The EGF-like proteins DLK1 and DLK2 function as inhibitory non-canonical ligands of NOTCH1 receptor that modulate each other's activities

• Published in: Biochimica et biophysica acta Vol. 1813; no. 6; pp. 1153 - 1164
• Main Authors: Sánchez-Solana, Beatriz, Nueda, María Luisa, Ruvira, María Desamparados, Ruiz-Hidalgo, María José, Monsalve, Eva María, Rivero, Samuel, García-Ramírez, José Javier, Díaz-Guerra, María José M., Baladrón, Victoriano, Laborda, Jorge
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01.06.2011
• Subjects: 3T3 Cells; 3T3-L1 Cells; Adipocytes - cytology; Adipocytes - metabolism; Animals; Binding, Competitive; Blotting, Western; Calcium-Binding Proteins - genetics; Calcium-Binding Proteins - metabolism; Cells, Cultured; DLK1; DLK2; Embryo, Mammalian - cytology; Fibroblasts - cytology; Fibroblasts - metabolism; HEK293 Cells; Humans; Immunoprecipitation; Intercellular Signaling Peptides and Proteins - genetics; Intercellular Signaling Peptides and Proteins - metabolism; Intracellular Signaling Peptides and Proteins - genetics; Intracellular Signaling Peptides and Proteins - metabolism; Membrane Proteins - genetics; Membrane Proteins - metabolism; Mice; Mice, Inbred BALB C; Mice, Knockout; NOTCH canonical ligand; NOTCH signaling; Protein Binding; Protein–protein interaction; Receptor, Notch1 - genetics; Receptor, Notch1 - metabolism; Serrate-Jagged Proteins; Signal Transduction; Two-Hybrid System Techniques; NOTCH signaling; NOTCH canonical ligand; DLK1; Protein–protein interaction; DLK2
• ISSN: 0167-4889; 0006-3002; 1879-2596
• DOI: 10.1016/j.bbamcr.2011.03.004

The protein DLK2, highly homologous to DLK1, belongs to the EGF-like family of membrane proteins, which includes NOTCH receptors and their DSL-ligands. The molecular mechanisms by which DLK proteins regulate cell differentiation and proliferation processes are not fully established yet. In previous reports, we demonstrated that DLK1 interacts with itself and with specific EGF-like repeats of the NOTCH1 extracellular region involved in the binding to NOTCH1 canonical ligands. Moreover, the interaction of DLK1 with NOTCH1 caused an inhibition of basal NOTCH signaling in preadipocytes and mesenchymal multipotent cells. In this work, we demonstrate, for the first time, that DLK2 interacts with itself, with DLK1, and with the same NOTCH1 receptor region as DLK1 does. We demonstrate also that the interaction of DLK2 with NOTCH1 similarly results in an inhibition of NOTCH signaling in preadipocytes and Mouse Embryo fibloblasts. In addition, we demonstrate that a membrane DLK1 variant, lacking the sequence recognized by the protease TACE, also inhibits NOTCH signaling. Furthermore, both DLK1 and DLK2 are able to decrease NOTCH activity also when triggered by specific NOTCH ligands. However, the decrease in NOTCH signaling induced by overexpression of Dlk2 is reversed by the overexpression of Dlk1, and viceversa. We conclude that DLK1 and DLK2 act as inhibitory non-canonical protein ligands for the NOTCH1 receptor that modulate NOTCH signaling. ► EGF-like protein DLK2 interacts with DLK1/Pref-1 and the EGF 10–15 region of NOTCH1 receptor. ► DLK2 protein inhibits NOTCH activity. ► DLK proteins compete with JAGGED1 and DLL4 canonical ligands for NOTCH receptor binding. ► The co-expresion of DLK1 and DLK2 results in higher levels of NOTCH activity.