Genetic studies have shown that ubiquitination and endocytosis of the Drosophila ligand Delta in signal-sending cells are required for activation of Notch signaling, but how these events promote Notch activation remains poorly understood. Here, we show that an ubiquitination-defective mutant of the murine Delta-homologue Dll1 is endocytosed but, in contrast to the wild-type Dll1, is unable to subsequently recycle back to the cell surface or to bind Notch1 efficiently. These results demonstrate that ubiquitination, although not required for endocytosis, is essential for Dll1 recycling and that recycling is required to acquire affinity for the receptor. On the other hand, a chimeric molecule encompassing the extracellular domain of Dll1 and the transmembrane/intracellular domain of Dll3, which contains no lysine, is endocytosed, recycled, and interacts with Notch1 but is unable to induce transendocytosis of the extracellular region of Notch1 or to signal. These observations suggest that the chimera uses an ubiquitination-independent signal to recycle, allowing it to acquire affinity for Notch1. Our results support the idea that ligand recycling determines its competence to bind efficiently to the receptor but that this is insufficient to allow it to perform transendocytosis, an event required for activation of Notch signaling. Finally, the present study indicates that Dll1 partially localizes to lipid microdomains, whereas both ubiquitination-defective Dll1 and the Dll1-3 chimera are excluded from these compartments, suggesting that these microdomains provide the environment necessary for Dll1 to activate Notch signaling.ubiquitination ͉ recycling ͉ transendocytosis ͉ membrane microdomains

The translation initiation factor complex eIF3f has an intrinsic deubiquitinase activity and regulates the Notch signaling pathway.

The Notch pathway is involved in cell-cell signaling during development and adulthood from invertebrates to higher eukaryotes. Activation of the Notch receptor by its ligands relies upon a multi-step processing. The extracellular part of the receptor is removed by a metalloprotease of the ADAM family and the remaining fragment is cleaved within its transmembrane domain by a presenilin-dependent ␥-secretase activity. ␥-Secretase processing of Notch has been shown to depend upon monoubiquitination as well as clathrin-mediated endocytosis (CME). We show here that AAK1, the adaptor-associated kinase 1, directly interacts with the membrane-tethered active form of Notch released by metalloprotease cleavage. Active AAK1 acts upstream of the ␥-secretase cleavage by stabilizing both the membrane-tethered activated form of Notch and its monoubiquitinated counterpart. We propose that AAK1 acts as an adaptor for Notch interaction with components of the clathrin-mediated pathway such as Eps15b. Moreover, transfected AAK1 increases the localization of activated Notch to Rab5-positive endocytic vesicles, while AAK1 depletion or overexpression of Numb, an inhibitor of the pathway, interferes with this localization. These results suggest that after ligand-induced activation of Notch, the membrane-tethered form can be directed to different endocytic pathways leading to distinct fates.The Notch gene encodes a receptor, matured by furin, and present at the cell surface as an heterodimer (1, 2). Notch activation is induced upon ligand binding and leads to its extracellular cleavage by TNF-␣-converting enzyme (TACE) 2 (3, 4), followed by a second cleavage within the transmembrane domain by a ␥-secretase complex. The released intracellular domain of Notch translocates to the nucleus and induces transcription of target genes together with the CSL and Mastermind cofactors. Ligand-induced activation of Notch is strictly regulated at several levels, both at the extracellular and the intracellular levels (5-7).An increasing number of data reveals that the activity of both Notch receptor and its ligands is regulated by internalization as well as ubiquitination events (8,9). A large repertoire of Notch pathway regulators has been identified, mainly in invertebrates, and conflicting data have emerged regarding Notch activity and trafficking (5). On one hand, endocytosis appears important for Notch activation in the signal-receiving cell (10 -13) while other studies point to an inhibitory effect (14 -18). Our data suggest that, in mammalian cells, both monoubiquitination and endocytosis are required before ␥-secretase cleavage of ligandactivated Notch (10).As the Notch pathway is conserved throughout evolution, we scanned the literature for genetic modulators of the pathway in invertebrates to further understand the intracellular events that control Notch activation. Loss of worm sel-5 (SEL for suppressor/enhancer of Lin12) results in suppression of the constitutive activity of lin-12(d) (equivalent to the metalloprotease-cleaved Notch or ⌬E c...

BACKGROUND: AKT, a critical effector of the phosphoinositide 3-kinase (PI3K) signalling cascade, is an intensely pursued therapeutic target in oncology. Two distinct classes of AKT inhibitors have been in clinical development, ATP-competitive and allosteric. Class-specific differences in drug activity are likely the result of differential structural and conformational requirements governing efficient target binding, which ultimately determine isoform-specific potency, selectivity profiles and activity against clinically relevant AKT mutant variants. METHODS: We have carried out a systematic evaluation of clinical AKT inhibitors using in vitro pharmacology, molecular profiling and biochemical assays together with structural modelling to better understand the context of drug-specific and drug-class-specific cell-killing activity. RESULTS: Our data demonstrate clear differences between ATP-competitive and allosteric AKT inhibitors, including differential effects on non-catalytic activity as measured by a novel functional readout. Surprisingly, we found that some mutations can cause drug resistance in an isoform-selective manner despite high structural conservation across AKT isoforms. Finally, we have derived drug-class-specific phosphoproteomic signatures and used them to identify effective drug combinations. CONCLUSIONS: These findings illustrate the utility of individual AKT inhibitors, both as drugs and as chemical probes, and the benefit of AKT inhibitor pharmacological diversity in providing a repertoire of context-specific therapeutic options.