Mechanisms of insect limb development

配体诱导质膜受体Notch的蛋白水解性切割和内化导致其激活。然而，非配体依赖的、稳定的Notch内化并不会导致激活。下游效应器区分这些Notch内化的两部分模式的机制尚不清楚。Nedd4是一种含有Hect结构域的E3泛素连接酶，针对含有PPSY基序的跨膜受体进行内吞作用。Deltex是Notch信号的正调控因子，编码一种可能的环指型泛素连接酶。我们发现Notch是泛素化的，并且被Nedd4以一种需要Notch胞内域中的PPSY基序的方式破坏稳定。Nedd4功能的丧失主要抑制Notch和Deltex突变体的表型，其过度激活减弱了Notch的活性。在组织培养细胞中，显性-负性形式的Nedd4阻断了Notch的稳态内化，并独立于配体结合激活了Notch信号，Deltex进一步加强了这一作用。Nedd4注定Deltex以一种依赖于Notch的方式降解。Nedd4通过促进Notch的内化以及Notch和Deltex的降解来拮抗Notch信号。Nedd4的这些功能对于保护未受刺激的细胞免受Notch信号的零星激活可能是重要的。

Ligand-induced proteolytic cleavage and internalization of the plasma membrane receptor Notch leads to its activation. Ligand-independent, steady state internalization of Notch, however, does not lead to activation. The mechanism by which downstream effectors discriminate these bipartite modes of Notch internalization is not understood. Nedd4 is a HECT domain-containing E3 ubiquitin ligase that targets transmembrane receptors containing the PPSY motif for endocytosis. Deltex is a positive regulator of Notch signaling and encodes a putative ubiquitin ligase of the ring finger type. We show that Notch is ubiquitinated, and destabilized by Nedd4 in a manner requiring the PPSY motif in the Notch intracellular domain. Loss of Nedd4 function dominantly suppresses the Notch and Deltex mutant phenotypes and its hyper activation attenuates Notch activity. In tissue culture cells, dominant-negative form of Nedd4 blocks steady-state Notch internalization and activates Notch signaling independently of ligand binding, and this effect was further potentiated by Deltex. Nedd4 destines Deltex to degradation in a Notch-dependent manner. Nedd4 antagonizes Notch signaling by promoting internalization of Notch and degradation of Notch and Deltex. Those functions of Nedd4 may be important for the protection of unstimulated cells from sporadic activation of Notch signaling.