The Notch ligand Delta1 is sequentially cleaved by an ADAM protease and γ-secretase

Abstract

Notch signaling is involved in numerous cell fate decisions in invertebrates and vertebrates. The Notch receptor is a type I transmembrane (TM) protein that undergoes two proteolytic steps after ligand binding, first by an ADAM (a distintegrin and metalloprotease) in the extracellular region, followed by ␥-secretasemediated cleavage inside the TM domain. We demonstrate here that the murine ligand Delta1 (Dll1) undergoes the same sequence of cleavages, in an apparently signal-independent manner. Identification of the ADAM-mediated shedding site localized 10 aa N-terminal to the TM domain has enabled us to generate a noncleavable mutant. Kuzbanian͞ADAM10 is involved in this processing event, but other proteases can probably substitute for it. We then show that Dll1 is part of a high-molecular-weight complex containing presenilin1 and undergoes further cleavage by a ␥-secretase-like activity, therefore releasing the intracellular domain that localizes in part to the nucleus. Using the sheddingresistant mutant, we demonstrate that this ␥-secretase cleavage depends on prior ectodomain shedding. Therefore Dll1 is a substrate for regulated intramembrane proteolysis, and its intracellular region possibly fulfills a specific function in the nucleus.

Regulation of Notch1 Signaling by Delta-like Ligand 1 Intracellular Domain through Physical Interaction

“…It has been previously reported that a truncated intracellular isoform of Delta evidences prominent nuclear localization (Bland et al, 2003;Ikeuchi and Sisodia, 2003;LaVoie and Selkoe, 2003;Six et al, 2003). The intracellular domain of Delta1 results in a non-proliferating senescent-like cell phenotype that depends on the expression of the cell cycle inhibitor, p21, and is suppressed by constitutively active Notch1 (Kolev et al, 2005).…”

“…Delta is cleaved sequentially by the RIP mechanism probably including ADAM and gamma-secretase (Ikeuchi and Sisodia, 2003;LaVoie and Selkoe, 2003;Qi et al, 1999;Six et al, 2003), and the cleaved Dll1-IC is released from the cell membrane and translocates to the nucleus, where it suppresses notch signaling through the disruption of Notch1-IC transcriptional complex. Henceforth, the results of this study may begin to shed some light on what may be a signal cross-talk mechanism of Notch and Delta after cleavage by gamma-secretase.…”

“…It was recently demonstrated that many single transmembranespanning proteins--including Notch, Delta and amyloid precursor protein (APP)--are substrates for gamma-secretase (Koo and Kopan, 2004). Delta has also been shown to be cleaved by ADAM protease and gamma-secretase to release an intracellular domain and thereby generate Dll1-IC, which is trafficked to the nucleus (Ikeuchi and Sisodia, 2003;LaVoie and Selkoe, 2003;Qi et al, 1999;Six et al, 2003). Indeed, several groups have produced evidence of the nuclear localization of the intracellular fragments of Notch ligands.…”

Delta expression in post‐mitotic neurons identifies distinct subsets of adult‐specific lineages in Drosophila

“…Kuzbanian and Neuralized are the central enzymes in the proteolytic and endocytosis-mediated processes, respectively, that regulate Delta expression at the cell surface (Lai et al, 2001;Mishra et al, 2002;reviewed in Le Borgne et al, 2005). Delta undergoes cleavage by Kuzbanian, resulting in shedding and clearance of the extracellular domain (Bland et al, 2003;Six et al, 2003), a process that ultimately downregulates its ligand activity. Thus, it is possible that elevated Delta levels occur where Kuzbanian expression is suppressed.…”

“…toma cells promote filipodia formation and process outgrowth (Franklin et al, 1999;De Joussineau et al, 2003), suggesting that Delta acts to mediate changes in neurite architecture. Furthermore, several recent studies have identified alternative mechanisms for Delta function through proteolytic and endocytosismediated processes that alter the structure and subcellular distribution of the Delta protein (Parks et al, 2000;Bland et al, 2003;LaVoie and Selkoe, 2003;Six et al, 2003). Altogether, these studies point to an additional level of activity of the Delta protein that remains to be elucidated.…”