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.
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.
Institut für Molekularbiologie, Medizinische Hochschule Hannover, Hannover, Germany.
Eike-Benjamin
EB
Braune
Karin
K
Schuster-Gossler
Marcin
M
Lyszkiewicz
Katrin
K
Serth
Kristina
K
Preusse
Johannes
J
Madlung
Boris
B
Macek
Andreas
A
Krueger
Achim
A
Gossler
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D030161:Avian Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001402:B-Lymphocytes / Q000166:cytology / Q000378:metabolism*; D019521:Body Patterning / Q000502:physiology*; D016466:CHO Cells; D002135:Calcium-Binding Proteins; D002645:Chickens; D006224:Cricetinae; D003412:Cricetulus; D005260:Female; D057809:HEK293 Cells; D006367:HeLa Cells; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D007739:L Cells; D008024:Ligands; D051379:Mice; D008817:Mice, Mutant Strains; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D011247:Pregnancy; D051880:Receptors, Notch / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D015398:Signal Transduction; D029867:Xenopus Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D030161:Avian Proteins; D002135:Calcium-Binding Proteins; C560106:DLL1 protein, Xenopus; C512925:Dlk1 protein, mouse; D036341:Intercellular Signaling Peptides and Proteins; D008024:Ligands; D051880:Receptors, Notch; D011994:Recombinant Proteins; D029867:Xenopus Proteins