authors.affiliation: Laboratory of Molecular and Cellular Cardiology, Dep. of Clinical Biochemistry and Pharmacology, Odense University Hospital (OUH), Winsloewparken 21 3rd, 5000 Odense C, Denmark; Dep. of Clinical Immunology (OUH), Denmark.
authors.forename: Sussi B
authors.initials: SB
authors.lastname: Mortensen
authors.affiliation: Department of Inorganic and Organic Chemistry and Biochemistry, Medical School, Regional Center for Biomedical Research, University of Castilla-La Mancha, Albacete, Spain.
authors.forename: Jorge
authors.initials: J
authors.lastname: Laborda
authors.affiliation: Department of Inorganic and Organic Chemistry and Biochemistry, Medical School, Regional Center for Biomedical Research, University of Castilla-La Mancha, Albacete, Spain.
authors.forename: Victoriano
authors.initials: V
authors.lastname: Baladrón
authors.affiliation: Laboratory of Molecular and Cellular Cardiology, Dep. of Clinical Biochemistry and Pharmacology, Odense University Hospital (OUH), Winsloewparken 21 3rd, 5000 Odense C, Denmark; Cardiovascular and Renal Research (University of Southern Denmark), OUH, Denmark; The Danish Centre for Regenerative Medicine (danishcrm@com), OUH, Denmark.
authors.forename: Søren P
authors.initials: SP
authors.lastname: Sheikh
authors.affiliation: Laboratory of Molecular and Cellular Cardiology, Dep. of Clinical Biochemistry and Pharmacology, Odense University Hospital (OUH), Winsloewparken 21 3rd, 5000 Odense C, Denmark; Clinical Institute (University of Southern Denmark), OUH, Denmark; The Danish Centre for Regenerative Medicine (danishcrm@com), OUH, Denmark. Electronic address: dandersen@health.sdu.dk.
authors.forename: Ditte C
authors.initials: DC
authors.lastname: Andersen
mesh_terms: D041721:3T3-L1 Cells; D000818:Animals; D002135:Calcium-Binding Proteins; D036341:Intercellular Signaling Peptides and Proteins / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D018345:Mice, Knockout; D017434:Protein Structure, Tertiary; D051881:Receptor, Notch1 / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction / Q000502:physiology*
chemical_list: D002135:Calcium-Binding Proteins; C512925:Dlk1 protein, mouse; D036341:Intercellular Signaling Peptides and Proteins; C497762:Notch1 protein, mouse; D051881:Receptor, Notch1
keywords: Delta-like 1 homolog (Dlk1); Non-canonical NOTCH signaling; Tissue development; Two mammalian hybrid assay