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Overview of Notch Signaling Pathways
The Notch family of receptors includes Notch-1, -2, -3, and -4 are highly conserved proteins with a wide range of physiological roles including regulating cell fate, proliferation, angiogenesis, cell survival, and the immune response. Like many other proteins associated with these processes, aberrant Notch activity is reported to have complex and context-dependent effects on tumorigenesis.
Upon translation, Notch undergoes several pre-processing steps during its transport to the membrane. These include the addition of O-fucose by O-fucosyltransferase 1 (POFUT1) in the endoplasmic reticulum and, in the Golgi, the addition of N-acetylglucosamine by any of three N-acetylglucosaminetransferases that in vertebrates include Lunatic Fringe (LFNG), Manic Fringe (MFNG), and Radical Fringe (RFNG). Also in the Golgi, Notch is cleaved by Furin to produce a heterodimer consisting of the Notch intracellular domain (NICD) and the Notch extracellular domain (NECD). The heterodimer is then transported to the membrane where it exists as a single pass transmembrane protein. Notch is thought to be in a cycling state that includes endocytosis and re-insertion into the membrane or it may be targeted for lysosomal degradation.
Notch is activated by a unique process that includes ligand binding and multistep proteolytic processing. Invertebrate, Notch ligands include Delta, Serrate, and Lag2 (DSL), while their DSL counterparts in mammals include Delta-like (DLL)-1, -3, -4, Jagged 1, and Jagged 2. Like Notch, DSL ligands are single pass transmembrane receptors and typical Notch activation includes direct cell-cell interaction (trans-activation). Subsequent to binding Notch, the intracellular domain of the Notch ligand is ubiquitinated via the E3 ligase Mind Bomb-1. This initiates endocytosis of the Notch ligand/NECD complex into the ligand-expressing cell. Common endocytic factors have been implicated in this process including Clathrin, Dynamin, Epsin, and Picalm. The mechanical forces generated by these endocytosis-related events may be important for the next steps in the Notch pathway that include sequential proteolytic cleavage of Notch. Notch is first cleaved by TACE/ADAM17 and then the gamma-Secretase complex that includes Presenilin, PSENEN/PEN-2, APH1, and Nicastrin. Whether gamma-Secretase cleavage occurs at the membrane or the endosomal compartment is still a matter of investigation. After its cleavage, the NICD is released into the cytosol and translocated to the nucleus.
Notch activity is primarily dependent on its ability to regulate gene transcription. Recombination Signal Binding Protein for Immunoglobulin Kappa J Region (RBPj) plays a crucial role in Notch-mediated gene transcription. RBPj is also known as CBF-1, or CSL based on its mammalian (CBF-1), Drosophila (Suppressor of Hairless), and C. elegans (Lag-2) orthologs. In the absence of Notch activation, CSL/RBPj acts as a transcriptional repressor in complex with a growing list of co-repressors, linker proteins, and enzymes such as histone deacetylases (HDACs). In the nucleus the NICD displaces transcriptional repressors and forms a complex with CSL/RBPj and Mastermind-like (MAML). MAML recruits transcriptional co-activators, such as the histone acetyltransferase p300, forming a Notch activator complex that culminates in the transcription of Notch target genes. The number of proteins associated with regulating the activator complex continues to grow. There are kinases that can directly phosphorylate and positively or negatively regulate the NICD. In addition, a range of DNA-binding factors and proteins that directly interact with the NICD exists that can either promote or inhibit transcription depending on the context. Turnover of the NICD is high and phosphorylation by CDK8 promotes recognition by the E3 ligase FBW7, resulting in NICD ubiquitination and proteasomal degradation. There appears to be a complex equilibrium in place, and the balance between of the opposing regulators that dictate the overall level of Notch activity.
Binding of Notch by DSL ligands and transcriptional activation involving CSL/RBPj is considered the canonical Notch pathway. However, descriptions of non-canonical signaling continue to be described. Several non-canonical Notch ligands have been identified that have varied effects including the inhibition or activation of the Notch pathways. In addition, it is evident that there is crosstalk between Notch and other signaling pathways, including Akt/mTOR, NF kappa B, Wnt/beta-Catenin, and others.