ITCH binds DTX

Genetic studies in Drosophila identified deltex as a positive regulator of Notch signaling, while the Drosophila homologue of ITCH (AIP4) was identified as a negative regulator of Notch signaling and named suppressor of deltex. ITCH and DTX1 interact and form a complex, as determined by co-immunoprecipitaion experiments in human embryonic kidney cell line HEK293 in which tagged recombinant human DTX1 and ITCH were expressed. It is not known whether this complex involves other proteins, but its formation is NOTCH-independent. Both DTX1 and ITCH are ubiquitin ligases. DTX1 is a RING-type ubiquitin ligase, while ITCH is a HECT-type ubiquitin ligase. The ubiquitin ligase activity of either protein is not needed for the formation of the DTX1:ITCH complex, and the inactive ITCH mutant co-immunoprecipitates more DTX1 than the wild-type ITCH, implicating the ubiquitin ligase activity of ITCH in DTX1 degradation.

在果蝇的遗传研究中，研究者鉴定出deltex为Notch信号通路的正向调控因子，而果蝇中ITCH（AIP4）的同源物被确认为Notch信号通路的负向调控因子，并被命名为deltex的抑制因子。通过在人类胚胎肾细胞系HEK293中进行的共免疫沉淀实验证实，ITCH与DTX1相互作用并形成复合物，其中标记的重构人DTX1和ITCH在细胞中得以表达。目前尚不清楚该复合物是否涉及其他蛋白质，但其形成过程与NOTCH无关。DTX1和ITCH均为泛素连接酶。DTX1是一种RING型泛素连接酶，而ITCH是一种HECT型泛素连接酶。这两种蛋白质的泛素连接酶活性并非形成DTX1:ITCH复合物的必需条件，且非活性ITCH突变体与野生型ITCH相比，能更多地与DTX1共免疫沉淀，这表明ITCH的泛素连接酶活性在DTX1降解中起到重要作用。