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
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
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DOI：
10.1128/mcb.00965-13
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发表时间：
2014-04-01
影响因子：
5.3
通讯作者：
Gossler, Achim
中科院分区：
文献类型：
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作者：
Braune, Eike-Benjamin;Schuster-Gossler, Karin;Gossler, Achim
Notch受体与Delta型和serrate型配体的相互作用是一种进化保守的机制，介导相邻细胞之间的直接通信，从而调节多种发育过程。受体和配体的翻译后修饰是正常Notch通路功能的关键。我们通过质谱分析在小鼠Notch配体DLL1的细胞内区域鉴定了两个丝氨酸和一个苏氨酸磷酸化位点。磷酸化需要DLL1的细胞膜结合，并顺序发生在两个丝氨酸残基上。一个丝氨酸残基的磷酸化很可能是由蛋白激酶B引起的，它会引发另一个丝氨酸的磷酸化。在DLL1变体中，所有三个鉴定的磷酸化丝氨酸/苏氨酸残基突变为丙氨酸和缬氨酸，比野生型DLL1更稳定，但在细胞表面的相对水平降低，并且在细胞外区域更有效地切割。此外，在体外共培养试验中，突变变体激活Notch1的效率明显低于野生型DLL1。然而，将内源性DLL1替换为磷酸化缺陷三重突变体的小鼠发育正常，这表明在体内生理条件下存在代偿机制。
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.