S/T Phosphorylation of DLL1 Is Required for Full Ligand Activity In Vitro but Dispensable for DLL1 Function In Vivo during Embryonic Patterning and Marginal Zone B Cell Development

Abstract

Interaction of Notch receptors with Delta- and Serrate-type ligands is an evolutionarily conserved mechanism that mediates direct communication between adjacent cells and thereby regulates multiple developmental processes. Posttranslational modifications of both receptors and ligands are pivotal for normal Notch pathway function. We have identified by mass spectrometric analysis two serine and one threonine phosphorylation sites in the intracellular domain of the mouse Notch ligand DLL1. Phosphorylation requires cell membrane association of DLL1 and occurs sequentially at the two serine residues. Phosphorylation of one serine residue most likely by protein kinase B primes phosphorylation of the other serine. A DLL1 variant, in which all three identified phosphorylated serine/threonine residues are mutated to alanine and valine, was more stable than wild-type DLL1 but had reduced relative levels on the cell surface and was more effectively cleaved in the extracellular domain. In addition, the mutant variant activated Notch1 significantly less efficient than wild-type DLL1 in a coculture assay in vitro. Mice, however, whose endogenous DLL1 was replaced with the phosphorylation-deficient triple mutant developed normally, suggesting compensatory mechanisms under physiological conditions in vivo.

SUPPLEMENTAL MATERIAL

Supplemental material for this article may be found at http://dx.doi.org/10.1128/MCB.00965-13.

ACKNOWLEDGMENTS

We thank C. Redeker for the Flag-DICD expressing CHO cell line, Alain Israël for HeLaN1 cells, David Ish-Horowicz, Chris Kintner, and Domingos Henrique for X-Delta1 and chicken DLL1 constructs and cDNAs, Michele Calos for attP, attB, and integrase plasmids, Gerry Weinmaster for the tagged-Notch expression vector, and Matthias Gaestel and Michael Stauber for helpful discussions and critical comments on the manuscript.

This study was supported by Deutsche Forschungsgemeinschaft grant GO449/10 to A.G. and by funding from the Cluster of Excellence (From Regenerative Biology to Reconstructive Therapy).